A pharmaceutical gastroretentive formulation for doxorubicin, a method of preparation thereof, and its use for treating malignancies

Abstract

The present disclosure provides a pharmaceutical formulation for Doxorubicin or its pharmaceutically acceptable salts, configured to enhance therapeutic cellular uptake while minimizing side effects. The formulation comprises a polymeric nano micelle including a therapeutically effective amount of Doxorubicin, monoterpene, and sodium alginate, wherein the pharmaceutical formulation is configured to provide Doxorubicin gastric targeting delivery system. The present disclosure further provide the use of the pharmaceutical formulation for treating malignances, and a method of preparation thereof.

Description

[0001] A PHARMACEUTICAL GASTRORETENTIVE FORMULATION FOR DOXORUBICIN, A METHOD OF PREPARATION THEREOF, AND ITS USE FOR TREATING MALIGNANCIES

[0002] TECHNICAL FIELD

[0003]

[0001] The present disclosure relates to drug formulations, and more particularly to a Doxorubicin (“DOX”) gastric targeting formulation by carvacrol coating, and nanoparticles encapsulation to enhance the cellular uptake of DOX and decrease side effects related to injection administration.

[0004] BACKGROUND

[0005]

[0002] Doxorubicin (“DOX”) is widely known as an anticancer and antibacterial drug that intercalates into DNA and inhibits topoisomerase-II-mediated DNA repair. Traditional administering through intravenous injection, DOX is used in the treatment of various malignancies, including stomach cancer. However, despite its therapeutic effectiveness, this route of administration is associated with numerous side effects, such as cough, fever, chills, skin irritation, anaphylaxis, azoospermia, dyspnea, heart disease, and injection pain. These side effects resulted from the unselective systemic distribution of DOX, due to its rapid distribution half-life of 3-5 minutes and extensive metabolism. Consequently, the development of oral delivery systems that can accurately target malignant tissues is essential for minimizing side effects and enhancing cellular uptake.

[0006]

[0003] Therefore, attempts in the prior art were made to develop oral delivery systems that target malignant tissues. For instance, the Chinese patent application published under number CN 112438944 discloses a gel pharmaceutical composition that would enhance drug delivery and therapeutic efficacy in cancer treatment, the pharmaceutical composition integrates a chemotherapeutic drug within a specialized gel matrix. This matrix includes natural polymers such as chitosan, dextran, cellulose, sodium alginate, and hyaluronic acid, alongside various synthetic polymers. Furthermore, the synthetic polymer component includes poly N-isopropylacrylamide copolymer, polyoxyethylene / polyoxypropylene,polyethylene glycol, polyvinyl alcohol, polylactic acid-glycolic acid copolymer, and polyethylene oxide sodium glycerophosphate.

[0007]

[0004] A study by Katuwavila et al. (2016) titled “Chitosan- Alginate Nanoparticle System Efficiently Delivers Doxorubicin to MCF-7 Cells” discloses the preparation of chitosanalginate nanoparticles encapsulating doxorubicin (DOX) through an ionic gelation method. The use of nanoparticles as effective carriers for targeted chemotherapy, in vitro studies showed two-phase drug release profile, demonstrating superior cumulative release and cytotoxicity against MCF-7 cells.

[0008]

[0005] A study by Akhlaq et al. (2023) titled “Carvacrol-Fabricated Chitosan Nanoparticles Synergistic Potential with Topoisomerase Inhibitor on Breast and Cervical Cancer Cells” discloses the anticancer effects of carvacrol-loaded chitosan nanoparticles in conjunction with topoisomerase inhibitors. The study indicates that the combination of Carvacrol-Chitosan-DOX leads to enhanced dose-dependent growth inhibition of both MCF-7 and HeEa cells, improving the efficacy of chemotherapeutics, addressing drug resistance issues, and promoting targeted delivery.

[0009] SUMMARY

[0010]

[0006] It is an object of the present disclosure to provide a pharmaceutical formulation for Doxubirucine (“DOX”) or any pharmaceutically acceptable salt thereof, the pharmaceutical formulation may include a therapeutically effective amount of DOX or any pharmaceutically acceptable salt thereof, monoterpenes, and alginate, wherein monoterpene may include Carvacrol (“CRV”), a hydrophobic volatile oil configured to enhance the cellular penetration of DOX. The alginates may include Sodium Alginate (“SA”) configured to decrease the amount of DOX by increasing sensitivity. The pharmaceutical formulation may further include any acceptable excipients.

