Systems and pharmaceutical compositions for treatment by direct injection of target cell populations

Abstract

Systems and methods are provided for delivering a therapeutic treatment to a target population of cells (including, but not limited to, tumors, eyeballs, pancreatic tissue, liver tissue, and lung tissue) in a subject. The system includes an injectable aqueous solution in a vial enclosed with a septum. The solution comprises particles containing a therapeutic agent and having a coating surrounding the therapeutic agent, the coating comprising chitosan to provide controlled release of the agent from the particles. The solution also comprises a chitosan polymer in the form of a polymer gel matrix, further providing controlled release of the particles from the aqueous gel environment. There is also provided a method of making a freeze-dried powder placed in a vial containing a chitosan polymer and chitosan-coated particles, the powder when mixed with water forming the above injectable aqueous solution with particles and chitosan gel.

Description

[0001] This application is a divisional application of the invention patent application filed on January 3, 2021, with application number 202180018850.2 and invention title "System and pharmaceutical composition for treatment by direct injection of target cell population".

[0002] Cross-references to related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 956,795, filed January 3, 2020, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0004] This invention relates to therapeutic compositions for injection into target cell populations to treat diseases and tumors, and more specifically to injectable aqueous solutions of chitosan gels and chitosan particles containing therapeutic agents, as well as methods for preparing and using such compositions. Background Technology

[0005] Tumors in the human body are usually treated by surgical removal. Tumor treatment is often urgent, especially for solid malignant tumors. These tumors include myeloid sarcoma, round cell sarcoma, melanosarcoma, spindle cell sarcoma, and papilloma. Other types of solid tumors are well-known to those skilled in the medical field.

[0006] Typically, tumors cannot be completely removed, and these solid tumors are considered inoperable. Inoperable solid tumors can be classified according to their location or size. Chemotherapy is often used to treat solid tumors to shrink their size, making them suitable for surgery.

[0007] Chemotherapy can be administered via three different routes: (1) intravenous (IV), (2) intra-arterial, and (3) intratumoral. Studies have shown that preoperative intravenous systemic chemotherapy is effective in reducing or shrinking solid tumors. Ferriere, JP et al. (1998), “Primary chemotherapy in breast cancer: correlation between tumor response and patient outcome,” American Journal of Clinical Oncology, Vol. 21, No. 2, pp. 117-120. Furthermore, the intravenous route provides simultaneous treatment throughout the entire organism, enabling treatment of metastatic cells (or micrometastases) throughout the body. However, one of the main challenges is delivering sufficient antitumor agents to the target site. Systemic chemotherapy can lead to severe side effects, which can be dose-limiting and intolerable to patients. These side effects reduce the use of particularly potent and effective drugs. According to the literature, most drugs are administered systemically at doses that do not provide optimal efficacy, within the limits of tolerable side effects (MTD—maximum tolerated dose).

[0008] This limitation on MTD not only affects treatment success but can also have counterproductive results, leading to more resistant tumors. Consider a specific solid tumor containing several populations of the same type of tumor cells, whose ability to tolerate specific dose levels of chemotherapeutic agents differs from one another. (Kinsella, AR; Smith, D.; and Pickard, M. (1997), “Resistance to chemotherapeutic antimetabolites: a function of salvage pathway involvement and cellular response to DNA damage,” British Journal of Cancer, Vol. 75, No. 7, p. 935). MTD can be a dose level capable of killing most, but not all, cells in a specific tumor. Therefore, not only do residual, more resistant cancer cells remain, but due to widespread proliferation, these more resistant cells will dominate a large portion of the tumor, presenting a more difficult challenge for future chemotherapy. Another obstacle is that many antitumor drugs can be period-sensitive. In other words, these anti-tumor drugs interact with cells only when the cells are in a specific phase of the cell cycle. Other cells that are not in the sensitive phase at the time of administration are preserved. Relatively short-duration intravenous administration, even at high dose intensities, may miss the sensitive phase for tumor cells. The treatment of many tumors can benefit from lower-dose, higher-frequency, or continuous dosing regimens, both in terms of efficacy and reduction of adverse event intensity.

[0009] Intratumoral injection is a promising alternative to chemotherapy and, at least conceptually, should be the most successful method. In this method, antitumor drugs are administered directly to the tumor, achieving high local concentrations and avoiding systemic side effects. It also offers virtually unlimited dosage flexibility.

[0010] Despite these advantages, intratumoral chemotherapy is not particularly effective. It has been suggested that this lack of efficacy reflects one or more of the following factors:

[0011] The density of tumor cells in a tumor is very high, so when a drug does not pass through blood vessels, this will prevent the drug from penetrating through the cells.

[0012] High interstitial fluid pressure prevents drugs from migrating into the interstitial fluid.

[0013] High density of cells and blood vessels leads to vasoconstriction. See Jain, RK (1999), “Transport of molecules, particles, and cells in solid tumors,” Annual Review of Biomedical Engineering, Vol. 1, No. 1, pp. 241-263.

[0014] Dosing regimens have been proposed to alleviate these problems by inducing apoptosis in tumors. See, for example, M. Flashner-Barak, U.S. Patent Application Serial No. 2002 / 0041888A1 and Serial No. 09 / 829,621.

[0015] Other possible reasons for intratumoral administration failure include: uneven diffusion of the drug throughout the tumor; and a lack of an effective dose for treating cells for a sufficiently long period when cells enter their sensitive phase of the cell cycle. The problem in intratumoral chemotherapy then boils down to maintaining sufficiently high concentrations of the chemotherapeutic agent for a sufficiently long period to allow it to diffuse throughout the tumor in order to achieve these goals. Gels, pastes, and microparticles have been used for intratumoral injection.

[0016] Chitosan is a non-toxic (LD50 > 16 g / kg), biodegradable natural polysaccharide derived from the exoskeleton of crustaceans. Chitosan is derived from chitin, a naturally abundant biopolymer found in the exoskeletons of crustaceans, insect epidermis, fungal cell walls, and mollusc shells. Chitin is composed of 2-acetamido-2-deoxy-β-D-glucose monomers (N-acetylglucosamine units) linked by β(1→4) bonds, and chitosan is a polymer of deacetylated α-(1,4)glucosamine units. It is typically obtained by demineralizing and deproteinizing crustacean shells or exoskeletons, followed by deacetylation of chitin with NaOH. Chitosan is a multifunctional material with good biocompatibility, no immunogenicity, and no skin irritation. In 2001, chitosan was approved as a GRAS (Generally Recognized As Safe) substance by the U.S. Food and Drug Administration (FDA). Chitosan is a widely used biomaterial with established safety in humans. It is used as a pharmaceutical excipient, a weight-loss supplement, and an experimental mucosal adjuvant, and is also used in FDA-approved hemostatic dressings. High molecular weight chitosan (>100 kDa), due to its long polymer chains, forms highly viscous solutions in mild aqueous solvents. Viscous solutions have been widely used to control the release of drugs and macromolecules in vivo because they hinder the diffusion and dispersal of these molecules after injection. Baldrick, P. (2010), “The safety of chitosan as an pharmaceutical excipient,” Regulatory Toxicology and Pharmacology, Vol. 56, No. 3, pp. 290-299.

[0017] Platinum-based drugs (such as cisplatin (cis-diamine dichloroplatin-II)) are among the most widely used chemotherapeutic agents and have shown efficacy against a variety of solid tumors outside the central nervous system, including testicular, ovarian, breast, colorectal, lung, and head and neck tumors. Due to the blood-brain barrier (BBB), systemically delivered cisplatin has difficulty penetrating normal brain tissue, resulting in plasma concentrations below 5% detectable in the brain after intravenous delivery. However, neovascularization within tumors allows for greater penetration than through the intact BBB, and therapeutic cisplatin levels have been detected in both primary and secondary brain tumors after systemic delivery, with lower levels detected in cerebral edema adjacent to the tumor. (Pérez, J. et al. (2019), “The effect of locally delivered cisplatin dependent on anintact immune function in an experimental glioma model,” Scientific Reports, Vol. 9, No. 1, p. 5632).

