Microsphere-drug combinations

Abstract

The present invention relates to a method for attaching an insoluble active pharmaceutical ingredient (API) to microspheres produced by thermally induced phase separation (TIPS), comprising: i) mixing the microspheres with an aqueous solution to form a first composition; ii) dissolving the insoluble API in the first solvent, followed by adding the insoluble API dissolved in the first solvent to the first composition to form a second composition; and iii) mixing the second composition. The present invention further relates to microspheres produced by thermally induced phase separation having an insoluble API attached to their surface, and compositions comprising said microspheres for use in therapy.

Description

[Technical field]

[0001] The present invention relates to a method for attaching insoluble active pharmaceutical ingredients (APIs) to thermally induced phase separation produced microspheres, as well as to thermally induced phase separation (TIPS) produced microspheres having insoluble APIs bound to their surface and uses thereof. [Background technology]

[0002] Thermally induced phase separation (TIPS) microspheres have been previously described in WO2008 / 155558. The resulting microsphere structures offer improved characteristics, such as mechanical strength and the ability to select pore size, as well as whether the microspheres are covered by the skin. TIPS microspheres provide a novel degradable scaffold structure that supports tissue growth and can deliver bioactive substances that promote tissue neogenesis. The microspheres utilized in the present invention can be manufactured by application of TIPS as disclosed in WO2008 / 155558, or by other suitable methods. The contents of WO2008 / 155558 are incorporated herein by reference, particularly with respect to the method used to manufacture TIPS microspheres. TIPS allows for the rapid formation of uniformly sized porous microspheres. TIPS microspheres are polymeric and biodegradable, and can be loaded with therapeutic agents for drug delivery applications. These microspheres can be easily delivered to the required location, they can conform to the irregular shapes of tissue cavities, they have predictable degradation times, they can deliver a wide range of drugs, and they are cost-effective to manufacture.

[0003] Cancer is one of the leading causes of death worldwide. Tumor resection is a common form of treatment, but positive surgical margins (defined as the histological presence of cancer cells in the ink border of the resection specimen) after resection are associated with a higher risk of biochemical recurrence due to residual tumor left behind in the patient. Factors for considering a more aggressive approach to surgical resection, which may more reliably obtain margins away from the tumor, include comorbidities, anatomical factors, and surgical experience. For prostate cancer, one of the most common cancers in men, radical prostatectomy is the standard treatment option for cancer confined to the prostate, but aggressive resection is associated with a higher risk of urinary incontinence and erectile dysfunction due to collateral tissue damage (vascular damage to the Santorini reticulum, epigastric or iliac vessels; nerve injury). Despite curative treatment for localized disease, approximately 50% of patients with high-risk prostate cancer develop biochemical recurrence after surgery within 5 years, which is associated with a poor prognosis (Briganti A, Karnes RJ, Gandaglia G, Spahn M, Gontero P, Tosco L, et al. Natural history of surgically treated high-risk prostate cancer. Urol Oncol. 2015 Apr;33(4):163.e7-13). Many of these patients require adjuvant or salvage (radiation) therapy or systemic hormone therapy, which carries a higher risk of functional impact on erectile function or urinary control. After failure of these therapies, men require third-line hormone or chemotherapy agents, which carry a higher risk of toxicity. Because it is not possible to completely remove the tumor or all residual cancer cells with 100% certainty, new multimodal therapies that safely allow targeted delivery of anticancer drugs during surgery immediately after tumor removal would be beneficial in improving outcomes and reducing the risk of recurrence and the requirement for further therapy.

[0004] Chemotherapy is a common cancer treatment, however clinical evidence of improved survival with systemic delivery of chemotherapy drugs is often associated with numerous drug-associated toxicities and non-specific distribution required to achieve therapeutic efficacy, with only a small fraction of the drug reaching the tumor.

[0005] Docetaxel (DTX) is one such type of chemotherapy drug. It is a taxane-based chemotherapy that binds to intracellular β-tubulin, inhibiting microtubule depolymerization, leading to mitotic arrest and activation of signaling pathways associated with apoptosis (Herbst RS, Khuri FR. Mode of action of docetaxel - a basis for combination with novel anticancer agents. Cancer Treat Rev. 2003 Oct;29(5):407-415).

[0006] Many tumor sites, such as the prostate, are anatomically accessible and therefore suitable targets for direct delivery, especially with the advent of laparoscopic and robotic techniques. However, modeling of direct docetaxel delivery to the prostate showed that it was unlikely to be effective because the directly delivered infusion would easily enter the urethra due to areas of tissue with high fluid conductivity (Raghavan R, Brady ML, Sampson JH. Delivering therapy to target: improving the odds for successful drug development. Ther Deliv. 2016 Jul;7(7):457-481).