[0011]

[0007] In some aspects of the present disclosure, the pharmaceutical formulation may include a polymeric nano micelle formation having DOX, CRV, and SA.

[0012]

[0008] In some aspects, a complexation ratio of DOX: CRV ranges from about 1 : 1 to about 0.065:1 by weight (mg / mg).

[0013]

[0009] In some aspects, the complexation ratio of DOX: CRV is 1:4 by weight (mg / mg).

[0010] In some aspects, the SA amount ranges from about 8 mg / ml to about 32 mg / ml.

[0014] [Oil] In some aspects, the SA amount is 16 mg / ml.

[0015]

[0012] In some aspects, the DOX, CRV, and SA are oriented in the polymeric nano micelle as hydrophilic core including DOX, hydrophobic middle layer including CRV and hydrophilic outer layer including SA.

[0016]

[0013] In some aspects, DOX and CRV may have a hydrophilic intramolecular binding, CRV and SA may have hydrophilic and hydrophobic intramolecular bindings, and SA and free DOX molecules may have a hydrophilic intramolecular binding.

[0017]

[0014] In some aspects, the hydrophilic and hydrophobic intramolecular bindings between the DOX-CRV-SA nano particles are configured to provide charge stabilization and neutralization for the formulation stimulating gastrointestinal epithelial cell adhesion.

[0018]

[0015] In some aspects, the pharmaceutical formulation may have a small particle size configured to increase the bioavailability of DOX.

[0019]

[0016] In some aspects, the pharmaceutical formulation may have a zeta- potential value of about -0.51 ± 0.67 mV.

[0020]

[0017] In some aspects, the pharmaceutical formulation may have polydispersity index (“PDI”) value of about 0.24 ± 0.13.

[0021]

[0018] In some aspects, the pharmaceutical formulation distribution and size of circular micellar formation may be less than or equal to 100 nm.

[0022]

[0019] Other aspects provide a drug delivery system for targeting gastric release including the pharmaceutical formulation of the present disclosure.

[0023]

[0020] In aspects of the present disclosure, the pharmaceutical formulation is an oral gel formulation.

[0024]

[0021] In some aspects, use of the pharmaceutical formulation for treating malignancies.

[0025]

[0022] In some aspects, malignancies may include stomach cancer.

[0026]

[0023] Aspects of the present disclosure further provide a method for preparing the pharmaceutical formulation, the method may include the steps of:

[0027] Mixing a proper amount of Doxorubicin and Carvacrol to provide an optimized complex and Doxorubicin coated particles with Carvacrol;

[0028] Adding the complex to a proper amount of Sodium Alginate solution to provide a mixture;Maintaining the mixture under high-speed sonication to provide an optimized nano micelle;

[0029] Freeze drying the nano micelle to provide a spongy powder; and

[0030] Inserting the spongy powder into hard-gelatin capsules.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032]

[0024] The present disclosure will now be described with reference to the accompanying drawings, which illustrate embodiments of the present disclosure, without however limiting the scope of protection thereto, and in which:

[0033]

[0025] FIG. 1 illustrates a line chart showing DOX standard calibration curve for a pharmaceutical formulation configured in accordance with one or more embodiments of the present disclosure, wherein values are expressed as mean ± SD.

[0034]

[0026] FIG. 2 illustrates a line chart showing DOX bound molecules per total CRV amount in a pharmaceutical formulation configured in accordance with one or more embodiments of the present disclosure, wherein values are expressed as mean ± SD.

[0035]

[0027] FIG. 3A illustrates a histogram showing particle size distribution curve of a first pharmaceutical formulation configured in accordance with one or more embodiments of the present disclosure, wherein values are expressed as mean ± SD.

[0036]

[0028] FIG. 3B illustrates a histogram showing particle size distribution curve of a second pharmaceutical formulation configured in accordance with one or more embodiments of the present disclosure, wherein values are expressed as mean ± SD.

[0037]

[0029] FIG. 3C illustrates a histogram showing particle size distribution curve of a third pharmaceutical formulation configured in accordance with one or more embodiments of the present disclosure, wherein values are expressed as mean ± SD.

[0038]

[0030] FIG. 4A illustrates floating analysis showing a first pharmaceutical formulation insoluble gel configured in accordance with one or more embodiments of the present disclosure, wherein such analysis is obtained after 8 hours in contact with 0.1N HC1.

