Systems and pharmaceutical compositions for treatment by direct injection of targeted population of cells

Abstract

To provide systems and methods for delivering a therapeutic treatment to a targeted population of cells of a subject.SOLUTION: The system includes an injectable aqueous solution in a vial enclosed with a septum. The injectable aqueous solution is formulated for injection into a targeted cell population. The solution includes particles containing a therapeutic agent and having a coating around the therapeutic agent, the coating including chitosan so as to provide controlled release of the agent from the particles. The solution further includes chitosan polymer in a form of a polymer gel matrix, further providing controlled release of the particles from the aqueous gel environment. Also provided are methods of manufacturing a lyophilized powder disposed within a vial containing chitosan polymer and chitosan coated particles, the powder forming the above-described injectable aqueous solution of particles and chitosan gel upon mixing with water. Also provided are methods of injecting the above-described injectable aqueous solution into a targeted population of cells, including but not limited to tumors, eyeballs, pancreatic tissue, liver tissue, and lung tissue, in order to provide localized delivery of a therapeutic agent.SELECTED DRAWING: Figure 12

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS 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.

[0002] The present invention relates to therapeutic compositions for injection into target cell populations for the treatment of diseases and tumors, and more particularly to injectable aqueous solutions of chitosan gels and chitosan particles containing therapeutic agents, and methods for preparing and using such compositions. [Background technology]

[0003] Human tumors are often treated by surgical resection. Tumor treatment is usually an urgent matter, especially for solid malignant tumors. These tumors include myeloid sarcoma, round cell sarcoma, melanotic sarcoma, spindle cell sarcoma, and papilloma. Other types of solid tumors are well known to those skilled in the medical arts.

[0004] Often, tumors are not completely resectable, and these solid tumors are considered inoperable. Inoperable solid tumors can be classified as such by their location or size. Chemotherapy is often used to treat solid tumors, reducing their size and making them operable.

[0005] Chemotherapy can be administered via three different routes: (1) systemic intravenous (IV), (2) intra-arterial, and (3) intratumoral. Systemic preoperative IV therapy has been shown to be effective in shrinking or reducing solid tumors. Ferriere, JP et al. (1998) Primary chemotherapy in breast cancer: correlation between tumor response and patient outcome, American Journal of Clinical Oncology, 21(2), 117-120. Furthermore, the IV route provides simultaneous treatment throughout the organism, ensuring that metastatic cells (or micrometastases) are treated systemically. However, one major problem is delivering sufficient amounts of antitumor agents to the target site. Systemic chemotherapy can be dose-limiting and can cause severe side effects that may be intolerable to patients. These side effects limit the use of particularly potent and effective drugs. According to the literature, most drugs are administered systemically at suboptimal doses, at the limit of acceptable side effects (MTD - maximum tolerated dose).

[0006] This limitation on the MTD not only impacts treatment success but can also have the counterproductive effect of creating more resistant tumors. It is hypothesized that within a given solid tumor, there exist several populations of tumor cells of the same type that differ in their ability to resist chemotherapeutic agents at a particular dose level (Kinsella, AR, Smith, D., & Pickard, M. (1997) Resistance to chemotherapeutic antimetabolites: a function of salvage pathway involvement and cellular response to DNA damage, British Journal of Cancer, 75(7), 935). The MTD may be the dose level that can kill most, but not all, cells within a given tumor. As a result, not only will a residual population of more resistant cancer cells remain, but due to their extensive proliferation, these more resistant cells will come to dominate the tumor, presenting a more difficult challenge for future chemotherapy treatments. Another obstacle is that many antitumor drugs may be phase sensitive, meaning they interact with cells only when they are in a specific phase of the cell cycle, sparing other cells that are not in the sensitive phase at the time of administration. Because IV administration is relatively short, even high dose intensities may miss sensitive phases of tumor cells. Treatment of many tumors may benefit from lower doses, more frequent, or continuous administration schedules, both in terms of efficacy and reduced intensity of adverse events.

[0007] Intratumoral injection is a promising alternative to chemotherapy and, at least conceptually, should represent the most successful approach. This method delivers antitumor drugs directly to the tumor, achieving high local concentrations and avoiding systemic side effects. It also offers nearly unlimited flexibility in dosage.

[0008] Despite these advantages, intratumoral chemotherapy has not been particularly effective. This lack of effectiveness is postulated to reflect one or more of the following factors:

[0009] The density of tumor cells within tumors is very high, thus preventing drug penetration through the cells when not via blood vessels.

[0010] Interstitial fluid pressure is high and prevents drugs from moving into the interstitial fluid.

[0011] The high density of cells and blood vessels causes the blood vessels to constrict themselves. See Jain, RK (1999) Transport of molecules, particles, and cells in solid tumors, Annual review of biomedical engineering, 1(1), 241-263.

[0012] Administration protocols have been proposed to alleviate these problems by inducing apoptosis in tumors, see, e.g., M. Flashner-Barak, U.S. Patent Application No. 2002 / 0041888A1, Application No. 09 / 829,621.

[0013] Other possible reasons for the failure of intratumoral administration include uneven distribution of the drug throughout the tumor and the lack of an effective dose long enough to treat cells as they enter their sensitive phase of the cycle. The problem of intratumoral chemotherapy then reduces to maintaining a high enough concentration of the chemotherapeutic agent throughout the tumor for a long enough period to achieve these goals. Intratumoral injections have been performed using gels, pastes, and microparticles.

[0014] Chitosan is a non-toxic (LD50 > 16 g / kg) biodegradable natural polysaccharide derived from the exoskeleton of crustaceans. Its source is chitin, a natural biopolymer found most abundantly in the exoskeletons of crustaceans and insect cuticles, fungal cell walls, and mollusk shells. Chitin consists of 2-acetamido-2-deoxy-β-D-glucose monomers (N-acetylglucosamine units) linked via β(1→4) bonds. Chitosan is a polymer of deacetylated α-(1,4)glucosamine units, typically obtained by deacetylating chitin with NaOH after demineralization and deproteinization of crustacean shells or exoskeletons. Chitosan is a multifunctional material with excellent biocompatibility, no immunogenicity, and no skin irritation. It was approved by the U.S. Food and Drug Administration (FDA) as a GRAS (Generally Recognized as Safe) substance in 2001. Chitosan is a widely used biomaterial with an established human safety profile. It is used as a pharmaceutical excipient, weight-loss supplement, experimental mucosal adjuvant, and in FDA-approved hemostatic dressings. High-molecular-weight chitosan (>100 kDa) forms highly viscous solutions in mild aqueous media due to its long polymer chains. Viscous solutions have been widely used to control the release of drugs and macromolecules in vivo because they hinder the diffusion and dispersion of these molecules after injection. Baldrick, P. (2010) The safety of chitosan as a pharmaceutical excipient, Regulatory toxicology and pharmacology, 56(3), 290-299.

[0015] Platinum-based drugs, such as cisplatin (cis-diamminedichloroplatinum-II), are among the most widely used chemotherapy agents and have demonstrated efficacy against a variety of solid neoplasms outside the central nervous system, including testicular, ovarian, breast, colorectal, lung, and head and neck tumors. Systemically delivered cisplatin has less than 5% of the plasma concentration detected in the brain after intravenous delivery, and penetration into normal brain tissue is poor due to the blood-brain barrier (BBB). However, tumor neovasculature is more permeable than an intact BBB, and therapeutic cisplatin levels have been detected in primary and secondary brain tumors, and to a lesser extent in the edematous brain adjacent to the tumor after systemic delivery. Perez, J. et al. (2019) The effect of locally delivered cisplatin is dependent on an intact immune function in an experimental glioma model, Scientific Reports, 9(1), 5632.

[0016] Cytokines, small proteins released by immune cells, enable them to communicate with one another. Cytokines have been investigated for some time as potential cancer treatments. However, despite their known efficacy and potential for use in combination with other immunotherapies, cytokines have yet to be successfully developed into effective cancer therapies. This failure likely reflects the high toxicity of cytokines to both healthy tissue and tumors, making them unsuitable for use in systemically administered therapies.

