Pharmaceutical composition for targeted radionuclide therapy

Abstract

A pharmaceutical composition for targeted radionuclide therapy comprising a plurality of nanostructures. Each respective nanostructure may comprise a hydrophilic inner cavity and an outer shell of PEG. The hydrophilic inner cavity may comprise a colloidal solution of Lu-177 DOTATAE and a plurality of high-atomic-number nanoparticles selected from a group consisting of gold nanoparticles, platinum nanoparticles and tantalum nanoparticles with a maximum diameter of 1 to 20 nm in a aqueous medium with a ratio (Lu-177 DOTATE: the plurality of high- atomic-number nanoparticles) of 1: 50-60. The outer shell of PEG may have a surface charge between -0.1 and -0.3 mV. The plurality of high-atomic-number nanoparticles may have a diameter of 20 nm. Each respective nanostructure may have a diameter between 50 and 200 nm. Each respective nanostructure may have a diameter of 150 nm. The aqueous medium may be selected from a group consisting water and acetone.

Description

PHARMACEUTICAL COMPOSITION FOR TARGETED RADIONUCLIDE THERAPYTECHNICAL FIELD

[0001] The present disclosure generally relates to an exemplary pharmaceutical composition for targeted radionuclide therapy, and more particularly to an exemplary pharmaceutical composition for targeted radionuclide therapy of neuroendocrine tumors (NET).BACKGROUND

[0002] NETs are rare neoplasms that arise from neuroendocrine cells, which are distributed widely throughout the body. As a result, NETs can develop in various organ systems, most commonly in the gastrointestinal tract, pancreas, and lungs. The combination of their diverse clinical presentations, variable symptoms, and rarity complicates timely diagnosis and effective management. First- line treatments for patients with NETs typically include surgical intervention, chemotherapy, and radiotherapy. However, these strategies often fall short of achieving complete tumor eradication which can be attributed to several factors, including the non- specificity of many existing anticancer agents and cytotoxic therapies, as well as the significant side effects associated with these treatments. In light of these challenges, there is a growing need for advanced therapeutic approaches. One promising option is targeted radionuclide therapy (TRT), a specialized modality within nuclear medicine.

[0003] TRT is a form of radiation therapy against NETs that involves the administration of a radionuclide — a radioactive substance — which is conjugated to a cell-targeting molecule specific to NETs, such as a monoclonal antibody or peptide. This NET-targeting molecule binds to receptors present on the surface of NET cells, facilitating their targeted destruction. To enhance the efficacy of TRT, high-atomic-number nanoparticles can be utilized incombination with targeted radionuclide within a biocompatible nano-carrier. This carrier is designed to simultaneously deliver both the targeted radionuclide and the high-atomic-number nanoparticles to NET tissues, which helps to improve the permeability and retention effect within the tumor environment. Furthermore, the inclusion of high-atomic-number nanoparticles in the treatment regimen can promote the release of Auger electrons, which possess a short range but high-energy photon radiation in the kiloelectron volt range. This mechanism increases the potency of the radiation delivered to NET cells while sparing adjacent healthy tissues.

[0004] Thereby there is a compelling need to develop a pharmaceutical composition that combines a targeted radionuclide effective against NETs, such as Lu- 177 DOTATATE, with high-atomic-number nanoparticles, including gold or platinum nanoparticle. This formulation should be encapsulated within a biocompatible, hydrophilic polymer carrier, such as polyethylene glycol (PEG), designed at the nanoscale. By utilizing high-atomic-number nanoparticles, the formulation can improve the absorption of both beta radiation and Auger electrons by NET cells, reducing the number of TRT sessions required for NET patients, leading to improved treatment outcomes and enhanced patient quality of life.SUMMARY

[0005] This summary is intended to provide an overview of the subject matter of the present disclosure, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. Its sole purpose is to present some concepts of one or more exemplary aspects in a simplified form as a prelude to the more detailed description that is presented later. The proper scope of the present disclosure may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0006] One or more exemplary embodiments describe an exemplary pharmaceutical composition for targeted radionuclide therapy. Exemplary pharmaceutical composition may comprise a plurality of nanostructures. Each respective exemplary nanostructure may comprise an exemplary hydrophilic inner cavity and an exemplary outer shell of PEG. An exemplary hydrophilic inner cavity may comprise an exemplary colloidal solution of Lu- 177 DOTATAE and an exemplary plurality of high-atomic-number nanoparticles in an aqueous medium with a ratio (Lu- 177 DOTATAE: exemplary plurality of high-atomic-number nanoparticles) of 1: 50-60. An exemplary high-atomic-number nanoparticle may be selected from the group consisting gold nanoparticles and platinum nanoparticles. An exemplary high-atomic-number nanoparticle may have a maximum diameter of 1 to 20 nm. An exemplary outer shell of PEG may have a surface charge between -0.1 and -0.3 mV.

[0007] In an exemplary embodiment, an exemplary plurality of high-atomic-number nanoparticles may have a diameter of 20 nm. In an exemplary embodiment, each respective exemplary nanostructure may have a diameter between 50 and 200 nm. In an exemplary embodiment, each respective exemplary nanostructure may have a diameter of 150 nm. In an exemplary embodiment, an exemplary aqueous medium is selected from a group consisting water and acetone.