[0039]

[0031] FIG. 4B illustrates floating analysis showing a second pharmaceutical formulation insoluble gel configured in accordance with one or more embodiments of the present disclosure, wherein such analysis is obtained after 8 hours in contact with 0.1N HC1.

[0032] FIG. 4C illustrates floating analysis showing a third pharmaceutical formulation insoluble gel configured in accordance with one or more embodiments of the present disclosure, wherein such analysis is obtained after 8 hours in contact with 0.1N HC1.

[0040]

[0033] FIG. 5 illustrates a spectrum showing Fourier Transform-Infrared (FT-IR) of DOX, CRV, SA, DOX-CRV complex, CRV-SA blank and the optimum DOX-CRV-SA polymeric nano micelle, wherein the nano micelle is configured in accordance with one or more embodiments of the present disclosure.

[0041]

[0034] FIG. 6 illustrates a scheme showing DOX, CRV, and SA orientation in the polymeric nano micelle configured in accordance with one or more embodiments of the present disclosure.

[0042]

[0035] FIG. 7A illustrates a diffractogram showing X-Ray analysis of DOX, wherein such diffractogram is obtained by nickel-filtered Cu-Kb reduction using Rigaku, Ultima IV device.

[0043]

[0036] FIG. 7B illustrates a diffractogram showing X-Ray analysis of CRV, wherein such diffractogram is obtained by nickel-filtered Cu-Kb reduction using Rigaku, Ultima IV device.

[0044]

[0037] FIG. 7C illustrates a diffractogram showing X-Ray analysis of SA, wherein such diffractogram is obtained by nickel-filtered Cu-Kb reduction using Rigaku, Ultima IV device.

[0045]

[0038] FIG. 7D illustrates a diffractogram showing X-Ray analysis of DOX-CRV complex configured in accordance with one or more embodiments of the present disclosure, wherein such diffractogram is obtained by nickel-filtered Cu-Kb reduction using Rigaku, Ultima IV device.

[0046]

[0039] FIG. 7A illustrates a diffractogram showing X-Ray analysis of DOX-CRV-SA polymeric nano micelle configured in accordance with one or more embodiments of the present disclosure, wherein such diffractogram is obtained by nickel-filtered Cu-Kb reduction using Rigaku, Ultima IV device.

[0047]

[0040] FIG. 8A illustrates a photomicrograph showing a scanning electron microscope image of DOX.

[0048]

[0041] FIG. 8B illustrates a photomicrograph showing a scanning electron microscope image of CRV.

[0042] FIG. 8C illustrates a photomicrograph showing a scanning electron microscope image of SA.

[0049]

[0043] FIG. 8D illustrates a photomicrograph showing a scanning electron microscope image of DOX-CRV complex configured in accordance with one or more embodiments of the present disclosure.

[0050]

[0044] FIG. 9 illustrates a photomicrograph showing a scanning electron microscope image of DOX-CRV-SA polymeric nano micelle configured in accordance with one or more embodiments of the present disclosure, wherein such photomicrograph is obtained in different magnifications of 1400X, 18000X, and 55000X shown in A, B, and C, respectively.

[0051]

[0045] FIG. 10A illustrates a photomicrograph showing a transmission electron microscope image of CRV-SA polymeric gel.

[0052]

[0046] FIG. 10B illustrates a photomicrograph showing a transmission electron microscope image of DOX-CRV-SA polymeric nano micelle configured in accordance with one or more embodiments of the present disclosure.

[0053]

[0047] FIG. 11 illustrates a chart showing DOX release analysis of DOX, DOX-CRV complex, DOX-SA gel, and the optimum DOX-CRV-SA polymeric nano micelle configured in accordance with one or more embodiments of the present disclosure.

[0054]

[0048] FIG. 12 illustrates a flow chart showing a method of preparing a pharmaceutical formulation for Doxorubicin of or any pharmaceutically acceptable salt thereof configured in accordance with embodiments of the present disclosure.

[0055] DETAILED DESCRIPTION

[0056]

[0049] Embodiments of the present disclosure provide a pharmaceutical formulation for Doxorubicin (“DOX”) or any pharmaceutically acceptable salt thereof, wherein the pharmaceutical formulation may include a therapeutically effective amount of DOX or any pharmaceutically acceptable salt thereof, monoterpenes, and alginates. Monoterpenes may include Carvacrol (“CRV”), a hydrophobic volatile oil configured to enhance the cellular penetration of DOX. The alginates may include Sodium Alginate (“SA”) configured to decrease the amount of DOX by increasing sensitivity of the cancer cells toward the DOX.The pharmaceutical formulation of the present disclosure may further include any acceptable excipients.