[0018] Cytokines are small proteins released by immune cells, allowing them to communicate with each other. Cytokines have been studied as potential cancer treatments for some time. However, despite their known potency and potential for use in conjunction with other immunotherapies, they have not yet been successfully developed into effective cancer treatments. This failure may reflect the high toxicity of cytokines to both healthy tissues and tumors, making them unsuitable for systemic administration.

[0019] Injecting cytokines directly into tumors could provide a way to confine their toxic effects within the tumor without harming healthy tissue, but previous attempts to do so resulted in proteins leaking from cancerous tissue and entering the body's circulation within minutes.

[0020] Cytokines are a broad class of loosely packed small proteins (approximately 5 kDa to 20 kDa) that play a crucial role in cell signaling. Cytokines are peptides and cannot cross the lipid bilayer into the cytoplasm. As immunomodulators, cytokines participate in autocrine, paracrine, and endocrine signaling. Their precise differentiation from hormones remains a part of ongoing research.

[0021] Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines are produced by a wide range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells; a given cytokine can be produced by more than one cell type. (Aznar, MA et al. (2017), “Intratumoral delivery of immunotherapy—act locally, think globally,” *The Journal of Immunology*, Vol. 198, No. 1, pp. 31-39.)

[0022] Cytokines function through receptors and are particularly important in the immune system, where they regulate the balance between humoral and cellular immune responses, and they modulate the maturation, growth, and responsiveness of specific cell populations. Some cytokines enhance or inhibit the effects of other cytokines in complex ways.

[0023] Cytokines include, but are not limited to, granulocyte colony-stimulating factor (G-CSF), interferon, interleukins (including IL-2, IL-7, and IL-12), and various chemokines.

[0024] Other immunomodulators are also being investigated, including imiquimod, cell membrane fractions from bacteria used for patients and others, synthetic cytosine phosphate-guanosine (CpG), oligodeoxynucleotides, and dextran.

[0025] Ocular vascular diseases are one of the leading causes of vision impairment and blindness worldwide. Intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) agents has revolutionized the treatment of common retinal diseases, including neovascular age-related macular degeneration (AMD), diabetic retinopathy, and retinal vein occlusion (RVO). Furthermore, encouraging results have been reported with intravitreal injection of anti-VEGF agents for the treatment of other ocular diseases, such as neovascular glaucoma, retinopathy of prematurity (ROP), and intraocular tumors.

[0026] Age-related macular degeneration (AMD) is a well-characterized and widely studied disease. It is currently considered the leading cause of visual impairment in patients over 60 years of age. Early AMD is marked by the formation of drusen, pigmentary changes in the macula, and mild to moderate vision loss. AMD exists in two forms: a "dry" form and a "wet" form. The "wet" form occurs less frequently but accounts for 90% of acute blindness caused by AMD. Risk factors are associated with the progression of AMD, and these risk factors are relevant to understanding how AMD develops: (1) advanced age and exposure to environmental factors induce high levels of oxidative stress that damage the macula, and (2) this damage leads to inflammation, which induces a vicious cycle that ultimately results in central vision loss.

[0027] There is neither a cure nor a treatment to prevent AMD. However, some treatments exist for the wet form of AMD. The introduction of anti-angiogenic drugs has brought about a major breakthrough in the treatment of the wet form; after two years of treatment, the functional prognosis has changed from almost certain blindness to a more than 90% chance of third-line visual improvement. However, even after this progress, treatment is far from perfect and there is still much room for improvement. (Hernández-Zimbrón, LF et al. (2018), “Age-Related Macular Degeneration: New Paradigms for Treatment and Management of AMD,” Oxidativemedicine and Cellular Longevity, 2018, 8374647.)

[0028] Reducing the treatment burden associated with routine intravitreal injections of anti-VEGF is of paramount importance. Neovascular AMD and diabetic retinopathy are chronic, relapsing diseases. Patients may require dozens of injections over many years of treatment. Adhering to such demanding protocols is challenging. Promising approaches include: (a) new hardware for delivering anti-VEGF drugs, (b) new drugs with longer-lasting biological effects, (c) new formulations for sustained-release anti-VEGF agents, and (d) gene therapy. Puliafito, CA et al. (2019), “Looking ahead in retinal disease management: highlights of the 2019 angiogenesis, exudation and degeneration symposium,” International Journal of Retina and Vitreous, Vol. 5, No. 1, p. 22.

[0029] Currently, several anti-VEGF drugs are available, including pegaptanib, ranibizumab, bevacizumab, and aflibercept. Although well-designed randomized clinical trials have demonstrated the efficacy of these agents in improving vision in various retinal diseases, each intravitreal injection carries the risk of post-injection and drug-class-related adverse events. The often-required repeated and long-term injections may increase the likelihood of ocular and systemic complications. (Falavarjani, KG et al. (2013), “Adverse events and complications associated withintravitreal injection of anti-VEGF agents: a review of literature,” Eye, Vol. 27, No. 7, p. 787.)

[0030] Specifically, the most common treatment for AMD is intravitreal bevacizumab injection. Injections are administered every three to four weeks and can be a source of surgical risks and inconvenience for patients.

[0031] Targeted drug delivery to other tissues, including but not limited to pancreatic, lung, and liver tissues, for local treatment of these tissues, and with potential benefits for treating diseases specific to these tissues, including pancreatitis, diabetes, brain cancer, lung cancer, and hepatitis. Summary of the Invention

[0032] In one embodiment of the invention, a composition is provided for formulation for delivery by injection to a target cell population of a subject. In this embodiment, the composition comprises an aqueous solution containing a chitosan gel and a plurality of particles containing a therapeutic agent and having a coating surrounding the therapeutic agent containing chitosan to provide controlled release of the agent from the particles.

[0033] One embodiment of the present invention provides a system for delivering a therapeutic treatment to a target cell population of a subject. In this embodiment, the system includes a vial sealed with a septum, the septum being permeable by a needle of an syringe used to administer the therapeutic treatment. In this embodiment, a therapeutic composition is placed in the vial, the therapeutic composition being provided for administering the therapeutic treatment and comprising an aqueous solution containing a chitosan gel and a plurality of particles embedded in the gel, the gel having a viscosity suitable for administration by injection. The particles in this embodiment contain a therapeutic agent and have a coating surrounding the therapeutic agent, the coating comprising chitosan to provide controlled release of the agent from the particles.

[0034] In further related embodiments, the aqueous solution also includes a compound selected from hydration promoters, particle adhesion inhibitors, particle aggregation inhibitors, and combinations thereof.

[0035] The hydration promoter is selected from ethylene glycol, propylene glycol, β-propylene glycol, glycerol, and combinations thereof.

[0036] The particle adhesion inhibitor is selected from HPMC, poloxamer, and combinations thereof.

[0037] The particle aggregation inhibitor is selected from monosaccharides, disaccharides, sugar alcohols, chlorinated monosaccharides, chlorinated disaccharides, and combinations thereof.

[0038] Alternatively or otherwise, the composition may also contain sodium tripolyphosphate.

[0039] In some embodiments, these particles are microparticles with an average diameter of 200 nm to 2000 nm. As a further alternative, these microparticles have an average diameter of 500 nm to 2000 nm.

[0040] Optionally, the aqueous solution also contains a free amount of therapeutic agent that is not coated with chitosan, which accounts for about 20% to about 80% by weight of the total amount of therapeutic agent in the aqueous solution.

[0041] Optionally, the therapeutic agent in the granules is an immunotherapeutic agent. As a further option, the therapeutic agent is selected from antibodies, cytokines, small molecule immunotherapeutic agents, and combinations thereof.

[0042] In a further related implementation, the therapeutic agent is a chemotherapeutic agent.

[0043] Optionally, the particles are in direct physical contact with the chitosan gel.

[0044] In another embodiment, the present invention provides a method for treating a target cell population in a subject. The method includes obtaining a system as described at the beginning of this section, loading an aqueous solution into a syringe, and injecting the aqueous solution into the target cell population using the syringe.