[0008] This Summary may introduce a number of concepts in a simplified format; the concepts are further disclosed within the “Detailed Description” section. This Summary is not intended to configure essential / key features of the claimed subject matter, nor is intended to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The novel features which are believed to be characteristic of the present disclosure, as to its structure, organization, use and method of operation, together with further objectives and advantages thereof, will be better understood from the following drawings in which anexemplary embodiment will now be illustrated by way of example. It is expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limits of the present disclosure. Exemplary embodiments will now be described by way of example in association with the accompanying drawings in which:

[0010] FIG. 1 illustrates a flowchart of exemplary method for synthesis of exemplary pharmaceutical composition, consistent with one or more exemplary embodiments of the present disclosure.

[0011] FIG. 2 illustrated diagram of dose (gray) as a function of distance from the nanoparticle surface for silver, gold, iron, gadolinium, palladium, platinum, tantalum, titanium along with their respective oxides in aqueous environment, consistent with one or more embodiment of the present disclosure.

[0012] FIG. 3 illustrated diagram of dose (gray) as a function of distance from the nanoparticle surface for silver, gold, iron, gadolinium, palladium, platinum, tantalum, titanium along with their respective oxides in PEG environment, consistent with one or more embodiment of the present disclosure.

[0013] FIG. 4 illustrated diagram of radiation dose enhancement factor as a function of distance from the nanoparticle surface for silver, gold, iron, gadolinium, palladium, platinum, tantalum, titanium along with their respective oxides in aqueous environment, consistent with one or more embodiment of the present disclosure.

[0014] FIG. 5 illustrated diagram of radiation dose enhancement factor as a function of distance from the nanoparticle surface for silver, gold, iron, gadolinium, palladium, platinum, tantalum, titanium along with their respective oxides in PEG environment, consistent with one or more embodiment of the present disclosure.

[0015] FIG. 6 illustrates DLS report of exemplary gold nanoparticle, consistent with one or more exemplary embodiment of the present disclosure.

[0016] FIG. 7 illustrates FESEM images of exemplary pharmaceutical composition, consistent with one or more exemplary embodiments of the present disclosure.

[0017] FIG. 8 illustrates DLS report of exemplary pharmaceutical composition with three replicates measurements, consistent with one or more exemplary embodiment of the present disclosure.

[0018] FIG. 9 illustrates Zeta potential report of exemplary pharmaceutical composition with three replicate measurements, consistent with one or more exemplary embodiment of the present disclosure.

[0019] FIG. 10 illustrates FTIR spectrum of exemplary pharmaceutical composition before and after loading Lu-177 DOTATATE, consistent with one or more exemplary embodiment of the present disclosure.

[0020] FIG. 11 illustrates UV / SPECT spectrum of exemplary pharmaceutical composition with three replicate measurements, consistent with one or more exemplary embodiment of the present disclosure.

[0021] FIG. 12 illustrates size distribution diagram of exemplary pharmaceutical composition with three replicate measurements, consistent with one or more exemplary embodiments of the present disclosure.

[0022] FIG. 13 illustrates PET scan of mice before treatment with exemplary pharmaceutical composition, consistent with one or more exemplary embodiments of the present disclosure.

[0023] FIG. 14 illustrates PET scan of mice before treatment with exemplary pharmaceutical composition, consistent with one or more exemplary embodiments of the present disclosure.DETAILED DESCRIPTION

[0024] In the following detailed description, numerous specific details are set forth by way of examples to provide a thorough understanding of the relevant teachings related to the exemplary embodiments. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and / or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings.

[0025] The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in one or more exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to the exemplary implementations will be plain to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure.

[0026] The present disclosure is not intended to be limited to the implementations shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0027] Disclosed herein is an exemplary pharmaceutical composition for ‘targeted radionuclide therapy’ against tumors. ‘Targeted radionuclide therapy’ (TRT) may refer to a therapeutic modality that employed ‘radionuclides’ which can bind preferentially to a particular receptor expressed on the surface of cancer cells. Upon binding to these targeted cancer cells, radionuclide emit ionizing radiation, leading to cell death while sparing adjacent healthy cells.

[0028] In an exemplary embodiment, ‘radionuclide’ may refer to a chemical element characterized by unstable nucleus that emits radiation during its transition into a stable form.Radionuclides serve a multifaceted role in oncology, employed in both therapeutic and diagnostic procedures.

[0029] In an exemplary embodiment, radionuclide may damage tumor cells through different mechanism, including but not limited to ‘Auger electron’, ‘P particle’ and ‘y photon’ release with half-life of 6 to 7 days. In an exemplary embodiment, ‘Auger electron’ may refer to low-energy electrons emitted from an atom following the ejection of an inner-shell electron. This process occurs because of ionization caused by incident radiation or interactions with high-energy photons. Due to low energy and limited range of Auger electrons, they can create localized damage for cancer treatment with minimal impact on surrounding healthy tissue.

[0030] In an exemplary embodiment, ‘P’particle’ may refer to subatomic particles emitted from nucleus of an unstable form of atom through beta decay. When these β particles interact with cancer cells, they ionize atoms and molecules within the cancerous cell, causing DNA disruption and cellular damage, ultimately leading to cell death or inhibition of cell division.

[0031] In an exemplary embodiment, ‘y photon’ or ‘y ray’ may refer to a kind of high-energy, short-wavelength electromagnetic radiation which are emitted during the radioactive decay of certain radionuclides and in nuclear reactions. As an ionizing radiation, γ photonspossess sufficient energy to penetrate various materials, including biological tissues (. e.g. cancerous tissue).