[0057]

[0050] In embodiments of the present disclosure, the pharmaceutical formulation may include a polymeric nano micelle formation having DOX, CRV, and SA.

[0058]

[0051] In some embodiments of the present disclosure, the complexation ratio of DOX:CRV ranges from about 1:4 to about 1:1 by weight (mg / mg).

[0059]

[0052] In some embodiments of the present disclosure, the complexation ratio of DOX:

[0060] CRV is preferably 3:4 by weight (mg / mg).

[0061]

[0053] In some embodiments of the present disclosure, the complexation ratio of DOX:

[0062] CRV is more preferably 1 :4 by weight (mg / mg).

[0063]

[0054] In some embodiments, the SA amount may be from about 8 mg / ml to about 32 mg / ml.

[0064]

[0055] In some embodiments, the SA amount is preferably from about 10 mg / ml to about 25 mg / ml.

[0065]

[0056] In some embodiments, the SA amount is more preferably from about 12 mg / ml to about 20 mg / ml.

[0066]

[0057] In some embodiments, the SA amount is more preferably about 16 mg / ml.

[0067]

[0058] In some embodiments, the DOX, CRV, and SA may be oriented in the polymeric nano micelle as a hydrophilic core including DOX, a hydrophobic middle layer including CRV and a hydrophilic outer layer including SA.

[0068]

[0059] In some embodiments, DOX and CRV may have a hydrophilic intramolecular binding, while CRV and SA may have hydrophilic and hydrophobic intramolecular bindings. Alternatively, SA and free DOX molecules may have a hydrophilic intramolecular binding.

[0069]

[0060] In some embodiments, the hydrophilic and hydrophobic intramolecular bindings between the DOX-CRV-SA nano particles are configured to provide charge stabilization and neutralization for the pharmaceutical formulation of the present disclosure, which may stimulate gastrointestinal epithelial cell adhesion.

[0070]

[0061] In some embodiments, the pharmaceutical formulation of the present disclosure may have a small particle size configured to increase the bioavailability of DOX.

[0062] In some embodiments, the pharmaceutical formulation may have a zeta- potential value of about -0.51V ± 0.67 mV.

[0071]

[0063] In some embodiments, the pharmaceutical formulation may have polydispersity index (PDI) value of about 0.24 ± 0.13.

[0072]

[0064] In some embodiments, the pharmaceutical formulation distribution and size of circular micellar formation may be less than or equal to 100 nm.

[0073]

[0065] In some embodiments, the DOX, CRV, and SA may be oriented in the polymeric nano micelle as hydrophilic core (DOX), hydrophobic middle layer (CRV) and hydrophilic outer layer (SA).

[0074]

[0066] In embodiments of the present disclosure, the pharmaceutical formulation is a gel for oral administration.

[0075]

[0067] Embodiments of the present disclosure further provide a drug delivery system for targeting gastric release including the pharmaceutical formulation of the present disclosure.

[0076]

[0068] Use of the pharmaceutical formulation of the present disclosure for treating malignancies.

[0077]

[0069] In some embodiments, the malignancies may include stomach cancer.

[0078]

[0070] Embodiments of the present disclosure provide a method for preparing the pharmaceutical formulation, the method may include the steps of:

[0079] Mixing a proper amount of DOX and CRV to provide an optimized complex and DOX coated particles with CRV (process block 12-1);

[0080] Adding the complex to a proper amount of SA solution to provide a mixture (process block 12-2);

[0081] Maintaining the mixture under high-speed sonication to provide an optimized nano micelle (process block 12-3);

[0082] Freeze drying the nano micelles to provide a spongy powder (process block 12-4); and

[0083] Inserting the spongy powder into hard-gelatin capsules (process block 12-5).

[0084]

[0071] The disclosure will be further illustrated on the basis of examples and a detailed description from which further features and advantages may be taken. It is to be noted that the following explanations are presented for the purpose of illustrating and descriptiononly; they are not intended to be exhaustive or to limit the disclosure to the precise form disclosed.