[0045] In one embodiment of the method, the target cell population includes tumors. In another embodiment, the target cell population is tissue from an organ. Optionally, the organ is selected from the eye, lung, pancreas, liver, kidney, brain, heart, thyroid gland, and pituitary gland.

[0046] In another embodiment, a system is provided for delivering a therapeutic treatment to a target cell population of a subject. This system includes a vial sealed with a septum, the septum being permeable by a needle of an syringe used to administer the therapeutic treatment. In this embodiment, a therapeutic composition is placed in the vial, the therapeutic composition being provided for administering the therapeutic treatment and comprising a formulated lyophilized precursor such that, upon mixing with water, the lyophilized precursor dissolves to provide an aqueous solution comprising a chitosan gel and a plurality of particles embedded within the gel, the gel having a viscosity suitable for administration by injection. The particles in this embodiment contain a therapeutic agent and have a coating surrounding the therapeutic agent, the coating comprising chitosan to provide controlled release of the therapeutic agent from the particles.

[0047] Another embodiment provides a lyophilization method for providing a system for delivering a therapeutic treatment to a target cell population. The method includes: (1) forming an aqueous solution comprising a chitosan gel and a plurality of particles containing a therapeutic agent and having a coating surrounding the therapeutic agent, the coating comprising chitosan to provide controlled release of the agent from the particles; (2) freezing the aqueous solution in a bath containing the aqueous alcohol solution at a temperature above a freezing temperature of the aqueous alcohol solution and up to -80°C to form a cryolayer precursor; (3) drying the cryolayer precursor to form an anhydrous powder with embedded particles; (4) containing the anhydrous powder in a vial sealed with a septum capable of being penetrated by a needle of an syringe used to administer the therapeutic treatment; and (5) adding water to the vial to dissolve the anhydrous powder.

[0048] Optionally, the aqueous solution in the freeze-drying method further includes a hydration promoter, a particle adhesion inhibitor, and a particle aggregation inhibitor.

[0049] In a further related embodiment of the freeze-drying method, the hydration promoter is selected from ethylene glycol, propylene glycol, β-propylene glycol, glycerol, and combinations thereof. Optionally, the particle adhesion inhibitor includes HPMC, poloxamer, and combinations thereof. In a related embodiment, the particle aggregation inhibitor is selected from monosaccharides, disaccharides, sugar alcohols, chlorinated monosaccharides, chlorinated disaccharides, and combinations thereof. Optionally, the composition also contains sodium tripolyphosphate. Optionally, the particles are microparticles with an average diameter of 200 nm to 2000 nm. As a further option, the particles are microparticles with an average diameter of 500 nm to 2000 nm.

[0050] In some embodiments of the invention, the particulate-based composition is formulated for delivery via intravitreal injection to treat eye conditions.

[0051] Optionally, the eye disease is age-related macular degeneration (AMD).

[0052] According to some implementation schemes, the therapeutic agent for intravitreal injection is selected from antibodies, cytokines, small molecule immunotherapeutic agents, chemotherapeutic agents, aptamers, and combinations thereof. In some implementation schemes, the therapeutic agent for intravitreal injection is bevacizumab. Attached Figure Description

[0053] The foregoing features of the embodiments will be more readily understood by referring to the following detailed description and the accompanying drawings, wherein:

[0054] Figure 1A An injectable chitosan formulation (PRV311) according to the invention is provided, which is frozen at -80°C and stored appropriately. Note that this formulation provides a clear aqueous solution. (Left) PRV311 (middle) and PRV111 (right) at room temperature. PRV111 is a similar product to PRV311, but it is manufactured by freezing in liquid nitrogen (-196°C) at a faster rate than by freezing in a freezer at -80°C.

[0055] Figure 1B An injectable chitosan formulation (PRV311) according to the invention, shown for comparison, was frozen at -80°C and stored at room temperature for five days.

[0056] Figure 1C An injectable chitosan formulation (PRV311) according to the invention, shown for comparison, was frozen in liquid nitrogen at -196°C and stored at room temperature for five days.

[0057] Figure 2 This is the Fourier transform infrared (FTIR) spectrum of injectable chitosan powder containing cisplatin internal standard, at 1400 cm⁻¹. -1 and 1560cm-1 It has a peak.

[0058] Figure 3 This is a photograph (2x magnification) of the matrix after being frozen in liquid nitrogen (-196°C) and subsequently freeze-dried. Note the multi-layered, dense, fabric-like structure.

[0059] Figure 4 This is a photograph (2x magnification) of the matrix after being frozen in a -80°C freezer and subsequently freeze-dried. Note the more porous, uniform monolayer polymer fibers.

[0060] Figure 5 This is a release profile of the drug from the microparticles in media at different pH levels. The powders were reconstituted in their respective media and placed in a dialysis bag with stirring for 72 hours. Samples were removed and the release percentages were shown, where the drug was released more rapidly at pH 6 (circles) due to faster degradation, and at pH 3 (triangles) the drug was released at a slower rate due to greater particle stability. A free cisplatin solution (squares) was used as a control.

[0061] Figure 6 A graph showing the change in mouse tumor volume over time for different treatments is provided. The black curve corresponds to untreated control tumors, the gray curve corresponds to intratumoral injection of placebo particles without drug, the green curve corresponds to intravenous injection of drug, the red curve corresponds to intratumoral injection of free drug, and the blue curve corresponds to injection of drug-encapsulated hydrogel PRV311.

[0062] Figure 7 This is a cross-section of lamb tissue observed under a fluorescence microscope after injection of PRV311 (a chitosan formulation according to an embodiment of the present invention). Here, the drug is labeled with fluorescein isothiocyanate (FITC), which appears green under the microscope.

[0063] Figure 8 This is a graph showing the change in FITC-labeled drug concentration in pig tongue tissue with tissue depth.

[0064] Figure 9 These are photographs showing the local distribution of intratumoral injection and FITC-labeled drugs using embodiments of the present invention.

[0065] Figure 10 This is a photograph showing the injection, according to an embodiment of the present invention, penetrating into the bovine brain.

[0066] Figure 11 This is a significantly enlarged diagram illustrating an embodiment of the invention configured for injection.

[0067] Figure 12This is a significantly enlarged diagram illustrating how the injectable solution PRV311, according to an embodiment of the present invention, constructs a polymer network within and around a tumor.

[0068] Figure 13A and Figure 13B This is a photomicrograph showing the PRV311 according to an embodiment of the present invention.

[0069] Figure 14 These are photographs illustrating some of the formulations evaluated by the inventors during the development of formulations suitable for reconstructing in seconds for clinical use, according to embodiments of the present invention. Note that the solutions are clear.

[0070] Figure 15A , Figure 15B and Figure 15C The study demonstrated complete corneal coverage and penetration when a formulation of PRV311 with a fluorescently labeled drug was injected into the eyes of sheep.

[0071] Figure 16 The injection of separately fluorescently labeled polymers (red) and drugs (green) illustrates how both bolus injection and controlled delivery are achieved.

[0072] Figure 17 The study showed that when fluorescently labeled PRV311 was injected into lung tissue, high local concentrations of the drug persisted for more than 24 hours after injection.

[0073] Figure 18 The image shows that injecting PRV311 into the liver of sheep again demonstrates a high local concentration of the drug.

[0074] Figure 19 The image shows a side view of a sheep liver injected with PRV311, illustrating how tissue type affects drug distribution patterns.

[0075] Figure 20 The study showed that local injection of PRV311 into the pancreas of sheep resulted in high local drug concentrations, and that tissue type affected the local drug distribution pattern. Detailed Implementation

[0076] Definitions. As used in this specification and the appended claims, the following terms shall have the meanings specified therein, unless the context requires otherwise:

[0077] "Subjects" include vertebrates, such as mammals, and further, such as humans.

[0078] A "polymer" is a molecule having at least 100 monomer units.