[0017] Injecting cytokines directly into tumors may offer a way to limit their toxic effects to the tumor and spare healthy tissue, but previous attempts to do so have resulted in the proteins leaking from cancerous tissue into the systemic circulation within minutes.

[0018] Cytokines are a broad and loose category of small proteins (approximately 5-20 kDa) that are important in cell signaling. Cytokines are peptides and cannot cross the lipid bilayer of cells into the cytoplasm. Cytokines are immunomodulators and are involved in autocrine, paracrine, and endocrine signaling. Their clear distinction from hormones is still part of ongoing research.

[0019] Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines are produced by a variety 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 particular cytokine may be produced by multiple cell types. Aznar, MA et al. (2017) Intratumoral delivery of immunotherapy—act locally, think globally, The Journal of Immunology, 198(1), 31-39.

[0020] Cytokines act through receptors and are particularly important in the immune system, regulating the balance between humoral and cell-based immune responses and modulating the maturation, growth, and responsiveness of specific cell populations. Some cytokines enhance or inhibit the actions of other cytokines in complex ways.

[0021] Cytokines include, but are not limited to, granulocyte colony-stimulating factor (G-CSF), interferons, interleukins including IL-2, IL-7, IL-12, and various chemokines.

[0022] Other immunomodulatory agents are also being investigated, including imiquimod, cell membrane fractions from bacteria, synthetic cytosine phosphate-guanosine (CpG), oligodeoxynucleotides, and glucans, which have been used by patients and others.

[0023] Ocular vascular disease is one of the leading causes of visual impairment and blindness worldwide. Intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) agents has revolutionized the treatment of common retinal diseases such as neovascular age-related macular degeneration (AMD), diabetic retinopathy, and retinal vein occlusion (RVO). Furthermore, promising results have been reported with intravitreal injection of anti-VEGF agents for other ocular diseases such as neovascular glaucoma, retinopathy of prematurity (ROP), and intraocular tumors.

[0024] Age-related macular degeneration (AMD) is a well-characterized and extensively studied disease. It is currently considered the leading cause of visual impairment in patients over 60 years of age. Early AMD is characterized by the formation of drusen, pigmentary changes in the macula, and mild to moderate vision loss. AMD exists in two forms: "dry" and "wet." Although less common, dry and wet forms account for 90% of acute AMD-related blindness cases. Risk factors are associated with AMD progression and are relevant to understanding how AMD develops: (1) aging and exposure to environmental factors induce high levels of oxidative stress that damage the macula, and (2) this damage, which leads to inflammation, triggers a vicious cycle that ultimately leads to central vision loss.

[0025] There is no cure or treatment to prevent AMD. However, several treatments are available for the wet form of AMD. Treatment of the wet form has made great strides with the introduction of antiangiogenic drugs; functional prognosis has changed from almost certain blindness to a greater than 90% chance of a three-line visual improvement after two years of treatment. Nevertheless, even after this advancement, treatment is far from perfect, and there is still ample room for improvement. Hernandez-Zimbron, LF et al. (2018) Age-Related Macular Degeneration: New Paradigms for Treatment and Management of AMD, Oxidative Medicine and Cellular Longevity, 2018, 8374647.

[0026] Reducing the treatment burden associated with regular intravitreal anti-VEGF injections is a priority. Neovascular AMD and diabetic retinopathy are chronic, relapsing disorders. Patients may require dozens of injections over years of treatment. Adherence to such rigorous regimens is difficult. Current promising approaches include (a) new hardware for delivering anti-VEGF drugs, (b) new pharmaceuticals with longer durability of biological effect, (c) new formulations of anti-VEGF agents for sustained release, 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, 5(1), 22.

[0027] Several anti-VEGF drugs are currently available, including pegaptanib, ranibizumab, bevacizumab, and aflibercept. Well-designed randomized clinical trials have demonstrated the efficacy of these drugs in improving vision in various retinal diseases; however, intravitreal injections each carry the risk of post-injection and drug-class-related adverse events. The commonly required repeated and prolonged injections may increase the likelihood of ocular and systemic complications. Falavarjani, KG et al. (2013) Adverse events and complications associated with intravitreal injection of anti-VEGF agents: a review of literature, Eye, 27(7), 787.

[0028] In particular, the most common treatment for AMD is intravitreal bevacizumab injections, which are administered every 3–4 weeks and can pose procedural risks and inconvenience to patients.

[0029] Targeted delivery of drugs to other tissues, including but not limited to pancreatic, lung, and liver tissue, localizes treatment to these tissues and has potential advantages in treating diseases specific to these tissues, including pancreatitis, diabetes, brain cancer, lung cancer, hepatitis, etc. Summary of the Invention [Means for solving the problem]

[0030] According to one embodiment of the present invention, there is provided a composition formulated for delivery by injection to a target population of cells in a subject. According to this embodiment, the composition comprises an aqueous solution comprising a chitosan gel and a plurality of particles, the particles comprising a therapeutic agent and having a coating around the therapeutic agent, the coating comprising chitosan to provide controlled release of the agent from the particles.

[0031] According to one embodiment of the present invention, a system for delivering a therapeutic treatment to a target population of cells in a subject is provided. According to this embodiment, the system includes a vial surrounded by a septum that is pierceable by the needle of a syringe used to administer the therapeutic treatment. According to this embodiment, a therapeutic composition is disposed in the vial, the therapeutic composition being provided for use in administering the therapeutic treatment and comprising an aqueous solution including a chitosan gel and a plurality of particles embedded in the gel, the gel having a viscosity suitable for administration by injection. According to this embodiment, the particles include a therapeutic agent and have a coating around the therapeutic agent, the coating including chitosan to provide controlled release of the agent from the particles.

[0032] According to a further related embodiment, the aqueous solution further comprises a compound selected from the group consisting of a hydration enhancer, a particle adhesion inhibitor, a particle aggregation inhibitor, and combinations thereof.

[0033] The hydration enhancer is selected from the group consisting of ethylene glycol, propylene glycol, beta-propylene glycol, glycerol, and combinations thereof.

[0034] The particle adhesion inhibitor is selected from the group consisting of HPMC, poloxamer, and combinations thereof.

[0035] The particle aggregation inhibitor is selected from the group consisting of monosaccharides, disaccharides, sugar alcohols, chlorinated monosaccharides, chlorinated disaccharides, and combinations thereof.

[0036] Alternatively, or in addition, the composition further comprises sodium tripolyphosphate.

[0037] According to some embodiments, the particles are microparticles having an average diameter between 200 nm and 2000 nm. As a further option, the microparticles have an average diameter between 500 nm and 2000 nm.

[0038] Optionally, the solution further comprises a free amount of therapeutic agent not coated with chitosan, wherein the free amount of said therapeutic agent comprises between about 20% and about 80% by weight of the total amount of therapeutic agent in the aqueous solution.

[0039] Also optionally, the therapeutic agent in the particle is an immunotherapeutic agent. Further alternatively, the therapeutic agent is selected from the group consisting of an antibody, a cytokine, a small molecule immunotherapeutic agent, and combinations thereof.

[0040] According to a further related embodiment, the therapeutic agent is a chemotherapeutic agent.

[0041] Optionally, the particles are in direct physical contact with the chitosan gel.

[0042] According to another embodiment, the present invention provides a method for treating a target population of cells in a subject, the method comprising obtaining a system as described at the beginning of this section, loading a syringe with an aqueous solution, and using the syringe to inject the aqueous solution into the target population of cells.

[0043] In a related embodiment of this method, the target population of cells comprises a tumor. In another related embodiment, the target population of cells is tissue within an organ. Optionally, the organ is selected from the group consisting of eye, lung, pancreas, liver, kidney, brain, heart, thyroid, and pituitary gland.

[0044] According to another embodiment, a system for delivering a therapeutic treatment to a target population of cells in a subject is provided, the system including a vial surrounded by a septum pierceable by the needle of a syringe used to administer the therapeutic treatment. According to this embodiment, a therapeutic composition is disposed in the vial, the composition provided for use in administering the therapeutic treatment, and includes a lyophilized precursor formulated to dissolve upon mixing with water to provide an aqueous solution including a chitosan gel and a plurality of particles embedded in the gel, the gel having a viscosity suitable for administration by injection. According to this embodiment, the particles include a therapeutic agent and have a coating around the therapeutic agent, the coating including chitosan to provide a controlled release of the therapeutic agent from the particles.