[0085] Example 1

[0086] Doxorubicin Calibration Curve

[0087]

[0072] Numerous stock solutions using different dilutions of DOX-98% (Sigma-Aldrich, USA) were prepared to create the calibration curve of DOX as shown in FIG. 1. A wavelength of 496 nm was applied to the prepared solutions in water at 30 mg / mL in a 1 cm cell, in comparison to a blank solution of water by UV spectrophotometric analysis (Thermo-Scientific, USA).

[0088]

[0073] Encapsulation efficiency, drug release, and adsorption isotherm were all analyzed using the calibration curve formula shown below. The concentration in the equation has an R2value of 0.990 and was measured in pg / mL.

[0089] Absorbance = 0.1212 * Concentration (pg / mL) — 0.0306

[0090] Example 2

[0091] Determination of the Maximum DOX-CRV Binding Capacity

[0092]

[0074] A plurality of DOX / CRV weight / molar ratios (mg / mg) ranging from about 1:1 to about 0.0625:1 were prepared, as illustrated in Table (1). The volumes of the DOX and CRV solutions were combined and placed into dialysis bags to create co -precipitate mixtures configured to optimize the maximum complexation ratio to be used in the pharmaceutical formulation of the present disclosure. To reach complexation equilibrium, the tightly packed bags were placed in plastic containers with 50 mL of distilled water and shaken overnight. Then, the precipitates obtained from combining DOX:CRV (mg / mg) different ratios were collected to calculate the binding capacity of DOX into CRV. The concentration of free DOX in the media surrounding the bags was used as a measurement parameter using UV spectrophotometry at 496 nm. Since the free DOX molecules that would flow through the dialysis bag holes had unique sizes, the analysis method was created (cut-off value was 12000 Daltons, Sigma-Aldrich, USA).Table (1) DOX and CRV amounts used to anticipate the DOX-CRV binding capacity.

[0093]

[0094]

[0075] FIG.2 illustrates the amount of DOX molecules bounded to CRV in accordance to the embodiments of the present disclosure. At low DOX ratios from about 0.0625:1 to about 0.25 : 1 , hydrogen bond / hydrophilic intramolecular bonds between the DOX (primary amine, hydroxyls, or carboxylic acid) and the phenolic hydroxyl of CRV are formed, resulting in linear increase of DOX:CRV complexation. On the other hand, at DOX:CRV weight ratios from about 0.25:1 to about 1:1, the complexation and binding capacity between DOX:CRV hit a plateau, resulting a statistically negligible change as the ration increased. This might be attributed to intramolecular hydrogen bond formation between DOX molecules, indicating the coverage of DOX molecules by CRV. Accordingly, the maximum capacity binding ratio of DOX:CRV found to be about 0.25: 1 (mg / mg)

[0095] Preparation of DOX:CRV Complex Assay

[0096]

[0076] DOX (about 1 mg) and CRV (about 4 mg) source materials were combined based on the DOX-CRV binding capacity. The mixture was then centrifuged, freeze-dried, and ground with a mortar and pestle to a fine powder. Additionally, about 30 mL of dimethyl sulfoxide (“DMSO”) was used, and about 50 mL centrifuge tube was centrifuged at about 3000 rpm for three minutes in order to analyze the complex powder of about 20 mg. The content of DOX in the supernatant was then determined at 496. The DOX assay per 1 mg of the dry formula was computed using the following formula:Extracted DOX amount

[0097] DOX assay, mg / mg = - - - - - Total powder amount

[0098]

[0077] The assay result of the selected DOX-CRV complex was 0.19±0.06 mg DOX / total complex weight (mg) after isolation, giving an added value to the complexation and isolation methods by having a minute drug and material loss percentage of 5% loss of the drug as the following formula:

[0099] Actual DOX Amount analysed at 496 nm

[0100] % Drug Loss 100

[0101]

[0102] Example 3

[0103] Encapsulation of DOX:CRV Complex

[0104]

[0078] To encapsulate the selected DOX-CRV complex ratio in a polymeric nano-micelle, the complex was maintained for about three minutes at 30 W and 25 KHz using a highspeed sonication machine (UP200St, Hielscher, Germany) in various volumes of SA-low molecular weight (200,000 g / mole) of 6% stock solution as shown in Table (2).

[0105] Table (2): Different DOX (0.4 pg / mL) nano micellar formulations for optimization.