[0079] The polymer "matrix" is a three-dimensional network of polymer molecules, selected from non-covalent entanglement, ionic crosslinking, covalent crosslinking, and combinations thereof.

[0080] A "gel" is a solution phase of a polymer matrix that swells in a solvent while maintaining entanglement and cross-linking.

[0081] "Particles" are groups of particles having an average diameter of about 200 nm to about 2000 nm. "Nanoparticles" are groups of particles having an average diameter of at least 1 nm to about 200 nm.

[0082] "Particle size" or "particle diameter" is the longest straight-axis length between two points on the surface of a particle.

[0083] "Pure chitosan" refers to chitosan that is not a chitosan salt.

[0084] "Unmodified chitosan" refers to chitosan that has not been chemically modified by adding functional groups or by linking it to a carrier.

[0085] "Unmodified therapeutic agents" refer to therapeutic agents that have not undergone chemical modification by adding functional groups or by linking to a carrier.

[0086] "Immunotherapy agents" are therapeutic agents that modulate the immune response. Immunotherapy agents can be biological drugs or small molecule drugs.

[0087] "Chemotherapy agents" refers to therapeutic agents that are small molecule drugs.

[0088] Aptamers are nucleic acids or modified nucleic acids that have been selected through in vitro selection methods for binding to biological targets. A notable example of an aptamer is the drug piperatanib (trade name: [omitted]). This drug combines with VEGF and is used to treat wet macular degeneration.

[0089] "Particle adhesion inhibitors" are additives that reduce the attractive force between the polymer matrix and the embedded particles. Therefore, the particles can move through the matrix at a faster rate compared to the absence of adhesion inhibitors.

[0090] "Particle aggregation inhibitors" are additives that reduce the tendency of particles embedded in a matrix to aggregate when the matrix is ​​subjected to freezing. Therefore, these particles are less likely to be damaged or destroyed when freezing occurs.

[0091] "Mucosal adhesion" materials are characterized by their ability to adhere to human mucosa.

[0092] The polymer matrix is ​​"porous" when a portion of its volume is void space. In some cases, void spaces can be accessed from the outer surface of the matrix, allowing substances such as particles present in the void spaces to migrate back and forth to the outer surface.

[0093] The “void” spaces in a polymer matrix are spaces not occupied by the polymer and allow particles and small molecules to move through these spaces.

[0094] "Mucosal tissue" refers to tissues with associated mucous membranes. Specifically, mucosal tissue includes the mucous membrane and the tissue beneath it.

[0095] For example, the "site in the mucosal tissue" where a cancerous tumor is present may involve not only the mucosa but also the tissue beneath it.

[0096] The polydispersity index (PDI), or simply dispersibility, is a measure of the size inhomogeneity of a group of particles (such as microparticles in a mixture).

[0097] "Zeta potential" (ZP) is a measure of the total charge a particle acquires in a given medium. ZP can be measured on a Zetasizer Nano instrument.

[0098] "Penetration" is the ability to pass through or penetrate mucous membranes, their underlying tissues, or both. "Biocompatibility" refers to the ability of a biomaterial to perform its desired function in a medical treatment without causing any significant undesirable local or systemic effects in the recipient or beneficiary of that treatment, but rather to produce the most appropriate beneficial cellular or tissue response in that particular situation, and to optimize the clinically relevant performance of that treatment.

[0099] "HPMC" refers to hydroxypropyl methylcellulose, also known as hydroxypropyl methylcellulose.

[0100] "Biodegradable" refers to the property of a material that can be broken down by biological processes, especially into harmless products.

[0101] "Thousands of counts per second" (Kcps) refers to the count rate (in thousands of counts per second (Kcps)). For example, a threshold can be set such that when the count rate of a sample is below 100, the measurement should be stopped, meaning that the sample concentration used for the measurement is too low. A sample with a suitable Kcps can be considered a stable sample with an ideal measurement concentration.

[0102] "Mesh" refers to a polymer matrix that adheres to the treatment area and contains elements bound therein, which are released from the mesh when it is applied to the treatment area.

[0103] "Therapeutic agent delivery system based on polymer matrix and microparticles" can also be called "drug delivery device" or "delivery patch".

[0104] Unless otherwise stated, the term "weight%" refers to the amount of a component of a system used to deliver a therapeutic agent, expressed as a weight percentage.

[0105] Unless otherwise stated, the “molar mass” of a polymer is intended to represent the number-average molar mass of the polymer molecules.

[0106] Cancer can develop in any organ and any tissue at any age. Once cancer is definitively diagnosed, treatment decisions become extremely important. While no single treatment works for all cancers, successful treatment must focus on both the primary tumor and its metastases. Historically, local and regional treatments (such as surgery or radiation) have been used in conjunction with systemic treatments (such as chemotherapy drugs) to treat cancer. Despite some success, conventional treatments have not achieved the desired therapeutic effects, and there has been a constant search for more effective treatments. Clearly, there is a significant unmet need for more effective cancer therapies.

[0107] One of the main uses of the embodiments of the present invention is for intratumoral injection in chemotherapy and immunotherapy, and data have shown that this is the best way to maintain a high concentration of the drug in the tumor and to drain some of the drug to the lymph nodes, thereby ensuring the most effective treatment of the tumor in a local and regional manner.

[0108] Intratumoral injection may be considered for any tumor, whether it is a primary lesion or a metastasis, that can be accessed percutaneously via direct injection or through specific procedures such as colonoscopy, cystoscopy, bronchoscopy, thoracoscopy, laparoscopy, or even surgery.

[0109] Currently, a large number of drugs are being investigated for their role in intratumoral therapy. These drugs include immune receptor agonists (such as Toll-like receptor (TLR) agonists and interferon gene-stimulating factor (STING) agonists, ICT mAbs, wild-type and genetically modified oncolytics (such as viruses and peptides), cytokines, and immune cells targeting various potential targets). Therefore, to support the clinical development of intratumoral strategies in humans, the inventors have developed an injectable system for the local delivery and retention of these drugs.

[0110] In addition, direct injection into the tumor reduces systemic exposure, off-target toxicity, and the amount of drug used, while inducing stronger antitumor activity in both the injected tumor lesion and distant, non-injected tumor lesions.

[0111] Systemic immunotherapy and systemic chemotherapy are commonly used, but both treatments expose the patient's entire body to the toxic side effects of the drugs. Systemic administration is dose-limited due to exposure to the bloodstream and other organs, and precautions must be taken to ensure the safety of such systemic exposure. Systemic delivery often results in harmful side effects from the body's reaction to toxic drugs. These side effects include neurotoxicity, nephrotoxicity, kidney failure, hair loss, nausea, and mucositis. Chemotherapy is also used as an alternative to surgery, in addition to radiation therapy, to treat anal tumors. Current standards of care for patients with anal canal squamous cell carcinoma, even those with small, localized tumors, use an initial simultaneous combination of chemotherapy and radiation therapy. When using chemotherapy, a temporary central venous catheter or a peripherally inserted central venous catheter may be used on the individual. Side effects of treatment include those typical of systemic chemotherapy. These side effects include nausea, hair loss, kidney damage, low blood cell counts, oral ulcers, and impaired immune system. Because chemotherapy is currently delivered systemically throughout the body, dose-limiting factors exist.

[0112] The therapeutic advantage of a drug can be increased by maximizing its efficacy and / or minimizing its side effects. The foundation for developing regional cancer drug therapies is achieving effective target tissue concentrations while minimizing systemic distribution and, consequently, minimizing toxicity. Existing, clinically used examples of regional chemotherapy include intra-arterial infusion for liver and kidney tumors, limb perfusion for melanoma and sarcoma, intrathecal administration for CNS tumors, and intraperitoneal administration for intra-abdominal tumors. Recently, direct intratumoral injection of pure ethanol for primary liver cancer has been developed. A major drawback associated with most cytotoxic chemotherapeutic agents is that these agents are strong vesicants and are therefore not ideal candidates for intratumoral administration unless the delivery technology can retain the drug locally within the tumor and prevent leakage into healthy tissue. Embodiments of the present invention utilize a combination of chitosan particles loaded with polymeric drugs and a combination of polymers to ensure drug retention within the tumor and minimize side effects.