[0007] In order to achieve localized targeted delivery and to mitigate the undesirable side effects associated with systemic delivery of chemotherapy drugs, various polymer-based drug delivery systems have been investigated in an attempt to achieve high therapeutic concentrations of chemotherapy at the site of malignancy. These include polymer nanoparticles, liposomes, dendrimers, and nanotubes. However, many nanoscale materials devised so far for anticancer drug delivery rely on intravenous administration, which requires targeting of drugs to tumor tissues at therapeutic concentrations. Moreover, their introduction into the systemic circulation can result in removal and sequestration by the reticuloendothelial system, and off-target tissue accumulation, both of which are major issues that need to be addressed when materials are developed within the nanoscale range.

[0008] The present invention can provide localized, controlled, and sustained drug release when implanted in or adjacent to a tumor, thereby increasing the potential for clinical efficacy and reducing collateral off-target tissue damage. A novel approach for adjuvant therapy allows for administration of a drug delivery depot system at the time of tumor resection. The main attributes of this system, intended to destroy residual cancer cells remaining after tumor resection, include: (i) minimally invasive delivery through suitable gauge needles or delivery ports; (ii) conforming to the shape of the tissue cavity created after tumor resection to maximize the surface area of ​​the drug in close proximity to the residual tumor cells; (iii) sustained delivery of therapeutic doses; (iv) retention of the product at the target site; and (v) ideal mobility for clinical use within a short time frame.

[0009] Therefore, the present invention relates to a novel controlled release drug delivery depot system that meets these target attributes by combining an API with existing, clinically approved, biodegradable, porous microspheres made by thermally induced phase separation (TIPS) that can be delivered and conform to the shape of the tissue cavity created immediately after tumor resection. The microspheres of the present invention further act as a scaffold to facilitate the tissue repair process after surgery. The method of the present invention further provides a novel, simple and stable loading regime that achieves a consistent amount of API loaded onto the TIPS microspheres that is compatible with minimally invasive delivery at the time of surgery, and secondly, the in vitro efficacy of the API-TIPS microsphere combination product. Summary of the Invention

[0010] According to a first aspect of the present invention, there is provided a method for attaching an insoluble active pharmaceutical ingredient (API) to microspheres produced by thermally induced phase separation (TIPS), comprising the steps of: i) mixing the microspheres with an aqueous solution to form a first composition; ii) dissolving the insoluble API in a first solvent, and subsequently adding the insoluble API dissolved in the first solvent to the first composition to form a second composition; and iii) mixing a second composition; The present invention provides a method comprising:

[0011] This simple and stable method was used to load insoluble APIs into TIPS microspheres. This approach offers advantages over other microsphere drug-device fabrication techniques, such as the solvent-emulsion evaporation process used to manufacture prior art PACLIMER® microspheres, including avoiding exposure of the API to solvents during the fabrication process, and high drug loading efficiency could be achieved. Moreover, the approach allows for preparation of the drug-device combination immediately prior to use, thus allowing the PLGA TIPS microspheres to be stored in a dry format, thereby extending their shelf life by avoiding degradation.

[0012] According to a second aspect of the present invention, there is provided a microsphere produced by a thermally induced phase separation process with an insoluble API bound to its surface. According to a third aspect of the invention there is provided a composition comprising the microspheres of the invention for use in therapy. According to a fourth aspect of the invention there is provided a composition comprising the microspheres of the invention for use in the treatment of cancer. According to a fifth aspect of the present invention there is provided the use of a composition comprising the microspheres of the present invention in the manufacture of a medicament for the treatment of cancer.

[0013] According to a sixth aspect of the present invention, there is provided a method of treating cancer comprising administering to a subject in need of such treatment a composition comprising the microspheres of the present invention. According to a seventh aspect of the present invention, there is provided a kit for treating cancer, comprising: i) TIPS microspheres provided in a sealed container; ii) an insoluble API dissolved in a first solvent; and iii) a carrier vehicle; A kit comprising:

[0014] The TIPS microspheres of the present invention provide localized delivery and sustained release formulation of API at the site of need and allow enhanced local concentration of API at the tumor site, thereby reducing the risk of disease recurrence caused by incomplete tumor resection by destroying residual tumor cells. Moreover, such a localized delivery system reduces the risk of loss of API into the circulation and off-target toxicities associated with chemotherapeutic agents delivered using conventional systemic delivery routes, such as neutropenia, leukopenia, neurological toxic effects, diarrhea, alopecia, asthenia, and nausea. [Brief description of the drawings]