[0106]

[0107] Optimization of DOX:CRV:SA Polymeric Nano Micelle:

[0108]

[0079] The pharmaceutical formulations of table (2) were analyzed based on zeta-sizer evaluation, particle size analysis, and floating analysis to determine the formulation of the present disclosure. The floating analysis was made using 0.1N HC1 to mimic simulated gastric fluid (“SGF”) to measure the insoluble gel's onset, coherence, and shape.

[0109]

[0080] Table (3) illustrates the analysis values for Fl, F2, and F3 of the present disclosure.

[0110] The F2 formulation showed the smallest particle size of 21.33 ± 0.51 nm, which is twice as small as the Fl and four times smaller than the F3 as shown in FIG. 3. Furthermore, F2 formulation results show nanoparticles charge stabilization and neutralization of -0.51 ± 0.67 mV and polydispersity index (PDI) of 0.24 ± 0.13, this might be contributed to formulation's hydrophobic and hydrophilic bindings between SA and CRV, hydrophilic SA and Free DOX, and hydrophilic DOX and CRV. The nanoparticles' neutral zetapotential charge and micellar shape may stimulate gastrointestinal epithelial cell adhesion, resulting in increased DOX cellular penetration and absorption.

[0111] Table (3) DOX (0.4 pg / mL) nano micellar formulations for optimization using the particle size analysis and zeta-potential results values.

[0112]

[0113] * For n=3 ± SD

[0114]

[0081] The analysis values were further validated by the floating capacity of the tested formulations. FIG. 4 and Table (4) illustrate floating analysis characterizations after 8hours of exposure to 0.1N HC, F2 found to be the most lasting, strongest, and lowest density insoluble gel when compared to the Fl and F3 formulations, these findings may be attributed to the conversion of alginate gel into the insoluble alginic acid in an acidic medium.

[0115] Table (4) Gel characterizations of the gel forming formulations in contact of 0.1N HC1.

[0116]

[0117]

[0082] Based on the optimization study of the various formulations, F2 showed the optimum results. Accordingly, DOX:CRV:SA nanoparticles were then freeze-dried to prevent the oils and other ingredients from breaking down. Then, the spongy powder may be inserted into hard-gelatin capsules. The efficacy of the nanoparticle encapsulation was analyzed and calculated for about 35 mg of powder and determined after about 3 minutes of spinning at about 3000 rpm in about 50 mL centrifuge tube, and about 45 mL of DMSO. Then, at 496 nm, the supernatant was examined for DOX amount and concentration. The following formula was used to calculate the DOX encapsulation efficiency per milligram of the F2 dry formula to be 88.60% ± 3.11.

[0118] Extracted DOX nanoparticles amount Encapsulation efficiency = - - - - - * 100

[0119] Total powder amount

[0120] Example 4

[0121] Physiochemical Characterizations of the DOX-CRV Complex and DOX Polymeric Nano Micelle Fourier Transform-Infrared (FT-IR)

[0083] Samples of DOX, CRV, SA, DOX-CRV complex and the optimum DOX-CRV-SA polymeric nano micelle, and blank CRV-SA polymeric gel (about 10 mg) were all subjected to FT-IR spectrum analysis (Thermo-Scientific, NicoletiSlO, USA) with resolution of 500-4000 cm1.

[0122]

[0084] The FT-IR spectrum of the samples illustrated in FIG. 5 provide proof to DOX:CRV:SA polymeric nano micelle formation of the present disclosure. The wide band at the range 3500-3000 cm'1in DOX-CRV spectrum may be contributed to the hydrogen intramolecular bond formation between DOX functionalities (hydroxyls, carboxylic acid, and amine) with the hydroxyl of the CRV. Moreover, DOX-CRV complex spectrum show aromatic and aromatic methyl bands at 1650-1600 cm1, which indicate the surface hydrophobic interaction resulted from CRV coating. On the other hand, DOX:CRV:SA spectrum shows the peaks of SA indicating DOX:CRV complex coating by SA, the blank spectrum further confirms the interaction nature of SA and CRV, which shows the ability of the cyclic carbon chain of SA to bind with the aromatic methyl of CRV.

[0123]

[0085] According to FT-IR spectra, a suggested orientation for DOX, CRV, and SA in the optimized polymeric nano micelle of the present disclosure is provided, the micelle formation of hydrophilic core (DOX), hydrophobic middle layer (CRV) and hydrophilic outer layer (SA) as shown in FIG. 6. This orientation supports the DOX long-acting system formation.