[0113] Intratumoral immunotherapy is a therapeutic strategy that aims to use the tumor as its own vaccine. Direct injection into the tumor yields high concentrations of immunostimulatory products in situ, along with small amounts of the drug. Local delivery of immunotherapy allows for a variety of combination therapies while preventing significant systemic exposure and off-target toxicity. Although the dominant epitopes of a given cancer are uncertain, an immune response against relevant neoantigens or tumor-associated antigens can be triggered without characterizing these antigens. This immunostimulation can locally induce a strong cancer immune initiation while simultaneously generating a systemic (ectopic) tumor response, thanks to the circulation of appropriately activated anti-tumor immune cells. In addition to addressing many current limitations in the development of cancer immunotherapy, intratumoral immunotherapy offers a unique opportunity to better understand the dynamics of cancer immunity by allowing for sequential and multifocal biopsies at each tumor injection. Marabelle, A. et al. (2018), “Starting the fight in the tumor: expert recommendations for the development of human intratumoral immunotherapy (HIT-IT)”, Annals of oncology: official journal of the European Society for Medical Oncology, Vol. 29, No. 11, pp. 2163-2174.

[0114] All five classes of immunotherapies face delivery challenges. Checkpoint inhibitors, cytokines, and agonist antibodies present similar delivery challenges. The success of these therapies depends on their interaction with target proteins. A major limitation to the use of these therapies is that they generate significant amounts of autoimmune activity, leading to side effects that limit the permissible dose. For this reason, a central goal in developing delivery technologies for these therapies is to achieve targeted and controlled release, enabling these therapies to work primarily in the desired cell types, thereby minimizing off-target effects.

[0115] The microenvironment in many solid tumors presents a challenge for the broad realization of all the immunotherapies discussed here. For example, the microenvironment of solid tumors can be categorized as immunologically “hot” (highly immunogenic) or “cold” (lowly immunogenic), characterized by high or low levels of cytotoxic lymphocyte infiltration within the tumor space, respectively. This key difference in microenvironmental composition suggests that tumors with high immunogenicity exhibit a stronger response to checkpoint inhibitors than those with low immunogenicity.

[0116] Delivery technologies can be used to modulate the immunogenicity of cold tumors. Furthermore, because delivery platforms can reduce the systemic toxicity of immunotherapy by limiting drug exposure to specific tissues, they can be used to deliver combinations of treatments that would otherwise be too toxic to be administered to patients.

[0117] Local delivery of immunotherapies using embodiments of the present invention allows for a variety of combination therapies while preventing significant systemic exposure and off-target toxicity. Although the dominant epitopes of a given cancer are uncertain, an immune response against relevant neoantigens or tumor-associated antigens can be triggered without characterizing these antigens. This immune stimulation can locally induce a strong cancer immune initiation while simultaneously generating a systemic (ectopic) tumor response due to the circulation of appropriately activated anti-tumor immune cells.

[0118] According to embodiments of the present invention, a composition comprising one or more of immunotherapy particles and chemotherapeutic particles can be injected into a tumor. The chemotherapeutic particles may contain chemotherapeutic agents, including but not limited to cisplatin and oxaliplatin, both of which, in addition to causing DNA damage in tumor cells, have been shown to activate dendritic cells and induce immune activity in the tumor.

[0119] When chemotherapy is delivered, embodiments of the present invention can cause immunologically cold tumors to become warm, and thus sensitize them to immunotherapy. The tumor-targeting immunotherapy particles and chemotherapy work synergistically to inhibit tumor growth and exhibit reduced toxicity compared to immunotherapy and chemotherapy alone (i.e., without the embodiments of the present invention).

[0120] We have discovered that microparticles can enable combination therapy strategies to sensitize tumors with low immunogenicity to immunotherapy. In addition to enabling combination therapy strategies, embodiments of the present invention can be designed to respond to the tumor microenvironment and increase penetration at those sites.

[0121] According to experiments by David Zaharoff et al., a mixture of chitosan and the cytokine IL-12 was effective for tumor regression in their mouse experiments (Zaharoff, DA et al. (2010), "Intratumoral immunotherapy of established solid tumors with chitosan / IL-12", J. Immunother., Vol. 33, p. 697). However, the inventors' data show that, according to embodiments of the present invention, the combination of chitosan matrix loaded with IL-12 particles exhibits a very high retention time (>10 days) while simultaneously releasing it in controlled high concentrations within the tumor. In various embodiments, the injectable cytokine can be mixed at the bedside for seconds for translational application in clinical use, unlike the laboratory experiments conducted in Zaharoff's work.

[0122] IL-12 is a potent antitumor cytokine that exhibits significant clinical toxicity after systemic administration. Zharoff hypothesized that intratumoral (it) administration of IL-12 co-formulated with the biodegradable polysaccharide chitosan could enhance its antitumor activity while limiting its systemic toxicity. Non-invasive imaging studies monitored local retention of IL-12 after itinerary injection with and without chitosan co-formulation. The antitumor efficacy of IL-12 alone and that of IL-12 co-formulated with chitosan (chitosan / IL-12) was evaluated in mice carrying established colorectal (MC32a) and pancreatic (Panc02) tumors. Other studies involving the depletion of immune cell subsets, tumor re-provocation, and CTL activity were designed to elucidate the mechanisms of regression and tumor-specific immunity. Co-formulation with chitosan increased local IL-12 retention from 1–2 days to 5–6 days. Weekly injections of IL-12 alone eradicated ≤10% of established MC32a and Panc02 tumors, while chitosan / IL-12 immunotherapy resulted in complete tumor regression in 80% to 100% of mice. Depletion of CD4+ or Gr-1+ cells had no effect on chitosan / IL-12-mediated tumor regression. However, depletion of CD8+ or NK cells completely eliminated antitumor activity. Chitosan / IL-12 immunotherapy generated systemic tumor-specific immunity because >80% of mice cured with chitosan / IL-12 immunotherapy were at least partially protected from tumor recurrence. Furthermore, CTLs from the spleen of cured mice cleaved targets loaded with MC32a and gp70 peptides. Chitosan / IL-12 immunotherapy increased local retention of IL-12 in the tumor microenvironment, eradicated established invasive mouse tumors, and generated systemic tumor-specific protective immunity. Chitosan / IL-12 is a well-tolerated and effective immunotherapy with considerable potential for clinical translation.

[0123] In some embodiments, particle-based formulations are provided for controlled drug delivery via intravitreal injection into the eye to treat ocular conditions, particularly age-related macular degeneration. In such embodiments, a biocompatible polymer forms a hydrogel matrix that forms a polymeric mesh that adheres to the ocular epithelium, where it implants drug particles into the tissue. These particles can degrade over an extended period (e.g., four months) to provide sustained drug release.

[0124] According to one embodiment of the present invention, the inventors have developed formulations for the oral and injectable delivery of poorly water-soluble pharmaceuticals and polymers. This formulation enables the conversion of liquid nanocrystal dispersions into solid dosage forms. These solid dosage forms comprise nanocrystals that readily recombine to their original size upon dissolution in water. Careful formulation is required to optimize the freezing rate, thereby reducing particle-particle aggregation. A critical freezing rate for drying the nanocrystals has been determined. Freeze-drying at freezing rates close to this critical value produces a dried powder with a bimodal particle size distribution after redispersibility. Furthermore, it has been found that the concentration of the pharmaceutical nanocrystals significantly affects the critical freezing rate, thereby influencing the redispersibility of the dried powder. The concept of a critical freezing rate is important for developing solid dosage forms of liquid nanocrystal dispersions.

[0125] Embodiments of the present invention provide a formulation that can be transported in powder form and can be rapidly, uniformly and consistently dissolved in sterile water for intratumoral injection at the patient's bedside.