[0045] According to another embodiment, there is provided a freeze-drying method for providing a system for delivering a therapeutic treatment to a targeted cell population, the method comprising: (1) forming an aqueous solution containing a chitosan gel and a plurality of particles, the particles containing a therapeutic agent and having a coating around the therapeutic agent, the coating comprising chitosan to provide controlled release of the agent from the particles; (2) freezing the initial aqueous solution in a bath containing an aqueous alcohol solution at a temperature above the freezing temperature of the aqueous alcohol solution, at most −80° C., to form a frozen layer precursor; (3) drying the frozen layer precursor to form an anhydrous powder having embedded particles; (4) containing the anhydrous powder in a vial surrounded by a septum pierceable by the needle of a syringe used to administer the therapeutic treatment; and (5) adding water to the container to dissolve the anhydrous powder.

[0046] Optionally, the aqueous solution in the freeze-drying process further comprises a hydration enhancer, a particle adhesion inhibitor, and a particle aggregation inhibitor.

[0047] According to further related embodiments of the freeze-drying method, the hydration enhancer is selected from the group consisting of ethylene glycol, propylene glycol, beta-propylene glycol, glycerol, and combinations thereof. Also optionally, the particle adhesion inhibitor includes HPMC, poloxamer, and combinations thereof. According to related embodiments, the particle aggregation inhibitor is selected from the group consisting of monosaccharides, disaccharides, sugar alcohols, chlorinated monosaccharides, chlorinated disaccharides, and combinations thereof. Optionally, the composition further comprises sodium tripolyphosphate. Also optionally, the particles are microparticles having an average diameter between 200 nm and 2000 nm. As a further option, the particles are microparticles having an average diameter between 500 nm and 2000 nm.

[0048] According to some embodiments of the present invention, the particle-based composition is formulated for delivery by intravitreal injection to treat an ocular condition.

[0049] Optionally, the eye disease is age-related macular degeneration (AMD).

[0050] According to some embodiments, the therapeutic agent for intravitreal injection is selected from the group consisting of an antibody, a cytokine, a small molecule immunotherapeutic, a chemotherapeutic, an aptamer, and combinations thereof. According to some embodiments, the therapeutic agent for intravitreal injection is bevacizumab.

[0051] The foregoing features of the embodiments will be more readily understood by reference to the following detailed description taken in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0052] [Figure 1A]Figure 1A shows an injectable chitosan formulation according to the present invention (PRV311) that has been frozen at -80°C and properly stored. Notably, the formulation provides a clear aqueous solution. (Left) vs. PRV311 at room temperature (center) and PRV111 (right). PRV111 is a similar product to PRV311, but is more quickly frozen in liquid nitrogen (-196°C) during manufacturing rather than in a -80°C freezer.

[0053] [Figure 1B] FIG. 1B shows, for comparison, an injectable chitosan formulation according to the present invention (PRV311) that was frozen at −80° C. and stored at room temperature for 5 days.

[0054] [Figure 1C] FIG. 1C shows, for comparison, an injectable chitosan formulation according to the present invention (PRV311) that was frozen in liquid nitrogen at −196° C. and stored at room temperature for 5 days.

[0055] [Figure 2] FIG. 2 shows the FTIR spectrum of the injectable chitosan powder containing cisplatin internal standard, with peaks at 1400 and 1560 cm −1 .

[0056] [Figure 3] Figure 3 shows a photograph of the matrix (2x zoom) when frozen in liquid nitrogen (-196 °C) and subsequently freeze-dried. Note the multi-layered, dense, dough-like structure.

[0057] [Figure 4] Figure 4 shows a photograph of the matrix (2x zoom) when frozen in a -80 °C chest freezer and subsequently lyophilized. Note the more porous and uniform single-layer polymer fibers.

[0058] [Figure 5]Figure 5 shows the release profiles of drug from microparticles in media at various pH levels. The powder was reconstituted in each medium and placed in a dialysis bag with stirring for 72 hours. Samples were taken and the release rates are shown. Microparticles at pH 6 (circles) were released faster due to faster degradation, while microparticles at pH 3 (triangles) were released at a slower rate due to greater particle stability. Free cisplatin solution (squares) was used as a control.

[0059] [Figure 6] Figure 6 shows a graph of the change in tumor volume over time in mice treated with different treatments. The black curve corresponds to the control untreated tumor, the gray curve corresponds to the intratumoral injection of drug-free placebo particles, the green curve corresponds to the intravenous injection of drug, the red curve corresponds to the intratumoral injection of free drug, and the blue curve corresponds to the injection of the drug-encapsulated hydrogel PRV311.

[0060] [Figure 7] 7 shows a cross-section of lamb tissue after injection with PRV311, a chitosan formulation according to an embodiment of the invention, viewed under a fluorescent microscope, where the drug is labeled with fluorescein isothiocyanate (FITC) and appears green under the microscope.

[0061] [Figure 8] FIG. 8 is a graph showing FITC-labeled drug concentration in pig tongue tissue as a function of tissue depth.

[0062] [Figure 9] FIG. 9 is a photograph showing intratumoral injection and local distribution of FITC-labeled drug using an embodiment of the present invention.

[0063] [Figure 10] FIG. 10 is a photograph showing the penetration of an injectable formulation according to an embodiment of the present invention into a bovine brain.

[0064] [Figure 11]FIG. 11 shows a greatly enlarged view of an embodiment of the present invention configured for injection.

[0065] [Figure 12] FIG. 12 is a greatly enlarged view showing how the injectable solution PRV311 builds a polymeric web within and around a tumor, according to an embodiment of the present invention.

[0066] [Figure 13] 13A and 13B are photomicrographs showing PRV311, according to an embodiment of the present invention.

[0067] [Figure 14] Figure 14 is a photograph showing some of the more than 80 formulations evaluated by the inventors in the course of developing a formulation suitable for reconstitution for clinical use within seconds, in accordance with an embodiment of the present invention.

[0068] [Figure 15] Figures 15A, 15B, and 15C show that a PRV311 formulation containing a fluorescently labeled drug was injected into sheep eyes, resulting in complete coverage and penetration of the cornea.

[0069] [Figure 16] FIG. 16 shows injection with separate fluorescent labels of polymer (red) and drug (green), illustrating how both bolus and controlled delivery can be achieved.

[0070] [Figure 17] FIG. 17 shows the injection of fluorescently labeled PRV311 into lung tissue, demonstrating high local concentrations of drug for over 24 hours after injection.

[0071] [Figure 18] FIG. 18 shows injection of PRV311 into the liver of a sheep, again demonstrating high local concentrations of the drug.

[0072] [Figure 19] FIG. 19 shows a lateral view of injection of PRV311 into sheep liver, demonstrating that tissue type influences drug distribution patterns.

[0073] [Figure 20] Figure 20 shows local injection of PRV311 into the ovine pancreas, demonstrating high local drug concentrations, and that tissue type influences the local drug distribution pattern. DETAILED DESCRIPTION OF THE INVENTION

[0074] Definitions. As used in this description and the accompanying claims, unless otherwise specified, the following terms shall have the meanings indicated.

[0075] A "subject" includes vertebrates, such as mammals, and even humans.

[0076] A "polymer" is a molecule having at least 100 monomer units.

[0077] A polymer "matrix" is a three-dimensional web of polymer molecules, the web being selected from the group consisting of non-covalently entangled, ionically crosslinked, covalently crosslinked, and combinations thereof.

[0078] A "gel" is a solution phase of a polymer matrix that swells in a solvent while retaining entanglements and crosslinks.

[0079] "Microparticles" are a set of particles having an average diameter of about 200 nm to about 2000 nm. "Nanoparticles" are a collection of particles having an average diameter of at least 1 nm to about 200 nm.

[0080] "Particle diameter" or "particle size" is the length of the longest linear axis between two points on the surface of a particle.

[0081] "Pure chitosan" is chitosan that is not a chitosan salt.