[0032] In an exemplary embodiment, the combination of a radionuclide and a high-atomic-number nanoparticle enhances the absorbed radiation dose in cancerous tissues, thereby increasing the effectiveness of TRT. In an exemplary embodiment, ‘high-atomic-number nanoparticle’ may refer to nanoparticles containing high number of protons in their nucleus, such as gold, platinum, and lead. The combination of a radionuclide and a high-atomic-number nanoparticle increases the absorbed radiation dose in cancerous tissue through several mechanisms, including but not limited to ‘photoelectric effect’ and ‘Auger effect’. In anexemplary embodiment, ‘photoelectric effect’ may refer to a mechanism in which γ photons or X-rays interact with high-atomic-number nanoparticles, resulting in the release of secondary electrons. In an exemplary embodiment, ‘Auger effect’ may refer to a process where an atom releases energy from an inner-shell electron transition by ejecting another electron instead of emitting an X-ray photon.

[0033] In an exemplary embodiment, radionuclides and high-atomic-number nanoparticles may be encapsulated within a ‘polymer’ shell for targeted delivery. In an exemplary embodiment, ‘polymer’ may refer to a large molecule composed of repeating structural subunits, typically linked together by covalent bonds. These subunits may be identical or different and result in the formation of a long chain or network structure.

[0034] In an exemplary embodiment, an exemplary pharmaceutical composition may comprise an exemplary plurality of nanostructures designed to bind specifically to ‘somatostatin receptor’. In an exemplary embodiment, ‘somatostatin receptor’ may refer to a G-protein coupled receptor which is abundantly expressed on the membranes of ‘neuroendocrine tumors’ to facilitates the targeted delivery of radionuclides and high-atomic-number nanoparticles to neuroendocrine tumors. In an exemplary embodiment, ‘neuroendocrine tumor’ may refer to a group of tumor cells that exhibit properties of both nerve cells and hormone-producing endocrine cells, and are commonly found in the gastrointestinal tract, pancreas, and lungs.

[0035] In an exemplary embodiment, an exemplary nanostructure may have a hydrophilic inner cavity, filled by an exemplary colloidal solution. In an exemplary embodiment, an exemplary colloidal solution may comprise Lu- 177 DOTATAE as a common radionuclide drug used against NETs. In an exemplary embodiment, Lu-177 DOTATAE may comprise Lu-177, a beta-emitting radionuclide with a half-life of 6 to 7 days. In an exemplary embodiment, Lu- 177 DOTATAE may further comprise DOTATATE, a synthetic peptide with capability tobind to somatostatin receptor of NETs. In an exemplary embodiment, an exemplary colloidal solution may further comprise an exemplary plurality of high-atomic-number nanoparticles. In an exemplary embodiment, an exemplary plurality of high-atomic-number nanoparticle may be selected from a group consisting gold and platinum. In an exemplary embodiment, exemplary colloidal solution may comprise Lu-177 DOTATATE and high-atomic-numbernanoparticles with a ratio (Lu-177 DOTATATE: exemplary plurality of high-atomic-number nanoparticles) of 1:50-60 in an exemplary aqueous solution. In an exemplary embodiment, high-atomic-number nanoparticles may have a maximum diameter of 1 to 20 nm. In an exemplary embodiment, an exemplary aqueous medium may be selected from a group consisting of water and acetone. In an exemplary embodiment, an exemplary nanostructure may comprise an outer shell of polyethylene glycol (PEG). In an exemplary embodiment, PEG may be a biocompatible polymer which may evade the immune system and prolongs exemplary nanostructures circulation time in the bloodstream, reducing clearance by the reticuloendothelial system (RES). In an exemplary embodiment, exemplary outer shell of PEG may have a surface charge between -0.1 and -0.3 mV. In an exemplary embodiment, an exemplary nanostructure may have a diameter between 50 and 200 nm.

[0036] FIG. 1 illustrates a flowchart of exemplary method 100 for synthesis of exemplary pharmaceutical composition, consistent with one or more exemplary embodiments of the present disclosure.

[0037] In one or more exemplary embodiments, exemplary method 100 may comprise: forming an exemplary PEG solution by dissolving PEG 4000 and PEG 6000 in acetone with a concentration between about 300 and 350 mg / mL (step 102); forming an exemplary colloidal solution by adding exemplary aqueous Lu- 177 DOTATAE solution with a concentration of 0.5 mM to exemplary aqueous gold nanoparticles solution with a concentration of about 25 mM (step 104); forming an exemplary primary mixture by adding exemplary colloidal solution toexemplary PEG solution (step 106); and forming exemplary pharmaceutical composition by dissolving polyvinyl alcohol (PVA) in exemplary primary mixture with a concentration of 1% (wt) (step 108).

[0038] In further detail with respect to step 102, step 102 may comprise forming an exemplary PEG solution by dissolving PEG 4000 and PEG 6000 in acetone with a concentration between about 300 and 350 mg / mL. In an exemplary embodiment, dissolving PEG 4000 and PEG 6000 in acetone with a concentration between about 300 and 350 mg / mL may include adding PEG 4000 powder and PEG 6000 powder with a ratio (PEG 4000 powder: PEG 6000 powder) of 1:2 to an exemplary acetone with a concentration of about 300-350 mg / mL (with respect to an exemplary final volume of an exemplary PEG solution), in an exemplary container. An exemplary container may include, but is not limited to, beakers, tins, flasks, tanks, bottles, buckets, basins, bowls, vials, tubes, barrels, cannisters, etc. In an exemplary embodiment, an exemplary water used for forming an exemplary PEG solution may include distilled water, double-distilled water, or ultrapure water. “Ultrapure water” may refer to a water that has been purified using a combination of ultrafiltration technologies and ultraviolet photo-oxidation system. In an exemplary embodiment, dissolving PEG 4000 powder and PEG 6000 powder with a weight ratio (PEG 4000 powder: PEG 6000 powder) of 1:2 in an exemplary acetone with a concentration of about 300 to 350 mg / mL in an exemplary first container may include dissolving PEG 4000 powder and PEG 6000 powder in exemplary acetone with a final concentration of about 300 to 350 mg / mL, in an exemplary first container, followed by stirring, e.g., using a magnetic stirrer or an agitation machine (with a speed between about 500 rpm and 600 rpm), for a time duration of about 30 minutes at a temperature level of about 35-40 °C and adding an exemplary polysorbate solution with a concentration of about 1% (wt) (with respect to an exemplary final volume of an exemplary polysorbate solution) till an exemplary clear yellow solution is achieved.