[0126] Embodiments of the present invention provide a system for delivering therapeutic agents to various tissues, particularly cancerous tumors. Various embodiments include chitosan and multiple particles embedded within a matrix.

[0127] Once the working formulation of the embodiments of the present invention is developed, chitosan microparticles can be synthesized at room temperature via ionogelation using sodium tripolyphosphate as a crosslinking agent. A separate formulation containing a polymer matrix comprising a particle adhesion inhibitor, a particle aggregation inhibitor, and a hydration promoter is added to the microparticle solution. The microparticles and matrix solution are dispensed into vials, transferred to a -80°C freezer, and frozen overnight. The vials are then lyophilized for 72 hours.

[0128] Some embodiments of the present invention for producing injectable chitosan powder containing cisplatin are based on the following:

[0129] 1. Add chitosan powder to acetic acid solution (0.186 w / w%) and stir until dissolved.

[0130] 2. In a separate container, add cisplatin (0.15 w / w%) to a sodium tripolyphosphate and salt solution. Dissolve the cisplatin by heating the solution to approximately 40°C and stirring.

[0131] a. All containers containing cisplatin were covered to avoid exposure.

[0132] 3. The contents of the cisplatin-sodium tripolyphosphate solution were then transferred to the chitosan solution.

[0133] a. Gently stir both solutions throughout this step.

[0134] b. Here, this step produces particles. Once a steady state is reached, the particle size / charge is collected.

[0135] 4. Prepare a sucralose aqueous solution (25 w / w%) and set aside for later use.

[0136] 5. In a separate screw-cap bottle, add the chitosan powder to a dilute acetic acid solution (1.0 w / w%). Then, add hydroxypropyl methylcellulose (0.1 w / w%) to the chitosan-acetic acid solution. Stir for 30 minutes.

[0137] 6. Transfer the sucralose solution to the chitosan-cisplatin microparticle solution. Then, transfer the patch matrix solution.

[0138] 7. Stir the final solution for 5 minutes, then dispense 5 mL of solution into each vial. Freeze the vials at -80°C for approximately 2 hours.

[0139] 8. Place the vial in a freeze dryer for 6 days.

[0140] The analytical certificate for the injectable chitosan powder containing cisplatin is shown in Table 1. The FTIR characterization of this sample is shown in... Figure 2 In the middle, 1400 can be seen. + 30cm -1 and 1560 + 30cm -1 The characteristic cisplatin peak at the location.

[0141] Table 1: Sample analysis certificate for injectable chitosan powder containing cisplatin:

[0142]

[0143] As summarized in Table 2, in order to characterize solubility relative to pH, injectable chitosan powder containing cisplatin was reconstructed in media at different pH values, and the release of cisplatin was monitored at 355 nm using a UV-Vis spectrometer.

[0144] Table 2: Relative solubility of injectable chitosan powder containing cisplatin in media of different pH values.

[0145] pH of the medium % transmitted light, λ = 355nm 5.5 (Pure water, control) 100 2.00 89.5 2.93 89.8 4.05 89.7 5.06 90.2 5.90 89.2 7.08 90.5 7.92 91.0

[0146] According to a first set of representative embodiments of the invention, a system is provided for the injectable local delivery of a therapeutic agent to a site within a tissue. The system comprises a polymer matrix capable of forming a gel in a solvent, i.e., a polymeric network structure, which facilitates the localization of embedded particles within the tissue. The gel matrix is ​​formed from a composition comprising chitosan, a hydration promoter, a particle adhesion inhibitor, and a particle aggregation inhibitor. A plurality of particles are embedded within the gel matrix. The gel matrix is ​​configured to open to allow drug-loaded particles to have a limited range of motion within the tissue. These particles contain a therapeutic agent and have a coating surrounding the therapeutic agent. The coating of the particles comprises chitosan to provide controlled release of the drug from the particles. Optionally, the hydration promoter is selected from ethylene glycol, propylene glycol, β-propylene glycol, glycerol, and combinations thereof. Also optionally, the particle adhesion inhibitor is a nonionic polymer, and as a further alternative, the nonionic polymer is HPMC or poloxamer. Alternatively, as an alternative, the particle aggregation inhibitor is selected from monosaccharides, disaccharides, sugar alcohols, chlorinated monosaccharides, chlorinated disaccharides, and combinations thereof. Optionally, these microparticles also contain sodium tripolyphosphate. Optionally, in this system, there is a free amount of therapeutic agent directly embedded in the matrix and not otherwise coated with chitosan, wherein this free amount of therapeutic agent constitutes 20% to 80% of the total amount of therapeutic agent in the system. Optionally, the chitosan in the matrix and the chitosan in the microparticles are pure chitosan. As a further option, the average diameter of the microparticles is about 500 nm to about 2000 nm.

[0147] The matrix is ​​configured to provide controlled release of microparticles through tissue. This system can be used to locally inject effective drugs with significant systemic toxicity into damaged tissues, such as cancerous tumors. The method for producing the invention further includes freezing the mixture at -80°C to form a cryolayer precursor. Finally, the method for producing the invention includes drying the cryolayer precursor to form a powder, which, upon hydration, forms a gel matrix in which microparticles are embedded. In some embodiments of the invention, the final product (the powder for reconstitution) is stable for up to 6 months only when stored with a desiccant, heat-sealed in a waterproof polyester film foil bag, and stored in a freezer at 2°C to 8°C. If these conditions are met, the system (sample PRV311, which contains chitosan nanoparticles within a network) can be reconstituted into a clear solution or a heterogeneous microparticle suspension. The solubility of PRV311 is further increased due to chain-breaking hydrolysis (e.g., freeze-thaw hydrolysis) occurring in the chitosan when frozen and when PRV311 is irradiated with gamma rays for use in patients. Properly stored PRV311 is shown in Figure 1A In this context, it is a clear solution / particulate suspension.

[0148] like Figure 1BAs shown, if PRV311 is stored at room temperature for several days before reconstruction, it cannot be fully reconstructed. Figure 1B The vial was stored at room temperature for five days before reconstruction, and it formed a non-uniform coarse-particle suspension that was unsuitable for injection. Figure 1C The vial shown in the image illustrates another formulation, PRV111, which was frozen faster with liquid nitrogen than PRV311 and also stored at room temperature for five days. Therefore, the treated PRV111 formulation also formed a non-uniform, coarse-particle suspension unsuitable for injection.

[0149] Unbound by theory, it is speculated that:

[0150] a) Even with the addition of aggregation inhibitors, particle-to-particle conformational aggregation in lyophilized powder formulations occurs slowly (driven by van der Waals forces), but this is not the case when the powder formulation is stored at 2°C.

[0151] At temperatures up to 8°C, lower temperatures result in greater dynamic stability of the system.

[0152] b) Some temperature-related factors cause a portion of the mesh to become insoluble.

[0153] PRV311 is prepared by dispensing the liquid product into vials and freezing the vials at -80°C for at least 8 hours. After reconstitution in at least 1 mL of medium, an appropriate volume of PRV311 is drawn using a Luer Lock syringe. PRV311 is injected directly into the tumor using a 18- to 30-gauge needle. Each vial of PRV311 contains 0.1 mg to 100 mg of the drug. Dosage limitations depend on the solubility of the encapsulated immunotherapeutic agent or small molecule in water. The frequency of administration depends on the treatment site, indication, and the user's judgment.

[0154] In some embodiments of the invention, a water-soluble polymer matrix is ​​formed from a composition comprising chitosan, a hydration promoter, a particulate adhesion inhibitor, and a particulate aggregation inhibitor. According to another set of representative embodiments of the invention, a method for manufacturing a therapeutic agent delivery system is provided. The method includes forming a first mixture having a plurality of particles. These particles contain a therapeutic agent and have a coating surrounding the therapeutic agent, the coating comprising chitosan. The method further includes forming a second mixture from components comprising the first mixture, chitosan, a hydration promoter, a particulate adhesion inhibitor, and a particulate aggregation inhibitor. The method further includes freezing the second mixture in a bath containing the aqueous alcohol solution at a temperature above a freezing temperature of the aqueous alcohol solution and at a temperature up to -80°C to form a frozen layer precursor. Finally, the method includes drying the frozen layer precursor to form a porous polymer matrix having particles embedded within the matrix. Optionally, the bath also contains dry ice. Furthermore, optionally, the alcohol in the aqueous alcohol solution is ethanol. As a further option, the aqueous alcohol solution is about 90% by weight of ethanol to about 99% by weight of ethanol. Optionally, the method further includes applying a second layer precursor to the cryolayer precursor to form a solid comprising the first and second layers. Optionally, the second layer comprises a therapeutic agent. Furthermore, optionally, drying is performed under vacuum.