[0082] "Unmodified chitosan" is chitosan that has not been chemically modified by the addition of functional groups or by binding to a carrier.

[0083] An "unmodified therapeutic agent" is a therapeutic agent that has not been chemically modified by the addition of a functional group or by attachment to a carrier.

[0084] An "immunotherapeutic agent" is a therapeutic agent that modulates the immune response. Immunotherapeutic agents can be biologic or small molecule drugs.

[0085] A "chemotherapeutic agent" is a therapeutic agent that is a small molecule drug.

[0086] An "aptamer" is a nucleic acid or modified nucleic acid that has been selected by in vitro selection methods for binding to a biological target. A notable example of an "aptamer" is the drug pegaptanib (trade name Macugen®), which binds to VEGF and is used to treat wet macular degeneration.

[0087] A "particulate adhesion inhibitor" is an additive that reduces the attractive forces between a polymer matrix and particles embedded therein. As a result, the particles are able to move through the matrix at a faster rate than they would in the absence of the adhesion inhibitor.

[0088] A "microparticle agglomeration inhibitor" is an additive that reduces the tendency of particles embedded in a matrix to agglomerate when the matrix is ​​exposed to freezing, thereby reducing the likelihood that the particles will be damaged or destroyed when freezing occurs.

[0089] "Mucouadhesive" materials are characterized as having the ability to adhere to mucous membranes of the human body.

[0090] A polymer matrix is ​​"porous" if a portion of its volume is void. In some cases, the void space is accessible from the outer surface of the matrix, allowing items residing in the void space, such as particulates, to migrate to and from the outer surface.

[0091] The "void" space in a polymer matrix is ​​the space not occupied by the polymer, allowing the movement of particulates and small molecules through the space.

[0092] "Mucosal tissue" is tissue that has an associated mucosa. In particular, mucosal tissue includes the mucosa and tissue underlying the mucosa.

[0093] For example, a "site within mucosal tissue" where a cancerous tumor is present may include not only the mucosa but also the tissue underlying the mucosa.

[0094] "Polydispersity index" (PDI) or simply "dispersity" is a measure of the heterogeneity in size of a set of particles, such as fine particles, in a mixture.

[0095] "Zeta potential" (ZP) is a measure of the overall charge a particle acquires in a particular medium. ZP can be measured with a Zetasizer Nano instrument.

[0096] "Penetration" is the ability to penetrate or penetrate the mucosa, the underlying tissue, or both. "Biocompatibility" refers to the ability of a biomaterial to perform its desired function with respect to a medical treatment without inducing significant undesirable local or systemic effects in the recipient or beneficiary of the treatment, but generating the beneficial cellular or tissue response most appropriate in that particular situation, optimizing the clinically relevant performance of the treatment.

[0097] "HPMC" refers to hydroxypropyl methylcellulose, also known as hypromellose.

[0098] "Biodegradable" refers to the property of a material that can be broken down by the action of living organisms, especially into harmless products.

[0099] "Kilocounts / second" (Kcps), the average count rate in kilocounts per second (kcps). For example, a threshold can be set so that the measurement should be stopped if the count rate of the sample is below 100, meaning the concentration of the sample is too low to measure. A sample with an appropriate Kcps can be considered a stable sample with an ideal concentration for measurement.

[0100] "Mesh" refers to a polymer matrix attached to the treated area and containing elements incorporated therein that are released from the mesh when applied to the treated area.

[0101] The "polymeric matrix and microparticle-based system for delivery of therapeutic agents" may also be referred to as a "drug delivery device" or a "delivery patch."

[0102] Unless otherwise specified, the term "wt %" refers to the amount of a component of a system for delivery of a therapeutic agent expressed as a weight percent.

[0103] Unless otherwise specified, the "molar mass" of a polymer is intended to mean the number-average molar mass of the polymer molecules.

[0104] Cancer can arise in any tissue in any organ at any age. Once a clear diagnosis of cancer is made, treatment decisions become paramount. While no single treatment approach is applicable to all cancers, successful treatment must target both the primary tumor and its metastases. Historically, local and regional therapies, such as surgery and radiation therapy, along with systemic therapies, such as chemotherapy drugs, have been used to treat cancer. Despite some success, traditional therapies have not been as effective as desired, and the search for more effective treatments continues. Clearly, there is a significant unmet need for more efficient cancer treatments.

[0105] One of the primary uses of embodiments of the present invention is for intratumoral injection of chemotherapy and immunotherapy, with data showing the best way to maintain a high concentration of drug within the tumor and drain some of the drug to the lymph nodes to ensure the most effective way to treat the tumor in a localized and regional manner.

[0106] Intratumoral injection may be considered for any tumor where the primary tumor or its metastases are accessible percutaneously by direct injection or by specific procedures such as colonoscopy, cystoscopy, bronchoscopy, thoracoscopy, colonoscopy, or even surgery.

[0107] Currently, there are numerous agents being investigated for their role in intratumoral therapy, including immunoreceptor agonists (Toll-like receptor (TLR) agonists, stimulator of interferon genes (STING) agonists, ICT mAbs, wild-type and genetically engineered oncolytic agents (e.g., viruses and peptides), cytokines and immune cells directed against a variety of potential targets. Therefore, to support the clinical development of intratumoral strategies in humans, we developed an injectable system for the local delivery and retention of these agents.

[0108] Furthermore, direct tumor injection reduces systemic exposure, off-target toxicity, and the amount of drug used, while inducing stronger antitumor activity in the injected tumor lesion and in distant, non-injected tumor lesions.

[0109] Systemic immunotherapy and systemic chemotherapy are often used, but they expose the patient's entire body to the toxic side effects of the drugs. Systemic administration is dose-limiting due to exposure within the bloodstream and other organs, and precautions must be taken to ensure safety of this systemic exposure. Systemic delivery often results in adverse side effects from toxic drugs reacting with the body. These include neurotoxicity, nephrotoxicity, renal failure, alopecia, nausea, and mucositis. Instead of surgery, chemotherapy in addition to radiation is also used to treat anal tumors. The current standard of care is to initially administer concurrent chemotherapy and radiation to patients with anal canal squamous cell carcinoma, even in cases with small, localized tumors. When chemotherapy is used, a temporary central venous catheter or a peripherally inserted central catheter can be used in individuals. Treatment-related side effects include those typical of systemic chemotherapy. These include nausea, alopecia, kidney damage, decreased blood counts, mouth sores, and a weakened immune system. Currently, chemotherapy is administered systemically, which is dose-limiting.

[0110] The therapeutic benefit of a drug can be increased by maximizing its efficacy and / or reducing its side effects. The basis for developing regional anticancer drug therapies is to achieve effective target tissue concentrations while minimizing systemic distribution and, therefore, toxicity. Examples of existing clinically used local chemotherapy include intra-arterial infusion for liver and kidney neoplasms, limb perfusion for melanoma and sarcoma, intrathecal administration for CNS neoplasms, and intraperitoneal administration for intraperitoneal neoplasms. Recently, direct intratumoral injection of pure ethanol for primary hepatocellular carcinoma has been developed. One major drawback associated with most cytotoxic chemotherapeutic agents is that they are potent cytotoxic agents and therefore not ideal candidates for intratumoral administration unless the delivery technique maintains the drug locally in the tumor and allows leakage into healthy tissue. Embodiments of the present invention use a combination of polymer-drug-loaded chitosan particles and a polymer combination to ensure drug retention in the tumor and reduced side effects.

[0111] Intratumoral immunotherapy is a therapeutic strategy that aims to use the tumor as its own vaccine. Direct injection into the tumor allows for the delivery of high concentrations of immunostimulatory products in situ while using small amounts of drug. Local delivery of immunotherapy allows for multiple combination therapies while preventing significant systemic exposure and off-target toxicity. Therefore, despite the uncertainty of the dominant epitopes of a given cancer, immune responses against relevant neoantigens or tumor-associated antigens can be elicited without the need for their characterization. Such immune stimulation can induce potent priming of cancer immunity locally while generating a systemic (abscopal) tumor response thanks to the circulating presence of appropriately activated antitumor immune cells. While addressing many of the current limitations of cancer immunotherapy development, intratumoral immunotherapy also offers a unique opportunity to better understand the dynamics of cancer immunity by enabling serial and multifocal biopsies with every 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, 29(11), 2163-2174.