[0039] In further detail with respect to step 104, step 104 may comprise forming an exemplary colloidal solution by adding exemplary aqueous Lu- 177 DOTATAE solution with a concentration of 0.5 mM to exemplary aqueous gold nanoparticles solution with a concentration of about 25 mM. In an exemplary embodiment, adding exemplary aqueous Lu-177 DOTATAE solution with a concentration of about 0.5 mM to exemplary aqueous gold nanoparticles solution with a concentration of about 25 mM may include adding exemplary aqueous Lu- 177 DOTATATE solution with a concentration of about 0.5 mM (with respect to an exemplary final volume of an exemplary colloidal solution) to exemplary aqueous gold nanoparticle solution with a concentration of about 25 mM (with respect to an exemplary final volume of an exemplary colloidal solution), in an exemplary second container. In an exemplary embodiment, adding exemplary aqueous Lu- 177 DOTATATE solution with a concentration of about 0.5 mM to exemplary aqueous gold nanoparticle solution with a concentration of 25 mM in an exemplary second container may include adding exemplary aqueous Lu- 177 DOTATATE solution with a concentration of about 0.5 mM to exemplary aqueous gold nanoparticle solution with a concentration of 25 mM in an exemplary second container, followed by ultrasonication with a power of 500 w, for a time duration of about 30 minutes at a temperature level of about 4 °C and stirring, e.g., using a magnetic stirrer or an agitation machine (with a speed between about 500 rpm and 600 rpm), for a time duration of about 2 hours at a temperature level of about 18-25 °C.

[0040] In further detail with respect to step 106, step 106 may comprise forming an exemplary primary mixture by adding exemplary colloidal solution to exemplary PEG solution. In an exemplary embodiment, forming an exemplary primary mixture by adding exemplary colloidal solution to exemplary PEG solution may include adding colloidal solution to exemplary PEG solution in a drop- wide manner, e.g., using a syringe pump (with a rate of about 20pL / min), followed by stirring, e.g., using a magnetic stirrer or an agitation machine(with a speed of about 1200 rpm), for a time duration of about 1 hours at a temperature level of about 4 °C till a clear red solution is achieved.

[0041] In further detail with respect to step 108, step 108 may comprise forming exemplary pharmaceutical composition by dissolving polyvinyl alcohol (PVA) in exemplary primary mixture with a concentration of 1% (wt). In an exemplary embodiment, dissolving PVA in an exemplary primary mixture with a concentration of about 1% (wt) may include adding PVA powder in an exemplary primary mixture with a concentration of about 1% (wt) (with respect to an exemplary final volume of an exemplary pharmaceutical composition), in a third exemplary container. In an exemplary embodiment, dissolving PVA in an exemplary primary mixture with a concentration of about 1% (wt) may include dissolving PVA in an exemplary primary mixture with a concentration of about 1% (wt), followed by ultrasonication with a power of 500 w, for a time duration of about 30 minutes at a temperature level of about 4 °C and stirring, e.g., using a magnetic stirrer or an agitation machine (with a speed between about 1200 rpm), for a time duration of about 12 hours at a temperature level of about 4°C till a clear pink solution is achieved which is exemplary pharmaceutical composition. In an exemplary embodiment, exemplary pharmaceutical composition may be kept at a temperature level of about 4 °C. In an exemplary embodiment, for extended time duration, exemplary pharmaceutical composition may be freeze-dried for a time duration of 48 hours, at a temperature level of about -20 °C.

[0042] While the foregoing has described what are considered to be the best mode and / or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings.

[0043] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0044] The scope of protection is limited solely by the claims that now follow. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed.

[0045] Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[0046] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0047] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0048] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study, except where specific meanings have otherwise been set forth herein. Relational terms such as “first” and “second” and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions.

[0049] The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it may be seen that various features are grouped together in various implementations. This is for purposes of streamlining the disclosure, and is not to be interpreted as reflecting an intention that the claimed implementations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed implementation. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter.

[0050] While various implementations have been described, the description is intended to be exemplary, rather than limiting and it will be apparent to those of ordinary skill in the art that many more implementations and implementations are possible that are within the scope of the implementations. Although many possible combinations of features are shown in the accompanying figures and discussed in this detailed description, many other combinations ofthe disclosed features are possible. Any feature of any implementation may be used in combination with or substituted for any other feature or element in any other implementation unless specifically restricted. Therefore, it will be understood that any of the features shown and / or discussed in the present disclosure may be implemented together in any suitable combination. Accordingly, the implementations are not to be restricted except in light of the attached claims and their equivalents. Also, various modifications and changes may be made within the scope of the attached claims.EXAMPLES

[0051] Hereinafter, one or more exemplary embodiments will be described further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples may be for illustrative purposes only and are not to be interpreted to limit the scope of the present disclosure.Example 1: Simulation of Exemplary Nanostructure

[0052] In this example, structure of exemplary nanostructure within human body was simulated using an exemplary software toolkit. The objective was to identify the optimal exemplary high-atomic-number nanoparticle and to predict the physio-chemical interaction of exemplary pharmaceutical composition on NET and healthy cells by investigation of the passage of radiation through DNA.