[0124] Powder X-ray Diffractometer (“PXRD”)

[0125]

[0086] X-ray diffraction (“PXRD”) was used to analyze the DOX, CRV, SA, DOX-CRV complex and the optimum DOX-CRV-SA polymeric nano micelle using a Rigaku, Ultima IV, Japan device. X-rays were generated by nickel -filtered Cu-Kb reduction at about 40 kV and about 40 mA. With a scanning speed of about 10 degrees per minute, the scan range (20) covered 5 to 70 degrees.

[0126]

[0087] FIGS. 7A-7E illustrate the diffractograms of DOX, CRV, SA, DOX-CRV complex, and the optimum DOX-CRV-SA polymeric nano micelle, respectively. DOX and CRV diffractograms show clear well-defined peaks to be correlated to the crystalline nature of DOX and CRV. On the other hand, the diffractogram of the SA shows wide peaks at 20values of 1.77 “and 5.77 “to be correlated to its amorphous nature. In a comparison to raw material diffractograms, DOX:CRV complex showed a wide peak at 20 value of 1.92° to be correlated to its amorphous nature as a result of complexation, while the optimum DOX- CRV-SA polymeric nano micelle diffractogram shows a wide peak at 20 value of 1.88°to be correlated to nano micellar formation.

[0127] Morphology and dispersion investigations

[0128]

[0088] The structure and particle distribution of powdered DOX, CRV, SA, DOX-CRV complex and the optimum DOX-CRV-SA polymeric nano micelle / gel, and blank CRV-SA polymeric gel were analyzed using a Scanning Electron Microscope (“SEM”) (JEM-F200, Jeol, Japan). The dry substance was examined at a voltage of 30 kV. The size and shape of the blank-polymeric nano micelle and the optimum DOX -polymeric nano micelle were further examined using a Transmission Electron Microscope (“TEM”) (JEM-F200, Jeol, Japan). TEM images were obtained by negative staining (phosphotungstic acid, 2%) with one drop of the substance applied to a copper grid at the proper dilution.

[0129]

[0089] The SEM images show the powder bed nature of DOX, CRV, and SA as illustrated in FIGS. 8A-8C, respectively. DOX-CRV complex image shows a pronounced change in powder bed resulting in film and pins structure, thus indicating the complexation formation as shown in FIG. 8D.

[0130]

[0090] FIG. 9 illustrates the SEM images of different magnifications of DOX:CRV:SA nano micelle of the present disclosure showing SA film formed to encapsulate the DOX- CRV complex, and white dots of the DOX or DOX-CRV further emphasize SA encapsulation.

[0131]

[0091] The TEM images of blank CRV-SA polymeric gel, and optimum DOX-CRV-SA polymeric nano micelle are illustrated in FIG. 10A and FIG. 10B, respectively. FIG. 10A shows the circular CRV coating by SA nanoparticles in size less than 100 nm. On the other hand, FIG. 10B confirms the circular and micellar formation of DOX-CRV nanoparticles coated with SA in size less than or equal to 100 nm. The size of the optimum DOX-CRV- SA polymeric nano micelle was 40-98 nm which was more than the size resulted using the zeta-sizer, wherein zeta-sizer measures the size of the DOX inner core, while the TEM image shows the overall molecular distribution and sizes of the final nano micelle.Example 5

[0132] DOX Release Analysis

[0133]

[0092] The release analysis experiments were conducted for DOX, DOX-CRV complex, DOX-SA gel, and the optimum DOX-CRV-SA polymeric nano micelle at 37.00±0.05°C and 75.00±0.05 rpm in a shaker. An equivalent amount of DOX concentration of about 0.4 pg / mL used for all samples. The samples were set up in dialysis bags with a cut-off value of 12,000 Daltons and about 300 mL of the release medium. For the first two hours, the formulations in the bags were immersed in 0.1 N HC1, SGF with a pH of 1.2. The groups were then immersed in a 6.8 pH phosphate buffer configured to simulate the fluid found in the proximal intestine for another two hours, this time period for each media is configured to imitate body conditions. The medium was completely changed to replicate sink body conditions after about one hour. The volume was doubled using DMSO to dilute samples, the dilution configured to prevent DOX precipitation. DOX release was then calculated by using UV spectrophotometry at 496 nm.