[0112] All five classes of immunotherapies face delivery challenges. Checkpoint inhibitors, cytokines, and agonist antibodies present similar delivery challenges. The success of these therapies relies on their interaction with target proteins. A major limitation of their use is that they produce substantial autoimmunity and other adverse effects that limit tolerated doses. Therefore, a central goal in developing delivery technologies for these therapies is to enable targeted, controlled release so that the therapy is primarily active in the desired cell type, thereby minimizing off-target effects.

[0113] The microenvironment in many solid tumors presents a challenge to the widespread implementation of all immunotherapy classes discussed here. For example, the microenvironment of solid tumors can be immunologically classified as either "hot" (highly immunogenic) or "cold" (lowly immunogenic), with high or low levels of cytotoxic lymphocyte infiltration within the tumor space, respectively. This important difference in the composition of the microenvironment suggests that highly immunogenic tumors will respond more strongly to checkpoint inhibitors than less immunogenic tumors.

[0114] Delivery technologies can be used to modulate immunogenicity in cold tumors. Furthermore, delivery platforms can also reduce the systemic toxicity of immunotherapies by limiting drug exposure to specific tissues, allowing them to be used to deliver therapeutic combinations that would otherwise be too toxic to administer to patients.

[0115] Local delivery of immunotherapy using embodiments of the present invention allows for multiple combination therapies while preventing significant systemic exposure and off-target toxicity. Despite the uncertainty of the dominant epitopes of a given cancer, immune responses can be elicited against relevant neoantigens or tumor-associated antigens without the need for their characterization. Such immune stimulation can induce potent priming of cancer immunity locally while generating a systemic (abscopal) tumor response thanks to the circulating presence of appropriately activated anti-tumor immune cells.

[0116] According to embodiments of the present invention, tumors can be injected with a composition comprising a combination of one or more immunotherapeutic particles and chemotherapy particles. The chemotherapy particles can include chemotherapeutic agents, including but not limited to cisplatin and oxaliplatin, which have been shown to activate dendritic cells and induce immune activity in tumors in addition to causing DNA damaging effects in tumor cells.

[0117] Embodiments of the present invention when delivering chemical agents can heat up immunologically cold tumors, thus making them susceptible to immunotherapy. Tumor-targeted immunotherapy particles and chemotherapy act synergistically to inhibit tumor growth and exhibit reduced toxicity compared to immunotherapy and chemotherapy alone, i.e., without the use of embodiments of the present invention.

[0118] It has been discovered that microparticles can enable combination treatment strategies to sensitize poorly immunogenic tumors to immunotherapy. In addition to enabling combination treatment strategies, embodiments of the present invention can be designed to respond to the tumor microenvironment and increase penetration at those sites.

[0119] According to David Zaharoff and others, a chitosan mixture with the cytokine IL-12 was effective in tumor regression in their mouse experiments (Zaharoff, DA, et al. (2010). Intratumoral immunotherapy of established solid tumors with chitosan / IL-12. J. Immunother., 33, 697). However, our data show that, according to embodiments of the present invention, the combination of a chitosan matrix loaded with IL-12 particles has a very high retention time (>10 days) along with controlled release in tumors at high concentrations. According to various embodiments, the injectable cytokine can be mixed at the bedside clinic within seconds for translational application, unlike the laboratory experiments performed in Zaharoff's study.

[0120] IL-12 is a potent antitumor cytokine that exhibits significant clinical toxicity after systemic administration. Zaharoff hypothesized that intratumoral (it) administration of IL-12 co-formulated with the biodegradable polysaccharide chitosan could enhance IL-12's antitumor activity while limiting systemic toxicity. Noninvasive imaging studies monitored the local retention of IL-12 after it injection, with or without chitosan co-formulation. The antitumor effects of IL-12 alone and IL-12 co-formulated with chitosan (chitosan / IL-12) were evaluated in mice bearing established colorectal (MC32a) and pancreatic (Panc02) tumors. Additional studies, including immune cell subset depletion, tumor rechallenge, 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 it injections of IL-12 alone eradicated less than 10% of established MC32a and Panc02 tumors, whereas chitosan / IL-12 immunotherapy induced complete tumor regression in 80% to 100% of mice. Depletion of CD4+ or Gr-1+ cells did not affect chitosan / IL-12-mediated tumor regression. However, depletion of CD8+ or NK cells completely abolished the antitumor activity. It chitosan / IL-12 immunotherapy generated systemic tumor-specific immunity, as more than 80% of mice cured by it chitosan / IL-12 immunotherapy were at least partially protected from tumor rechallenge. Furthermore, CTLs from the spleens of cured mice lysed targets loaded with MC32a and gp70 peptides. Chitosan / IL-12 immunotherapy increased local retention of IL-12 in the tumor microenvironment, eradicated established aggressive murine tumors, and generated systemic tumor-specific protective immunity. Chitosan / IL-12 is a well-tolerated and effective immunotherapy with considerable potential for clinical translation.

[0121] According to some embodiments, particle-based formulations are provided for the controlled delivery of drugs via intravitreal injection into the eye to treat ocular conditions, particularly age-related macular degeneration. According to such embodiments, the biocompatible polymer forms a hydrogel matrix that forms a polymer mesh that adheres to the ocular epithelium, thereby implanting the drug particles into the tissue. The particles may degrade over an extended period of time, e.g., four months, to provide sustained release of the drug.

[0122] In accordance with one embodiment of the present invention, the inventors have developed a formulation for oral and injectable delivery of poorly water-soluble drugs and polymers. This formulation allows for the conversion of liquid nanocrystal dispersions into solid dosage forms. The solid dosage forms contain nanocrystals that can easily reconstitute to their original size upon dissolution in water. Careful formulation is required to optimize the freezing rate and reduce interparticle aggregation. The critical freezing rate for drying nanocrystals has been determined. Freeze-drying at freezing rates close to the critical value produces dry powders with bimodal particle size distributions after redispersion. Furthermore, it has been found that the drug nanocrystal concentration significantly affects the critical freezing rate and, therefore, the redispersibility of the dried powder. The concept of critical freezing rate is important for the development of solid dosage forms from liquid nanocrystal dispersions.

[0123] Embodiments of the present invention provide a formulation that can be shipped in powder form and can be rapidly, uniformly, and consistently dissolved in sterile water for intratumoral injection at the patient's bedside.

[0124] Embodiments of the present invention provide a system for the delivery of therapeutic agents to various tissues, particularly cancerous tumors. Various embodiments include chitosan and a plurality of particles embedded within the matrix.

[0125] Once a working formulation of an embodiment of the present invention was developed, chitosan microparticles were synthesized at room temperature using ionic gelation with sodium tripolyphosphate as a crosslinker. A separate formulation of a polymer matrix containing a particle adhesion inhibitor, a particle aggregation inhibitor, and a hydration promoter was added to the microparticle solution. The microparticle and matrix solution were dispensed into vials, transferred to a -80°C freezer, and frozen overnight. The vials were then lyophilized for 72 hours.

[0126] Some embodiments of the present invention for producing an injectable chitosan powder containing cisplatin are based on the following protocol: 1. Add chitosan powder to the acetic acid solution (0.186 w / w%) and stir to dissolve. 2. In a separate container, add cisplatin (0.15 w / w%) to sodium tripolyphosphate and saline. Heat the solution to approximately 40°C and stir to dissolve the cisplatin. 3. All containers containing cisplatin were protected from light exposure, and the contents of the cisplatin-sodium tripolyphosphate solution were transferred to the chitosan solution. a. Both solutions were gently stirred throughout this process. b. Now in this process, fine particles are produced. Once steady state is reached, the particle size / charge is collected. 4. A sucralose solution in water (25 w / w%) was prepared and set aside for later use. 5. In a separate screw-top bottle, add the chitosan powder to the dilute acetic acid solution (1.0 w / w%). Next, add the hydroxypropyl methylcellulose (0.1 w / w%) to the chitosan-acetic acid solution. Stir for 30 minutes. 6. The sucralose solution was transferred to the chitosan-cisplatin microparticle solution, followed by the patch matrix solution. 7. The final solution was stirred for 5 minutes, after which 5 mL of solution was dispensed. The vials were frozen in a -80°C freezer for approximately 2 hours. 8. The vials were placed in a freeze dryer for 6 days.