[0155] In some embodiments of the invention, an ultra-low temperature freezer at -80°C is used to freeze the aqueous solution. In other embodiments, liquid nitrogen is used to freeze the solution. Comparison of the freezing method using an ultra-low temperature freezer at -80°C with the liquid nitrogen freezing method yields surprising results. Figure 3 As shown, when frozen and freeze-dried in liquid nitrogen (-196°C), a multi-layered, dense, fabric-like structure is obtained. In contrast, as... Figure 4 As shown, when frozen at -80°C, more porous and uniform monolayer polymer fibers are obtained. The structural differences between the solutions produced by the two methods have a significant impact on the control and timing of the release of the encapsulated therapeutic agent. In some embodiments containing cisplatin, the inventors discovered through the product development process that a specific combination of polymer and excipient is required for the cisplatin network to function properly. During development, the agglomeration and aggregation of nanoparticles within the network hindered the penetration of cisplatin-containing nanoparticles from the network into the tissue. Studies have shown that the addition of a combination of excipient and polymer leads to the complete release of nanoparticles from the network. It is currently unclear why the inclusion of this combination leads to the desired release and penetration; however, through microscopic analysis, it is clear that the inclusion of this combination reacts with the nanoparticle and network structure in such a way that microparticle “colonies” are formed within the pores of the network structure. (See...) Figure 3Unlike typical large and non-uniform aggregates or clusters, these “colonies” are nearly uniform in size and remain small enough to be released from the network and penetrate into the tissue. The structure of the network also changes when a polymeric excipient combination is included. This inclusion produces a more crystalline structure with pores that both accommodate and facilitate the release of microparticles. The aforementioned function attributed to the polymeric excipient combination is reported to be a result of the polymer’s ability to open cell connections within the tissue; in tests conducted for this purpose, the polymeric excipient combination was initially included in the composition of the network. However, no effects of this polymeric excipient combination other than altering cells have been previously reported or observed, including its effect on the network structure and its role in “colonies” and preventing microparticle aggregation. The combined effects of the polymers and excipients used are synergistic, as their combined effect on release and penetration is far greater than the sum of their individual effects.

[0156] Release curves of the particles at different pH values ​​are shown below. Figure 5 The powders were reconstituted in their respective media and placed inside dialysis bags with stirring for 72 hours. Samples were removed and the percentage of release was shown, where the drug was released more rapidly due to faster degradation when the particles were at pH 6 (circles), and at a slower rate due to greater particle stability when the particles were at pH 3 (triangles). Free cisplatin solution (squares) was used as a control. In this graph, it is clear that lower pH slows the release of the drug from the particles.

[0157] According to some embodiments of the present invention, the polymer excipient composition comprises chitosan, hydroxypropyl methylcellulose, and propylene glycol.

[0158] In some embodiments of the present invention, the hydration promoter is selected from ethylene glycol, propylene glycol, β-propylene glycol, glycerol, and combinations thereof.

[0159] In some embodiments of the present invention, the particulate adhesion inhibitor is a nonionic polymer.

[0160] In some embodiments of the present invention, the nonionic polymer is HPMC or poloxamer.

[0161] In some embodiments of the present invention, the particle aggregation inhibitor is selected from monosaccharides, disaccharides, sugar alcohols, chlorinated monosaccharides, chlorinated disaccharides, and combinations thereof.

[0162] In some embodiments of the present invention, the particles further comprise sodium tripolyphosphate.

[0163] In some embodiments of the invention, the system further comprises a free amount of therapeutic agent that is directly embedded in the matrix and not otherwise coated with chitosan, wherein the free amount of therapeutic agent constitutes 20% to 80% by weight of the total amount of therapeutic agent in the system.

[0164] In some embodiments of the present invention, the chitosan in the matrix and the chitosan in the particles are unmodified chitosan.

[0165] In some embodiments of the present invention, the average diameter of the particles is about 0.5 μm to about 2 μm.

[0166] In some embodiments of the invention, the therapeutic agent is an antibody, such as an immunotherapeutic agent, or a small molecule, such as a chemotherapeutic agent.

[0167] In some embodiments of the invention, the invention includes microparticles for targeted delivery of a therapeutic agent, the microparticles containing an unmodified therapeutic agent and unmodified chitosan.

[0168] In some embodiments of the invention, the microparticles are embedded in a matrix so that they are directly surrounded by and in contact with the matrix.

[0169] In some embodiments of the invention, systems for delivering therapeutic agents based on polymer matrices and microparticles are provided, which are improved by adding hydration promoters to the matrix. Exemplary hydration promoters include hygroscopic compounds such as glycols, for example ethylene glycol, propylene glycol, β-propylene glycol, and glycerol. Exemplary concentration ranges of the amount of hydration promoter include about 0.001 wt% to about 10 wt%, about 0.01 wt% to about 5 wt%, and about 0.1 wt% to about 1 wt%.

[0170] Without being bound by any particular theory, it is believed that the hydration accelerator increases the moisture absorption of the delivery system. This increased hydration allows the microparticles to be rapidly released and permeate from the matrix. It is also believed that the hydration accelerator improves uniformity and durability by acting as a cryoprotectant during the manufacturing process of the delivery system. Similarly, without being bound by any particular theory, it is believed that the hydration accelerator acts as a “spacer” between ice crystals and matrix polymer molecules to ensure a uniform freezing mode. The resulting structure is more flexible, uniform, and durable compared to the absence of the hydration accelerator.

[0171] In another set of representative embodiments, an improved delivery device is provided by adding an adhesion inhibitor. Without wishing to be bound by any particular theory, it is believed that particle mobility is affected when the matrix and particles are made of materials with polar or ionicly charged moieties, such as chitosan. Taking chitosan as an example, it is believed that the interaction between the acetyl and amine moieties of the polymer causes the particles to adhere to the matrix and inhibits the release of these particles.

[0172] Studies have found that incorporating adhesion inhibitors can reduce matrix-particle adhesion. Unbound from any particular theory, adhesion inhibitors are believed to act as "spacers" between the chitosan in the particles and the chitosan in the matrix bulk, releasing the particles and allowing for improved drug release profiles. Representative exemplary adhesion inhibitors include nonionic polymers, such as hydroxypropyl methylcellulose (HPMC). Depending on the application, the molar mass of the nonionic polymer can be from about 1 kDa to about 200,000 kDa, while its viscosity can vary from about 10 cps to 100,000 cps. In representative embodiments, the molar mass of the nonionic polymer is from about 10 kDa to 30 kDa, and its viscosity is from about 10 cps to about 100 cps. Depending on the application, the amount of adhesion inhibitor can be from about 0.1 wt% to about 99 wt%. In some embodiments, the amount of adhesion inhibitor is from about 0.1 wt% to about 25 wt%.

[0173] In another set of representative embodiments, an improved delivery device is disclosed by adding an aggregation inhibitor. Methods of manufacturing these delivery devices include a freezing step, during which ice crystals can form within a matrix. Such crystals can force particles closer together, creating particle aggregates in which the particles are damaged or destroyed. Again, not wishing to be bound by any particular theory, it is believed that the aggregation inhibitor acts as a cryoprotectant by forming crystalline microstructures that prevent particle aggregation. Carbohydrates and carbohydrate derivatives provide exemplary types of aggregation inhibitors, including monosaccharides, disaccharides, sugar alcohols, chlorinated monosaccharides, and chlorinated disaccharides such as sucralose. Depending on the application, the amount of aggregation inhibitor in the patch can range from about 0.1% by weight to about 50% by weight. In some embodiments, the amount of aggregation inhibitor is from about 1% by weight to about 10% by weight.