[0134]

[0093] FIG. 11 illustrates the release analysis percentage during the time period of about 8 hours, the results show a sustained-release pattern of DOX:CRV:SA polymeric nano micelle of the present disclosure in the gastric media in comparison to the DOX, DOX- CRV complex and DOX-SA gel by 32%, 21% and 18% lower total % of gastric release, respectively. Furthermore, in the intestinal-colonic release period, the nano micelle of the present disclosure shows faster release rate comparing to the gastric release rate, this may be attributed to the conversion of SA from the insoluble alginic acid to the soluble alginate.

[0135] Example 6

[0136] Statistical Analysis

[0137]

[0094] The one-way ANOVA was used for the statistical analysis, and Tukey's Multiple Comparison Test was used afterwards. The results were expressed as mean ± standard deviations (“SD”). In the statistical analysis, which was carried out with Graph Pad Prism 4.0 software (Graph Pad Software San Diego, USA), a P-value of 0.05 was considered statistically significant.

[0095] While embodiments of the present disclosure have been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various additions, omissions, and modifications can be made without departing from the spirit and scope thereof.

[0138]

[0096] In describing and claiming the present invention, the following terminology was used.

[0139]

[0097] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0140]

[0098] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0141]

[0099] As used herein, the term “about”, when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0142] Y1

Claims

CLAIMS1. A pharmaceutical formulation for Doxorubicin or any pharmaceutically acceptable salt thereof, the pharmaceutical formulation comprises a therapeutically effective amount of Doxorubicin or any pharmaceutically acceptable salt thereof, monoterpenes, alginate, and any acceptable excipient, wherein monoterpene comprises Carvacrol, the alginate comprises sodium alginate.

2. The pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation comprises a polymeric nano micelle having Doxorubicin, Carvacrol, and Sodium Alginate.

3. The pharmaceutical formulation of claim 2, wherein a complexation ratio of Doxorubicin: Carvacrol ranges from about 1:1 to about 0.065:1 by weight (mg / mg).

4. The pharmaceutical formulation of claim 3, wherein the complexation ratio of Doxorubicin: Carvacrol is 1:4 by weight (mg / mg).

5. The pharmaceutical formulation of claim 2, wherein the sodium alginate amount ranges from about 8 mg / ml to about 32 mg / ml.

6. The pharmaceutical formulation of claim 5, wherein the sodium alginate amount is 16 mg / ml.

7. The pharmaceutical formulation of claim 2, wherein the Doxorubicin, Carvacrol, and sodium alginate are oriented in the polymeric nano micelle as hydrophilic core comprising Doxorubicin, hydrophobic middle layer comprising Carvacrol, and hydrophilic outer layer comprising sodium alginate.

8. The pharmaceutical formulation of claim 1, wherein Doxorubicin and Carvacrol have a hydrophilic binding, Carvacrol and sodium alginate have hydrophilic and hydrophobic intramolecular bindings, and sodium alginate and free Doxorubicin molecules have a hydrophilic intramolecular binding.

9. The pharmaceutical formulation of claim 8, wherein the hydrophilic and hydrophobic intramolecular bindings between the Doxorubicin, Carvacrol, and sodium alginate nano particles are configured to provide charge stabilization and neutralization for the pharmaceutical formulation, thus stimulating gastrointestinal epithelial cell adhesion.

10. The pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation has a small particle size configured to increase the bioavailability of Doxorubicin.

11. The pharmaceutical formulation of claim 1 , wherein the pharmaceutical formulation has a zeta- potential value of about -0.51 ± 0.67 mV.

12. The pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation has a polydispersity index value of about 0.24 ± 0.13.

13. The pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation distribution and size of circular nano micellar formation is less than or equal to 100 nm.

14. A drug delivery system for targeting gastric release comprising the pharmaceutical formulation of claim 1.

15. The pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation is an oral gel formulation.

16. Use of the pharmaceutical formulation of claim 1 for treating malignancies.

17. The use of claim 16, wherein the malignancies comprise stomach cancer.

18. A method for preparing the pharmaceutical formulation of claim 1, wherein the method comprises the steps of:Mixing a proper amount of Doxorubicin and Carvacrol to provide an optimized complex of Doxorubicin coated particles with Carvacrol;Adding the complex to a proper amount of Sodium Alginate solution to provide a mixture; Maintaining the mixture under high-speed sonication to provide an optimized nano micelle; Freeze drying the nano micelles to provide a spongy powder; andInserting the spongy powder into hard-gelatin capsules.

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