[0127] A sample certificate of analysis of injectable chitosan powder containing cisplatin is shown in Table 1. The FTIR characteristics of the sample are shown in Figure 2, where the FTIR spectrum at 1400 + 30 cm -1 and 1560+30cm -1 A characteristic cisplatin peak is observed. [Table 1]

[0128] To characterize the solubility with respect to pH, as summarized in Table 2, injectable chitosan powder containing cisplatin was reconstituted in various pH media and the release of cisplatin was monitored at 355 nm in a UV-visible spectrometer. [Table 2]

[0129] According to a first set of exemplary embodiments of the present invention, a system for injectable localized delivery of a therapeutic agent to a site within tissue is provided. The system includes a polymer matrix capable of forming a gel, i.e., a polymer web-like structure, in a solvent that contributes to localizing embedded particles within the tissue. The gel matrix is ​​formed by a composition including chitosan, a hydration enhancer, a microparticle adhesion inhibitor, and a microparticle aggregation inhibitor. Multiple microparticles are embedded within the gel matrix. The gel matrix is ​​configured to open to allow the drug-loaded particles a limited range of movement within the tissue. The microparticles include a therapeutic agent and have a coating around the therapeutic agent. The microparticle coating includes chitosan to provide controlled release of the agent from the microparticles. Optionally, the hydration enhancer is selected from the group consisting of ethylene glycol, propylene glycol, beta-propylene glycol, glycerol, and combinations thereof. Also optionally, the microparticle adhesion inhibitor is a non-ionic polymer, and as a further option, the non-ionic polymer is HPMC or poloxamer. Further optionally, the microparticle aggregation inhibitor is selected from the group consisting of monosaccharides, disaccharides, sugar alcohols, chlorinated monosaccharides, chlorinated disaccharides, and combinations thereof. Also optionally, the microparticles further comprise sodium tripolyphosphate. Optionally, the system includes a free amount of therapeutic agent embedded directly in the matrix and not coated with chitosan, wherein the free amount of therapeutic agent comprises 20-80% 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 microparticles have an average diameter of about 500 nm to about 2000 nm.

[0130] The matrix is ​​configured to provide controlled release of microparticles through tissue. This system can be used to locally inject potent drugs with significant systemic toxic side effects into damaged tissue, such as cancerous tumors. The method for manufacturing the present invention further includes freezing the mixture at -80°C to form a frozen layer precursor. Finally, the method for manufacturing the present invention includes drying the frozen layer precursor to form a powder that, upon hydration, forms a gel matrix with microparticles embedded within the matrix. According to some embodiments of the present invention, the final product (powder for reconstitution) is stable for more than six months, provided that it is stored with a desiccant, heat-sealed in a watertight Mylar foil pouch, and stored in a refrigerator at 2-8°C. When these conditions are met, the system (sample PRV311 containing chitosan nanoparticles within the mesh) can be reconstituted into either a clear solution or a heterogeneous microparticle suspension. The solubility of PRV311 is further increased by chain fragmentation hydrolysis (e.g., freeze-thaw hydrolysis) that occurs in chitosan when frozen in the mesh and when PRV311 is gamma-irradiated for patient use. Properly stored PRV311 is shown in Figure 1A and is a clear solution / particulate suspension.

[0131] As shown in Figure 1B, PRV311 does not reconstitute properly if stored at room temperature for several days before reconstitution. The vial in Figure 1B was stored at room temperature for 5 days before reconstitution and forms a heterogeneous, coarse suspension unsuitable for injection. Figure 1C shows another formulation, PRV111, frozen more rapidly than PRV311 in liquid nitrogen and stored at room temperature for 5 days. This processed PRV111 formulation also forms a heterogeneous, coarse suspension unsuitable for injection.

[0132] Without being bound by theory, it is assumed that: a) Even with the addition of aggregation inhibitors, particle-particle conformational aggregation occurs slowly (driven by van der Waals forces) in the lyophilized powder formulation, but storing the powder formulation at 2-8°C improves the kinetic stability of the system; b) Temperature-related factors cause parts of the mesh to become insoluble.

[0133] PRV311 is prepared by dispensing the liquid product into vials and freezing the vials at -80°C ambient temperature for at least 8 hours. After reconstitution with at least 1 mL of vehicle, the appropriate amount of PRV311 is withdrawn with a LuerLock syringe. PRV311 is injected directly into the tumor using an 18-30 gauge needle. Each PRV311 vial can contain 0.1-100 mg of the drug. Dosage limits depend on the aqueous solubility of the encapsulated immunotherapy or small molecule. The frequency of administration varies depending on the treatment site, indication, and at the discretion of the administrator.

[0134] According to some embodiments of the present invention, there is a water-soluble polymer matrix formed by a composition including chitosan, a hydration promoter, a microparticle adhesion inhibitor, and a microparticle aggregation inhibitor. According to yet another set of exemplary embodiments of the present invention, a method for manufacturing a therapeutic drug delivery system is provided. The method includes forming a first mixture with a plurality of microparticles. The microparticles include a therapeutic drug and have a coating including chitosan around the therapeutic drug. The method also includes forming a second mixture from the first mixture, chitosan, a hydration promoter, a microparticle adhesion inhibitor, and a microparticle aggregation inhibitor. The method further includes freezing the second mixture in a bath containing an aqueous alcohol solution at a temperature above the freezing temperature of the aqueous alcohol solution, 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 microparticles embedded within the matrix. Optionally, the bath further includes dry ice. Optionally, the alcohol in the aqueous alcohol solution is ethanol. As a further option, the aqueous alcohol solution is about 90% ethanol by weight to about 99% ethanol by weight. Optionally, the method further comprises applying a second layer precursor to the frozen layer precursor to form a solid comprising the first layer and the second layer. Optionally, the second layer comprises a therapeutic agent. Also, optionally, drying is performed under vacuum.

[0135] According to some embodiments of the present invention, the aqueous solution is frozen using a -80°C deep freezer. In other embodiments, the solution is frozen using liquid nitrogen. Surprising results were obtained when comparing the -80°C deep freezer freezing method with the liquid nitrogen freezing method. As shown in Figure 3, freezing in liquid nitrogen (-196°C) followed by freeze-drying results in a multi-layered, dense, fabric-like structure. In contrast, freezing at -80°C results in a more porous, uniform, single-layered polymer fiber, as shown in Figure 4. The structural differences between the solutions resulting from the two methods significantly impacted the control and timing of the release of the encapsulated therapeutic agent. In certain embodiments containing cisplatin, the inventors discovered through product development that a specific combination of polymers and excipients was required for the cisplatin mesh to function properly. During development, clumping and aggregation of nanoparticles within the mesh hindered the penetration of cisplatin-containing nanoparticles from the mesh into tissue. The addition of a combination of excipients and polymers was found to ensure complete release of the nanoparticles from the mesh. The reason why the inclusion of the combination resulted in ideal release and penetration was not immediately clear; however, microscopic analysis revealed that the inclusions reacted with the nanoparticles and mesh structure, forming "colonies" of microparticles within the pores of the mesh structure (see Figure 3). Unlike the usual heterogeneous aggregation or clumping that occurs in one large mass, the "colonies" were nearly uniform in size and remained small enough to break free from the mesh and penetrate the tissue. When the polymer excipient combination was included, the structure of the mesh was also altered. The inclusions resulted in a more crystalline structure with pores that retained the microparticles and allowed for easier release. The aforementioned functionality attributed to the polymer excipient combination has been reported to be the result of the polymer's ability to open cellular junctions within tissue; these were initially included in the mesh's construction during testing for this purpose. However, their effects beyond cellular changes, including both their effects on the mesh's structure and their prevention of microparticle "colonization" and aggregation, have not previously been reported or observed.The effect of polymers and excipients used in combination is synergistic, and their combined effect on release and permeation is much greater than the sum of their individual effects.