[0053] To conduct internal dosimetry using exemplary software toolkit, an exemplary voxel-based human phantoms representing a 43 -year-old woman, who is 167 cm tall and weighs 59 kg, was utilized. An exemplary tumor of endocrine origin was simulated, characterized by the configuration of seven nested spheres with an overall diameter of 1 cm, positioned at a depth of 1 cm in the left breast. The structural characteristics of exemplary nanostructure were also modeled, incorporating polyethylene glycol (PEG) (formulationdefined as H-(O-CH2-CH2) n-OH with n equal to 135), with a molecular weight of 6000 g / mol. Additionally, exemplary gold nanoparticles were represented with a diameter of 45 nm and a concentration of 50 parts per million (ppm).

[0054] In a comparative study, various forms of radionuclide including Lu- 177 in its elemental form (Lu- 177), radionuclide with outer shell of PEG (Lu-177+PEG), colloidal solution of radionuclide and gold nanoparticle (Lu-177+GNP), and exemplary nanostructure were assessed. All forms of radionuclide were maintained at a concentration of 50 ppm and were simulate to accumulate within exemplary tumor using exemplary software toolkit.

[0055] Subsequently, adsorbed radiation doses within exemplary tumor and 6 other healthy organs were measured. Table 1 below presents the absorbed radiation dose of Lu- 177, Lu-177+PEG, Lu-177+GNP, and exemplary nanostructure in various tissues including exemplary tumor, kidney cortex, kidney medulla, kidney pelvis, breast adipose, breast glandular, liver, spleen, heart wall, heart content, and thyroid.Table 1: Absorbed radiation dose of Lu-177, Lu-177+PEG, Lu-177+GNP, and exemplary nanostructure in exemplary tumor, kidney cortex, kidney medulla, kidney pelvis, breast adipose, breast glandular, liver, spleen, heart wall, heart content, and thyroid, consistent with one or more exemplary embodiments of the present disclosure.

[0056] To identify the optimal high-atomic-number nanoparticle in exemplary nanostructure, simulations were conducted using various nanoparticles, including silver, gold, iron, gadolinium, palladium, platinum, tantalum, titanium along with their respective oxides using an exemplary software toolkit within an aqueous environment, which serves as an analog for soft tissues. The radiation dose enhancement factor was measured over a distance from the nanoparticle surface, extending to 10 μm, which approximates the average diameter of a cell.

[0057] FIG. 2 illustrated diagram 200 of dose (gray) as a function of distance from the nanoparticle surface for silver, gold, iron, gadolinium, palladium, platinum, tantalum, titanium along with their respective oxides in aqueous environment, consistent with one or more embodiment of the present disclosure. Referring to the FIG. 2, an increased in the dose was observed for all nanoparticles tested which may lead to auger electron release and secondary ionization. Notably, gold, platinum, and tantalum exhibited the highest radiation dose enhancement factors, respectively, indicating their superior performance when used with Lu-177.

[0058] FIG. 3 illustrated diagram 300 of dose (gray) as a function of distance from the nanoparticle surface for silver, gold, iron, gadolinium, palladium, platinum, tantalum, titanium along with their respective oxides in PEG environment, consistent with one or moreembodiment of the present disclosure. Referring to the FIG. 3, an increased in dose was observed in PEG environment for all nanoparticles tested with gold, platinum, and tantalum exhibited the highest radiation dose enhancement factors.

[0059] comparison of the radiation dose enhancement factor diagrams in aqueous and PEG environments demonstrates that exemplary outer shell of PEG may increase the absorbed radiation dose in tumor tissue due to higher density of PEG compared to water and higher photon-electron interactions. Simulations conducted using an exemplary software toolkit indicated that the elemental form of nanoparticles yields a superior radiation dose enhancement factor when compared to their oxide counterparts. Table 2 below shows the number of secondary electrons released from nanoparticles of silver, gold, iron, gadolinium, palladium, platinum, tantalum, titanium along with their respective oxides in aqueous environment. Table 2: number of secondary electrons released from nanoparticles of silver, gold, iron, gadolinium, palladium, platinum, tantalum, titanium along with their respective oxides in aqueous environment, consistent with one or more exemplary embodiments of the present disclosure.

[0060] The absorbance of photoelectrons by exemplary high-atomic-number nanoparticles may facilitate the release of additional Auger electrons, thereby enhancing the effectiveness of radiopharmaceutical therapy. These Auger electrons, characterized by lower energy, shorter range, and higher linear energy transfer, have the capacity to disrupt NETs while minimizing damage to the surrounding healthy cells

[0061] When energy levels equal to or exceeding 17.5 kV are imparted to the backbone of DNA, single strand breaks (SSBs) may occur. If two SSBs occur in proximity to each other, they can lead to the formation of a double strand break (DSB), which is significantly more detrimental to cell. Utilizing an exemplary software toolkit, the incidence of SSBs and DSBs induced by various nanoparticles — including gold, platinum, silver, tantalum, iron, and palladium — in an aqueous environment has been predicted. Table 3 presented below illustrates the number of direct and indirect SSBs and DSBs caused by nanoparticles of silver, gold, iron, gadolinium, palladium, platinum, tantalum, titanium along with their respective oxides in aqueous environment.Table 3: the number of direct and indirect SSD and DSB caused by nanoparticles of silver, gold, iron, gadolinium, palladium, platinum, tantalum, titanium along with their respective oxides in aqueous environment, consistent with one or more exemplary embodiments of the present disclosure.