[0174] In another set of representative embodiments, improved pure chitosan microparticles are provided. Conventional chitosan particles are made from chitosan salts (e.g., chitosan chloride and chitosan glutamate) characterized by high deacetylation and charged moieties. It has been found that better results are provided if the particles are made from pure chitosan, a material characterized by being non-salt, i.e., the amino groups of the material are unprotonated and have at least 70% deacetylation. Specifically, these particles are characterized by a larger diameter than conventional particles. In some embodiments, the average diameter of the pure chitosan particles ranges from about 200 nm to about 2000 nm. In other embodiments, the average diameter ranges from about 500 nm to about 2000 nm, and in yet another embodiment, it ranges from 500 nm to 1000 nm. In a further improvement, improved chitosan microparticles are provided by adding sodium tripolyphosphate (STPP). Without being bound by any particular theory, it is believed that STPP functions as a crosslinking agent, forming particles by acting as a counterion to the positively charged amine groups on chitosan. This electrostatic interaction forms ionic bonds that support the particle structure. Again, without being bound by any particular theory, it is believed that the presence of sodium as a positive counterion makes STPP a more effective crosslinking agent than other TPP salts.

[0175] It was also found that when the gel matrix contains a free amount of therapeutic agent that is directly embedded in the matrix and not otherwise coated with chitosan in the particles, the device is more therapeutically effective than comparative matrices containing only a free amount of therapeutic agent or only a chitosan-coated therapeutic agent. In a representative embodiment, the free amount of therapeutic agent constitutes 20% to 80% of the total amount of therapeutic agent in the delivery system.

[0176] Example:

[0177] Injectable formulations reconstituteable from common media (such as water for injection USP, 0.12% saline USP, and 0.9% saline USP) were tested in the following examples:

[0178] Example 1: In vivo study in mice

[0179] like Figure 6As shown, intratumoral injection of PRV311 into mice with transplanted cancer cells eliminated significant tumor growth (blue curve). Here, the PRV311 composition contains microparticles loaded with the anti-tumor cytokine IL-12. In comparative experiments, injection of control saline solution (black curve) and placebo particles (gray curve) into tumors showed minimal effect on tumor growth. Similarly, intravenous injection of IL-12 alone (green curve) did not show a significant change in tumor growth rate compared to the control. It should be noted that, in contrast to the near elimination of tumor growth with IL-12-loaded PRV311 (blue curve), intratumoral injection of IL-12 alone (red curve) only delayed tumor growth. It is speculated that the polymeric network of the injected PRV311 results in local retention of the PRV311-containing microparticles, thereby enhancing efficacy.

[0180] Example 2: Study of an isolated tongue

[0181] 500 μL of PRV311 was injected into the pig's tongue using a 23G subcutaneous Luer-Lock needle. The drug remained localized due to the polymer mesh. Figure 8 As shown, the drug concentration remains approximately uniform across each tissue cross-section and forms a bell-shaped curve (similar to an injection sphere).

[0182] Example 3: Bovine brain

[0183] Inject 200 μL of PRV311 into the bovine brain using a 26-gauge needle. Figure 10 As shown, tissue sections were prepared for imaging via a microscope.

[0184] For the injected volume, the approximate size of the diffusion is 9mm high × 5mm long. Figure 10 The red color seen is a Cy5 fluorophore dye attached to the chitosan polymer, which illustrates the movement of particles and the network. The green color represents the encapsulated fluorophore, mimicking the diffusion of encapsulated drugs. The network within the injectable formulation keeps the fluorophore localized and concentrated.

[0185] Example 4: Drug delivery to sheep cornea

[0186] Studies in sheep have shown that PRV311 can be delivered into the vitreous humor via the cornea. Figure 15A , Figure 15B and Figure 15C PRV311 is injected into the vitreous humor of the eye, approximately 5 mm below the iris. Figure 16 The red image in the upper right corner shows how Cy5-tagged chitosan can fully penetrate the retina, choroid, and sclera. Figure 16The green image in the lower right corner shows a similar distribution of FITC-labeled free drug during the same injection process.

[0187] This supports PRV311 as an alternative form of existing treatment for AMD. Data collected to date shows:

[0188] • Delivering tagged particles into the cornea and vitreous fluid

[0189] Bevacizumab sustained release for up to 4 months

[0190] Encapsulation enhances the stability and absorption of bevacizumab.

[0191] Example 5: Delivery of drug to sheep lungs

[0192] Studies in sheep have shown that PRV311 is able to deliver the drug to lung tissue, where it remains localized. Figure 17 The drug is kept at a high concentration for 24 hours after injection, and then frozen to prepare for sectioning.

[0193] Example 6: Drug delivery to sheep liver

[0194] like Figure 17 and Figure 18 As shown, PRV311 is able to deliver the drug to liver tissue, where it remains localized. The drug is maintained at a high concentration for 24 hours after injection, and then frozen for sectioning. Figure 19 As shown in the side view, tissue type affects drug distribution patterns.

[0195] Example 7: Drug delivery to sheep pancreas

[0196] like Figure 20 As shown, PRV311 is able to deliver the drug into pancreatic tissue, where it remains localized. The drug is maintained at a high concentration for 24 hours after injection, and then frozen for sectioning.

[0197] The embodiments of the present invention described above are intended to be exemplary only; many variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to fall within the scope of the invention as defined by any of the appended claims.

Claims

1. A pharmaceutical composition for intratumoral injection, comprising: a lyophilized powder that can be reconstituted within 30 seconds to form a uniform, viscous aqueous solution; the aqueous solution comprising a chitosan gel matrix and a plurality of microparticles embedded therein; the microparticles comprising a therapeutic agent and a chitosan coating; wherein the microparticles form uniform colonies within pores of the chitosan gel matrix to prevent microparticle aggregation and provide controlled release; and wherein the combination of the gel matrix and chitosan coating prolongs the retention time of the therapeutic agent in the tumor to at least 10 days following intratumoral injection.

2. The composition of claim 1, wherein the microparticles have an average diameter of 500 nm to 2000 nm.

3. The composition of claim 1, wherein the composition further comprises sodium tripolyphosphate as a crosslinking agent.

4. The composition of claim 1, further comprising: a hydration promoter selected from ethylene glycol, propylene glycol, beta- propylene glycol, or glycerol; a particle adhesion inhibitor selected from HPMC or poloxamer; and a particle aggregation inhibitor selected from a sugar or sugar alcohol.

5. The composition of any one of the preceding claims, wherein the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent, a cytokine, or a combination thereof.

6. The composition of claim 5, wherein the cytokine is IL-12 and the composition allows for retention of IL-12 in the tumor for more than 10 days.

7. The composition of claim 5, wherein the chemotherapeutic agent is cisplatin or oxaliplatin.

8. The composition of any one of the preceding claims, wherein the composition transforms a cold tumor immunologically to a hot tumor, thereby enhancing sensitivity to immunotherapy.

9. Use of the composition of any one of claims 1-8 for treating cancer by intratumoral injection, wherein the composition induces systemic anti-tumor immunity while reducing systemic toxicity as compared to systemic administration of the therapeutic agent.

10. A system for delivering a therapeutic treatment to a target cell population of a subject, the system comprising: a vial closed with a septum that can be penetrated by a needle of a syringe used to administer the therapeutic treatment; a therapeutic composition disposed in the vial, the therapeutic composition provided for administering the therapeutic treatment and comprising an aqueous solution comprising a chitosan gel and a plurality of particles embedded in the gel, the gel having a viscosity that makes it suitable for administration by injection; the particles containing a therapeutic agent and having a coating surrounding the therapeutic agent; and the coating comprising chitosan so as to provide controlled release of the agent from the particles.

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