[0136] The release profiles of particles at various pH levels are shown in Figure 5. The powders were reconstituted in their respective media and placed in dialysis bags with stirring for 72 hours. Samples were taken and the release rates are shown. Microparticles at pH 6 (circles) were released faster due to faster degradation, while microparticles at pH 3 (triangles) were released at a slower rate due to greater particle stability. A free cisplatin solution (squares) was used as a control. It is clear from this figure that lower pH results in slower drug release from the microparticles.

[0137] According to some embodiments of the invention, the polymer excipient combination comprises chitosan, hypromellose, and propylene glycol. According to some embodiments of the invention, the hydration enhancer is selected from the group consisting of ethylene glycol, propylene glycol, beta-propylene glycol, glycerol, and combinations thereof.

[0138] According to some embodiments of the present invention, the microparticle adhesion inhibitor is a non-ionic polymer.

[0139] According to some embodiments of the invention, the non-ionic polymer is HPMC or poloxamer.

[0140] According to some embodiments of the present invention, the microparticle aggregation inhibitor is selected from the group consisting of monosaccharides, disaccharides, sugar alcohols, chlorinated monosaccharides, chlorinated disaccharides, and combinations thereof.

[0141] According to some embodiments of the present invention, the microparticles further comprise sodium tripolyphosphate.

[0142] In some embodiments of the invention, the system further comprises a free amount of a therapeutic agent embedded directly in the matrix and not otherwise coated with chitosan, wherein the free amount of the therapeutic agent comprises 20-80% of the total weight of the therapeutic agent in the system.

[0143] According to some embodiments of the present invention, the chitosan in the matrix and the chitosan in the microparticles is unmodified chitosan.

[0144] According to some embodiments of the present invention, the microparticles have an average diameter of about 0.5 μm to about 2 μm.

[0145] According to 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.

[0146] According to some embodiments of the present invention, the present invention comprises a microparticle for targeted delivery of a therapeutic agent, the microparticle comprising an unmodified therapeutic agent and unmodified chitosan.

[0147] According to some embodiments of the present invention, the microparticles are embedded within the matrix such that they are directly surrounded by and in contact with the matrix.

[0148] Some embodiments of the present invention provide a system for therapeutic agent delivery based on a polymer matrix and microparticles, which is improved by the addition of a hydration enhancer to the matrix. Examples of hydration enhancers include hygroscopic compounds such as glycols, e.g., ethylene glycol, propylene glycol, beta-propylene glycol, and glycerol. Exemplary concentration ranges for the amount of hydration enhancer include about 0.001 to about 10% by weight, about 0.01 to about 5% by weight, and about 0.1 to about 1% by weight.

[0149] Without wishing to be bound by any particular theory, it is believed that the hydration enhancer increases water absorption by the delivery system. This increased hydration allows for rapid release and penetration of the microparticles from the matrix. It is also believed that the hydration enhancer improves uniformity and durability by acting as an antifreeze agent during the manufacturing process of the delivery system. Again, without being bound by any particular theory, it is believed that the hydration enhancer acts as a "spacer" between the ice crystals and the matrix polymer molecules, ensuring a uniform freezing pattern. The resulting structure is more flexible, uniform, and durable than without the hydration enhancer.

[0150] In another set of exemplary embodiments, improved delivery devices are provided by the addition of adhesion inhibitors. Without wishing to be bound by any particular theory, it is believed that particle mobility is reduced when the matrix and particles are made of materials with polar or ionically charged moieties, such as chitosan. In the case of chitosan, it is believed that interactions between the acetyl and amine moieties of the polymer cause the particles to adhere to the matrix and inhibit their release.

[0151] It has been found that the inclusion of an adhesion inhibitor can reduce adhesion between the matrix and the particles. Without being bound by theory, it is believed that the adhesion inhibitor acts as a "spacer" between the chitosan of the particles and the chitosan of the matrix body, releasing the particles and improving the drug release profile. Representative examples of adhesion inhibitors include nonionic polymers such as hydroxypropyl methylcellulose (HPMC). Depending on the application, the molar mass of the nonionic polymer ranges from about 1 kDa to about 200,000 kDa, and its viscosity ranges from about 10 cps to about 100,000 cps. According to representative embodiments, the molar mass of the nonionic polymer ranges from about 10 kDa to about 30 kDa, and its viscosity ranges from about 10 cps to about 100 cps. Depending on the application, the amount of adhesion inhibitor can range from about 0.1% to about 99% by weight. According to some embodiments, the amount of adhesion inhibitor is from about 0.1% to about 25% by weight.

[0152] According to a further set of exemplary embodiments, a delivery device improved by the addition of an aggregation inhibitor is disclosed. The method for manufacturing the delivery device includes a freezing step, which can result in the formation of ice crystals within the matrix. These crystals can push microparticles together, creating particle aggregates where particles are damaged or destroyed. Again, without wishing to be bound by theory, it is believed that the aggregation inhibitor exerts its cryoprotective effect by forming a crystalline microstructure that prevents particle aggregation. Exemplary types of aggregation inhibitors include carbohydrates and carbohydrate derivatives, 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 to about 50% by weight. According to some embodiments, the amount of aggregation inhibitor is from about 1 to about 10% by weight.

[0153] According to another set of exemplary embodiments, improved pure chitosan microparticles are provided. Conventional chitosan particles are characterized by a high degree of deacetylation and are made from chitosan salts bearing charged moieties, such as chitosan chloride and chitosan glutamate. It has been discovered that better results are obtained when the particles are made from pure chitosan. This is a material that is not a salt, i.e., its amine groups are not protonated and is characterized by a degree of deacetylation of at least 70%. In particular, the particles are characterized by a larger diameter than conventional particles. According to some embodiments, the average diameter of the pure chitosan particles can range from about 200 to about 2000 nanometers. According to other embodiments, the average diameter is in the range of about 500 to about 2000 nanometers, and according to additional embodiments, the average diameter is in the range of 500 to 1000 nm. In a further refinement, improved chitosan microparticles are provided by the addition of sodium tripolyphosphate (STPP). Without wishing to be bound by any particular theory, it is believed that STPP functions as a cross-linking agent to form particles by acting as a negative counterion to the positively charged amine groups on chitosan. This electrostatic interaction forms ionic bonds that support the particle structure. Also, without wishing to be bound by any particular theory, it is believed that the presence of sodium as a positive counterion makes STPP a more effective cross-linking agent than other TPP salts.

[0154] It has been found that when the gel matrix contains a free amount of therapeutic agent, embedded directly in the matrix and not coated with chitosan in particles, the device is more therapeutically effective than a comparable matrix containing only the free amount of therapeutic agent or only the therapeutic agent coated with chitosan. In exemplary embodiments, the free amount of therapeutic agent constitutes between 20-80% of the total amount of therapeutic agent in the delivery system. [Example]

[0155] Injectable formulations that can be reconstituted with common vehicles such as water for injection USP, 0.12% saline USP, and 0.9% saline USP were tested as follows: Example 1: In vivo mouse studies

[0156] As shown in Figure 6, intratumoral injection of PRV311 in mice implanted with cancer cells substantially eliminated tumor growth (blue curve). Here, the PRV311 composition contained microparticles loaded with the antitumor cytokine IL-12. In comparative experiments, injection of control saline (black curve) or placebo particles (gray curve) into tumors showed minimal effect on tumor growth. Similarly, intravenous injection of IL-12 alone (green curve) showed no significant change in tumor growth rate compared to the control. Notably, intratumoral injection of IL-12 alone (red curve) merely slowed tumor growth, in contrast to the near elimination of tumor growth by IL-12-loaded PRV311 (blue curve). It is hypothesized that the polymer mesh of injected PRV311 locally retains the PRV311-containing microparticles, enhancing their efficacy. Example 2: In vitro tongue studies

[0157] Using a 23G subcutaneous Luer-lock needle, 500 µL of PRV311 was injected into the pig tongue. Due to the polymer mesh, the drug remained localized. As shown in Figure 8, the drug concentration remained nearly uniform across the tissue cross-section, forming a bell-curve-like shape (resembling an injection sphere). Example 3: Bovine brain

[0158] PRV311 (200 μL) was injected into the bovine brain with a 26-gauge needle, and the tissue was sectioned for microscopic imaging, as shown in Figure 10.