[0062] Table 4 presented below illustrated the number of direst and indirect SSDs and DSBs caused by nanoparticles of silver, gold, iron, gadolinium, palladium, platinum, tantalum, titanium along with their respective oxides in PEG environment.Table 4: the number of direct and indirect SSD and DSB caused by nanoparticles of silver, gold, iron, gadolinium, palladium, platinum, tantalum, titanium along with their respective oxides in PEG environment, consistent with one or more exemplary embodiments of the present disclosure.

[0063] According to the data presented in Tables 3 and 4, the incorporation of exemplary high-atomic-number nanoparticles is associated with an increase in direct and indirect SSB and DSB in both aqueous and PEG environments. Indirect SSB and DSB can occur due to various reactive oxygen species (ROS), generated by Auger electrons, with hydroxyl radicals being the most critical among them.

[0064] FIG. 4 illustrated diagram 400 of radiation dose enhancement factor as a function of distance from the nanoparticle surface for silver, gold, iron, gadolinium, palladium, platinum, tantalum, titanium along with their respective oxides in aqueous environment, consistent with one or more embodiment of the present disclosure. FIG. 5 illustrated diagram 500 of radiation dose enhancement factor as a function of distance from the nanoparticle surface for silver, gold, iron, gadolinium, palladium, platinum, tantalum, titanium along with their respective oxides in PEG environment, consistent with one or more embodiment of the present disclosure. Comparing FIG. 4 and FIG. 5, it is understood that dose enhancement factor in PEG environment is higher that aqueous environment which indicated that usage of a PEG shell around an exemplary nanostructure may increase absorbed dose in cancerous tissue, leading to a more effective treatment.

[0065] Based on the results obtained from simulations conducted using an exemplary software toolkit, gold nanoparticles have demonstrated significant potential to enhance the absorbed radiation dose and to induce effective damage and cytotoxicity within NETs Example 2: The Optimal Method for Synthesis of Pharmaceutical Composition

[0066] In this example, exemplary pharmaceutical composition was synthesized based on an exemplary process similar to method 100. To synthesize exemplary pharmaceutical composition, first an exemplary PEG solution with a concentration of about 300 to 350 mg / mL may be prepared in a first solution tank. In an exemplary embodiment, an exemplary PEG solution may be prepared by dissolving PEG 6000 powder and PEG 4000 powder in a ratio (PEG 6000: PEG 4000) of 1:2 in acetone such that the final concentration of PEG may be about 300 to 350 mg / mL by final volume of PEG solution, followed by stirring at about 500-600 rpm for about 40 minutes at a temperature level of about 18-25 °C. For example, to prepare about 5 mL of 300-350 mg / mL PEG solution, 1 g of PEG 6000 (with a molecular weight: 6000) and 0.5 g of PEG 4000 (with a molecular weight: 4000) may be dissolved in 5 mL acetone using a magnet stirrer with a speed range of between 500 rpm and 600 rpm until a clear solution is achieved. Then, about 50 µL of a nonionic surfactant (such as polysorbate) with a final concentration (with respect to the final volume of polysorbate solution) of about 1% (wt) may be added to an exemplary clear solution to achieve exemplary PEG solution.

[0067] On the other hand, exemplary gold nanoparticles were prepared by standard protocol of Turkevich. FIG. 6 illustrates DLS report 600 of exemplary gold nanoparticle, consistent with one or more exemplary embodiment of the present disclosure. Size and morphology of exemplary gold nanoparticle was examined using DLS, revealing spherical shape of exemplary gold nanoparticles with an average dimension less than 10 nm.

[0068] Also, an exemplary Lu- 177 DOTATATE solution with a concentration of about 1 mM was prepared in a second solution tank. In an exemplary embodiment, an exemplary Lu-177 DOTATATE solution may be prepared by dissolving Lu- 177 DOTATAE powder in double distilled water such that the final concentration of Lu- 177 DOTATAE may be about 1 nM by final volume of Lu- 177 DOTATATE solution, followed by stirring at about 500-600 rpm for about 40 minutes at a temperature level of about 18-25 °C. For example, to prepareabout 1 mL of ImM Lu- 177 DOT AT ATE solution, 1 mg of Lu- 177 DOT AT ATE (with a molecular weight: 1000) may be dissolved in 1 mL double distilled water using a magnet stirrer with a speed range of between 500 rpm and 600 rpm until a clear solution is achieved.

[0069] in an exemplary third solution tank, 1 mL of 50 mM gold nanoparticle solution was added to 1 mL of 1 mM Lu- 177 DOT AT ATE solution, followed by sonication in an ultrasonic bath with power of 500 w, for about 30 minutes, at a temperature level of about 2-6 °C and subsequently, stirring at about 500-600 rpm for about 2 hours, at temperature level of about 18-25 °C to achieve an exemplary colloidal solution.

[0070] Then, exemplary 2 mL of colloidal solution is added to 5 mL of exemplary PEG solution using a syringe pump in a drop-wise manner with a rate of about 20 µL / minute,followed by stirring at about 1000-1200 rpm for about 40 minutes at a temperature level of about 2-6 °C, until a clear red solution is achieved.