[0159] The approximate dimensions of the spread were 9 mm high x 5 mm long for the injected volume. The red in Figure 10 shows the Cy5 fluorophore dye attached to the chitosan polymer, demonstrating the movement of the microparticles and the mesh. The green shows the encapsulated fluorophore, which models the spread of the encapsulated drug. The mesh of the injectable keeps the fluorophore localized and concentrated. Example 4: Drug delivery to the sheep cornea

[0160] A study conducted in sheep showed that PRV311 can deliver drugs through the cornea into the vitreous fluid (Figures 15A, 15B, and 15C). PRV311 was injected into the vitreous approximately 5 mm below the iris. The red image in the upper right of Figure 16 shows how Cy5-labeled chitosan completely penetrates the retina, choroid, and sclera. The green image in the lower right of Figure 16 shows a similar distribution of FITC-labeled free drug during the same injection.

[0161] This supports PRV311 in providing an alternative to existing treatments for AMD. Data collected to date shows: Delivery of labeled particles to the cornea and vitreous fluid 4-month sustained release of bevacizumab Encapsulation improves the stability and absorption of bevacizumab Example 5: Pulmonary drug delivery in sheep

[0162] Studies conducted in sheep showed that PRV311 was able to deliver the drug to lung tissue, where it remained localized (Figure 17). The drug remained at high concentrations for more than 24 hours after injection before being frozen in preparation for sectioning. Example 6: Drug delivery to the liver in sheep

[0163] As shown in Figures 17 and 18, PRV311 is able to deliver the drug to liver tissue, where it remains localized. The drug remained at high concentrations for more than 24 hours after injection before being frozen in preparation for sectioning. As can be seen from the side view in Figure 19, tissue type influences the drug distribution pattern. Example 7: Drug delivery to the pancreas in sheep

[0164] PRV311 is able to deliver drugs to pancreatic tissue where they remain localized, as shown in Figure 20. The drug remained at high concentrations for more than 24 hours after injection before being frozen in preparation for sectioning.

[0165] The embodiments of the present invention described above are intended to be merely illustrative; many variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention, as defined in the appended claims.

Claims

1. 1. A system for providing therapeutic treatment to a target population of cells in a subject, comprising: a vial surrounded by a septum that is pierceable by the needle of a syringe used to administer the therapeutic treatment; a therapeutic composition disposed in a vial, the composition being provided for use in administering a therapeutic treatment and comprising an aqueous solution including a chitosan gel and a plurality of particles embedded in the gel, the gel having a viscosity suitable for administration by injection; Particles that include a therapeutic agent and have a coating around the therapeutic agent; and a coating comprising chitosan to provide controlled release of the drug from the particles; A system including:

2. The aqueous solution further comprises a compound selected from the group consisting of a hydration enhancer, a particle adhesion inhibitor, a particle aggregation inhibitor, and combinations thereof, (a) the hydration enhancer is selected from the group consisting of ethylene glycol, propylene glycol, beta-propylene glycol, glycerol, and combinations thereof; (b) the particle adhesion inhibitor is selected from the group consisting of HPMC, poloxamer, and combinations thereof; and (c) the particle aggregation inhibitor is selected from the group consisting of monosaccharides, disaccharides, sugar alcohols, chlorinated monosaccharides, chlorinated disaccharides, and combinations thereof.

3. The system of claim 1 or 2, wherein the aqueous solution further comprises sodium tripolyphosphate.

4. The system according to claim 1 or 2, wherein the particles are microparticles having an average diameter of 200 nm to 2000 nm.

5. The system according to claim 1 or 2, wherein the particles are microparticles having an average diameter of 500 nm to 2000 nm.

6. 6. The system of any one of claims 1 to 5, wherein the solution further comprises a free amount of therapeutic agent that is not coated with chitosan, wherein the free amount of therapeutic agent comprises between about 20% and about 80% by weight of the total amount of therapeutic agent in the aqueous solution.

7. The system according to any one of claims 1 to 6, wherein the therapeutic agent is an immunotherapeutic agent.

8. The system of claim 7 , wherein the immunotherapeutic agent is selected from the group consisting of an antibody, a cytokine, a small molecule immunotherapeutic agent, and combinations thereof.

9. The system according to any one of claims 1 to 6, wherein the therapeutic agent is a chemotherapeutic agent.

10. The system of claim 1 , wherein the particles are in direct physical contact with the chitosan gel.

11. 1. A method for treating a target population of cells in a subject, comprising: Obtaining the system of claim 1; Loading the aqueous solution into a syringe; injecting said aqueous solution into a target population of cells using a syringe.

12. The method of claim 11 , wherein the target population of cells comprises a tumor.

13. The method of claim 11 , wherein the target population of cells is tissue within an organ.

14. 14. The method of claim 13, wherein the organ is selected from the group consisting of the eye, lung, pancreas, liver, kidney, brain, heart, thyroid, and pituitary gland.

15. 1. A system for providing therapeutic treatment to a target population of cells in a subject, comprising: a vial surrounded by a septum that is pierceable by the needle of a syringe used to administer the therapeutic treatment; a therapeutic composition disposed in a vial, the composition being provided for use in administering a therapeutic treatment and comprising a lyophilized precursor formulated to dissolve upon mixing with water to provide an aqueous solution comprising a chitosan gel and a plurality of particles embedded in the gel, the gel having a viscosity suitable for administration by injection; Particles that include a therapeutic agent and have a coating around the therapeutic agent; and a coating comprising chitosan to provide controlled release of the drug from the particles; A system including:

16. 1. A freeze-drying method for providing a system for providing a therapeutic treatment to a target population of cells in a subject, comprising: forming an aqueous solution comprising a chitosan gel and a plurality of particles, the particles comprising a therapeutic agent and having a coating around the therapeutic agent, the coating comprising chitosan to provide a controlled release of the agent from the particles; freezing the initial aqueous solution in a bath containing the aqueous alcohol solution at a temperature higher than the freezing temperature of the aqueous alcohol solution, at most −80° C., to form a frozen layer precursor; drying the frozen layer precursor to form an anhydrous powder with embedded particles; containing an anhydrous powder in a vial surrounded by a septum pierceable by the needle of a syringe used to administer the therapeutic treatment; and The freeze-drying process involves adding water to a container to dissolve the anhydrous powder.

17. the anhydrous powder further comprises a compound selected from the group consisting of a hydration enhancer, a particle adhesion inhibitor, and a particle aggregation inhibitor, and combinations thereof; the hydration enhancer is selected from the group consisting of ethylene glycol, propylene glycol, beta-propylene glycol, glycerol, and combinations thereof; the particle adhesion inhibitor is selected from the group consisting of hydroxypropyl methylcellulose, poloxamer, and combinations thereof; and 17. The freeze-drying method of claim 16, wherein the particle aggregation inhibitor is selected from the group consisting of monosaccharides, disaccharides, sugar alcohols, chlorinated monosaccharides, chlorinated disaccharides, and combinations thereof.

18. 17. The freeze-drying method of claim 16, wherein the anhydrous powder further comprises sodium tripolyphosphate.

19. The freeze-drying method according to claim 16, wherein the particles are microparticles having an average diameter of 200 nm to 2000 nm.

20. The freeze-drying method according to claim 16, wherein the particles are microparticles having an average diameter of 500 nm to 2000 nm.

21. 10. The system of claim 1, configured to provide therapeutic treatment of an ocular condition by intravitreal injection.

22. 22. The system of claim 21, wherein the therapeutic agent is selected from the group consisting of an antibody, a cytokine, a small molecule immunotherapeutic, a chemotherapeutic, an aptamer, and combinations thereof.

23. 22. The system of claim 21, wherein the eye condition is age-related macular degeneration.

24. 24. The system of claim 23, wherein the therapeutic agent is bevacizumab.

Images