[0071] Separately, an exemplary PVA solution with a concentration of about 1% (wt) was prepared in a fourth solution tank. In an exemplary embodiment, an exemplary PVA solution may be prepared by dissolving PVA powder in double distilled water such that the final concentration of PVA may be about 1% (wt) by final volume of PVA solution, followed by stirring at about 500-600 rpm for about 40 minutes at a temperature level of about 18-25 °C. For example, to prepare about 3 mL of 1% (wt) PVA solution, 3 mg of PVA (with a molecular weight: 72000) may be dissolved in 3 mL double distilled water using a magnet stirrer with a speed range of between 500 rpm and 600 rpm until a clear solution is achieved.

[0072] In the next step, 3 mL of PVA solution with a concentration of 1 % (wt) was added to clear red solution using a syringe pump in a drop-wise manner with a rate of about 20 µL / minute, followed by sonication for about 30 minutes at a temperature level of about 2-6 °C, and then stirring at about 1000-1200 rpm for about 12 hours at a temperature level of about 2- 6 °C until a clear pink solution is achieved. Achieved clear pink solution is liquid form ofexemplary pharmaceutical composition which should be stored at about 2-6 °C and may be used for intravenous injections. To store for more extended time, liquid form of exemplary pharmaceutical composition may be transformed into powder form by freeze-drying for about 48 hours at about -20°C. Powder form of exemplary pharmaceutical composition should be kept at about -20°C and may be dissolved in normal saline solution to be prepared for intravenous injections.Example 3: Characterization of Pharmaceutical Composition

[0073] In this example, exemplary produced pharmaceutical composition in “Example 2” was characterized by field emission scanning electron microscopy (FESEM), dynamic light scattering (DLS), Zeta potential, Fourier transform infrared (FTIR), and ultraviolet spectroscopy (UV / SPECT).

[0074] FIG. 7 illustrates FESEM images 700 of exemplary pharmaceutical composition, consistent with one or more exemplary embodiments of the present disclosure. Size and morphology of each respective nanostructure of an exemplary pharmaceutical composition was examined using FESEM, revealing spherical shape of nanostructure with an average dimension less than 50 nm. An exemplary pharmaceutical composition characterized by a diameter between 5 and 200 nm may have an elevated surface-to-volume ratio, which may enhance its potential for cellular interactions and effective delivery of radionuclide. Also, spherical morphology of an exemplary pharmaceutical composition may exhibit a symmetrical shape, which facilitate smooth transit within the vascular system, potentially minimizing accumulation in the liver and spleen.

[0075] FIG. 8 illustrates DLS report 800 of exemplary pharmaceutical composition with three replicates measurements, consistent with one or more exemplary embodiment of the present disclosure. The average size of each respective nanostructure of an exemplary pharmaceutical composition was further determined by DLS, yielding a Z-average diameterbetween 48 and 170 nm. The Z-average diameter of each respective nanostructure of an exemplary pharmaceutical composition may suggest that immune and renal system are unlikely to eliminate an exemplary nanostructure from bloodstream of the patient.

[0076] FIG. 9 illustrates Zeta potential report 900 of exemplary pharmaceutical composition with three replicate measurements, consistent with one or more exemplary embodiment of the present disclosure. Zeta potential of each respective nanostructure of an exemplary pharmaceutical composition was examined using Zeta potential technique, revealing average Zeta potential between -0.1 and -0.3 mV. A slightly negative Zeta potential indicates that each respective nanostructure of an exemplary pharmaceutical composition may exhibit a propensity for accumulation in neoplastic tissues, such as those associated with NET, in addition to proper radionuclide release. Moreover, the achieved Zeta potential contributed significantly to the prolonged physical and microbiological stability of exemplary pharmaceutical composition.

[0077] FIG. 10 illustrates FTIR spectrum 1000 of exemplary pharmaceutical composition before and after loading Lu-177 DOTATATE, consistent with one or more exemplary embodiment of the present disclosure. In FTIR spectroscopy analysis, acetone was selected as solvent for conducting tests on an exemplary pharmaceutical composition both prior to and subsequent to loading of Lu- 177 DOTATATE. Looking at the FTIR spectrum of an exemplary pharmaceutical composition, all peaks of Lu- 177 DOTATATE, PEG and exemplary gold nanoparticle are observed with infinitesimal variations (which are acceptable due to changes in special configuration) which indicated that Lu- 177 DOTATATE, PEG and exemplary gold nanoparticle are present in final structure of an exemplary pharmaceutical composition. Moreover, in FTIR spectrum of an exemplary pharmaceutical composition a distinct peak at 3400 cm-1is observed, attributed to H-0 stretching which indicated the presence of PEG or amine and hydroxyl functional group of DOTATATE peptide. Additionally, a broad peakbetween 1400 and 1600 cm-1attributed to N-H and C=0 stretching, was evident which indicated the presence of DOTATATE peptide of Lu- 177 DOT AT ATE. Furthermore, peaks indicative of the presence of PEG were observed at 1100 cm-1(attributed to C-O-C stretching).

[0078] FIG. 11 illustrates UV / SPECT spectrum 1100 of exemplary pharmaceutical composition with three replicate measurements, consistent with one or more exemplary embodiment of the present disclosure, measures absorbance over a wavelength range of 200 to 400 nm, with specific absorbance recorded at 266 nm at predetermined time intervals. This analysis utilized a 12 kDa dialysis bag within a shaker incubator to simulate physiological conditions representative of human body. Based on these UV / SPECT spectra, a drug release pattern was achieved which indicates that release of exemplary pharmaceutical composition follows a first-order pattern, characterized by an initial rapid release of Lu- 177 DOTATATE in the first hours, followed by a gradual decrease in the release rate. This release pattern is favorable for radionuclide therapy, as an extended-release period may increase the absorbed radiation dose in healthy tissues, potentially causing harm to the surrounding tissues near NETs.

[0079] An exemplary pharmaceutical composition may have a pH of 7 which exhibits minimal aggregation, resulting in an extended duration of presence within the vein and facilitates a more controlled release of the radionuclide.

[0080] FIG. 12 illustrates size distribution diagram 1200 of exemplary pharmaceutical composition with three replicate measurements, consistent with one or more exemplary embodiments of the present disclosure. This value indicates a high level of homogeneity among exemplary nanostructures within exemplary pharmaceutical composition.Example 4: in-vitro Evaluation of Pharmaceutical Composition

[0081] In this example, cytotoxicity of exemplary produced pharmaceutical composition in “Example 2” was characterized by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide” assay (MTT), and Alamar Blue assay (AB). In MTT and AB assay, T-47D cell line was used as a model cell line of breast cancer and MCF10A cell line was used as healthy breast.

[0082] Table 5 below shows the output of MTT assay for T-47D in a 96-well plate at varying concentration of exemplary pharmaceutical composition between 540 and 50 µg / µL.Table 6 below shows the output of MTT assay for MCF10A in a 96-well plate at varying concentration of exemplary pharmaceutical composition between 540 and 50 µg / µL.Table 5: absorbance values measured at 570 nm for T-47D at varying concentration of exemplary pharmaceutical composition between 540 and 50 µg / µL, consistent with one or more exemplary embodiments of the present disclosure.Table 6: absorbance values measured at 570 nm for MCF10A at varying concentration of exemplary pharmaceutical composition between 540 and 50 µg / µL, consistent with one or more exemplary embodiments of the present disclosure.Comparing Table 5 and Table 6, it is observed that exemplary pharmaceutical composition shows a higher lethal effect on T47D cell line. Also, lethal effect of exemplary pharmaceutical composition has increased by increase in concentration. To confirm acquired data from MTT and also minimize margin of error, AB assay was also conducted. Table 7 below shows the fluorescence values measured of AB assay for T-47D in a 96-wll plate at varying concentration of exemplary pharmaceutical composition between 540 and 50 µg / µL.Table 7: fluorescence values measured at 560-590 nm for T-47D at varying concentration of exemplary pharmaceutical composition between 540 and 50 µg / µL, consistent with one or more exemplary embodiments of the present disclosure.Table 8 below shows the absorbance values measured of AB assay for T-47D in a 96-well plate at varying concentration of exemplary pharmaceutical composition between 540 and 50 µg / µL.Table 8: absorbance values measured at 560-590 nm for T-47D at varying concentration of exemplary pharmaceutical composition between 540 and 50 µg / µL, consistent with one or more exemplary embodiments of the present disclosure.Example 5: Animal Experiment of Pharmaceutical Composition

[0083] In this example, animal experiment of exemplary produced pharmaceutical composition in “Example 2” was conducted on 10 female Nude mice, in which T47D cancer was induced. To initiate the development of T47D cancer in the mice, a total of 5 × 106T47D cells were cultured according to standard protocols and subsequently injected intravenously into the mice to induce tumor formation. Following the induction of tumor development, a positron emission tomography (PET) scan was performed to accurately identify the tumor’s location. Subsequently, exemplary pharmaceutical composition was administered intravenously at a precise concentration and volume, calibrated according to the body mass of each individual mouse. After an observation period of approximately two months, a repeat PET scan was conducted to evaluate the efficacy of exemplary pharmaceutical composition in reduction of size of the induced tumors in the murine subjects.

[0084] FIG. 13 illustrates PET scan 1300 of mice before treatment with exemplary pharmaceutical composition, consistent with one or more exemplary embodiments of the present disclosure.

[0085] FIG. 14 illustrates PET scan 1400 of mice before treatment with exemplary pharmaceutical composition, consistent with one or more exemplary embodiments of the present disclosure.

[0086] A comparative analysis of FIG. 13 and FIG. 14 reveals a significant reduction in tumor size (black area) following treatment with exemplary pharmaceutical composition, underscoring the potential efficacy of the exemplary pharmaceutical formulation for TRT while minimizing adverse side. It is also evident that usage of exemplary high-atomic-number nanoparticle has enhanced tumor uptake of exemplary pharmaceutical composition, leading to increase in absorbed radiation.

Claims

What is claimed is:

1. A pharmaceutical composition for targeted radionuclide therapy comprising a plurality of nanostructures, each respective nanostructure comprising:A hydrophilic inner cavity comprising a colloidal solution of Lu-177 DOTATATE and a plurality of high-atomic-number nanoparticles selected from the group consisting of gold nanoparticles, platinum nanoparticles and tantalum nanoparticles with a maximum diameter of 1 to 20 nm in an aqueous medium, wherein the colloidal solution comprises Lu-177 DOTATATE and the plurality of high-atomic-number nanoparticles with a ratio (Lu-177 DOTATATE: the plurality of high-atomic-number nanoparticles) of 1:50 to 1:60; andan outer shell of polyethylene glycol (PEG) encapsulating the colloidal solution, the outer shell of PEG with a surface charge between -0.1 and -0.3 mV.

2. The pharmaceutical composition of claim 1, wherein the plurality of high-atomic-number nanoparticles has a diameter of 20 nm.

3. The pharmaceutical composition of claim 1, wherein each respective nanostructure has a diameter between 50 and 200 nm.

4. The pharmaceutical composition of claim 3, wherein each respective nanostructure has a diameter of 150 nm.

5. The pharmaceutical composition of claim 1, wherein the aqueous medium is selected from a group consisting water and acetone.

Images