[0015] [Figure 1]Figure 1 illustrates an example of the loading process of DTX to TIPS microspheres. (i) 5 mg of PLGA TIPS microspheres in a 20 ml clear Type 1B borosilicate glass vial was sealed with a butyl injection stopper. (ii) 4.5 ml of ultrapure water was added to the vial. (iii) The vial was vortexed for 10 seconds. (iv) 0.5 ml of docetaxel 0.1% (w / v) in ethanol was added using a 1 ml syringe with a 25 G needle through the rubber stopper while inverting the vial. (v) The vial was then vortexed for 10 seconds and placed on a roller mixer (IKA® Roller 6 Digital; 60 rpm) at room temperature for a scheduled time. [Figure 2-1] Figure 2 shows the immobilization of taxanes onto PLGA TIPS microspheres. (a) The amount of DTX loaded onto the microspheres was indirectly quantified by calculating the amount of DTX remaining in solution (measured by UV absorption) at various time points during the loading phase. (b) Representative scanning electron microscopy (SEM) images of unloaded control TIPS microspheres and (c) DTX-TIPS microspheres loaded with 500 μg of DTX. (d) SEM image demonstrating the time-dependent adsorption of DTX to the surface of TIPS microspheres. (e) Nitrogen (N1s) X-ray electron spectroscopy high energy resolution spectra obtained on the surface of control TIPS microspheres or DTX-TIPS microspheres. [Figure 2-2] Figure 2 shows immobilization of taxanes onto PLGA TIPS microspheres. (f) DTX loading rates for dry TIPS PLGA microspheres, "pre-wet" TIPS PLGA microspheres and PLGA polymer solid microparticles. [Figure 2-3]FIG. 2 shows immobilization of taxanes onto PLGA TIPS microspheres. (g) TIPS microspheres were loaded with docetaxel, paclitaxel and cabazitaxel via antisolvent precipitation. Scanning electron microscopy of the microspheres revealed the presence of crystalline material on the surface of the microspheres incubated in paclitaxel or cabazitaxel solutions compared to unloaded control TIPS microspheres. (h) Docetaxel showed a faster loading rate onto the microspheres compared to cabazitaxel. Approximately 80% of docetaxel was loaded from solution onto the microspheres within 60 minutes after mixing began. At the same time point, approximately 60% of cabazitaxel was loaded from solution onto the microspheres. No further increase in docetaxel loading was detected during the entire loading phase over 60 minutes. [Diagram 3] Figure 3 shows that excipients present in paclitaxel for injectable formulations (e.g., Taxol®) interfere with the antisolvent precipitation process and prevent loading of the drug onto the surface of the microspheres. Scanning electron microscopy of the microspheres revealed the presence of less crystalline material on the surface of the microspheres incubated in injectable formulations of docetaxel and paclitaxel compared to TIPS microspheres incubated with pure formulations of docetaxel and paclitaxel without added excipients. [Figure 4] FIG. 4 shows that (a) injectable docetaxel (e.g., Taxotere®) is formulated in a vehicle consisting of a 1:1 mixture of Tween® 80 and ethanol, which is further diluted with saline prior to administration. (b) injectable paclitaxel (e.g., Taxol®) is formulated in a vehicle consisting of a 1:1 mixture of Cremophor EL and ethanol, which is diluted 5-20 times with saline prior to administration. Excipients present in the injectable formulation interfere with the antisolvent precipitation process and prevent loading of the drug onto the surface of the microspheres. [Diagram 5] FIG. 5 shows that scanning electron microscopy of the microspheres revealed a similar distribution of crystalline material on the surface of the "wet" and "dry" microspheres. [Figure 6] FIG. 6 shows that the cumulative release of docetaxel showed similar release rate trends for the "wet" and "dry" docetaxel-loaded TIPS microspheres over the first 72 hours. [Figure 7] FIG. 7 shows that the diameter of the spheroids calculated from the acquired images showed a significant reduction in size compared to both the starting size of the spheroids and the control spheroids measured at the same time points, producing a similar effect with perfusate recovered from "wet" and "dry" docetaxel-loaded TIPS microspheres. [Figure 8] FIG. 8 shows SEM images confirming the presence of comparable quantities of docetaxel on the surface of TIPS microspheres after one month of storage. [Figure 9] FIG. 9 shows (a) the cumulative release of DTX from DTX-TIPS microspheres measured using UV absorption at 229 nm. (b) Colony formation assay performed by incubating PC3 cells for 48 h with perfusate collected every other day from DTX-TIPS microspheres over a 12-day period. Cells exposed to perfusate collected from various time points were re-plated in Petri dishes and stained colonies were counted after 2 weeks of incubation. (c) Quantification of cells showing morphological changes associated with apoptosis after incubation of PC3 cells for 48 h with perfusate from DTX-TIPS microspheres collected on days 1, 5, and 10. Cell morphology was evaluated after 48 h of incubation in fresh complete medium and after 5 and 10 days of incubation. [Figure 10]FIG. 10 shows (a) 3D spheroid cultures of PC3 cells incubated in perfusate collected from DTX-TIPS or no-additive control TIPS microspheres for 12 days. The medium was replaced with the collected perfusate at the corresponding time points. The diameter and volume of the spheroids were calculated from images acquired throughout the incubation period. Spheroids incubated with perfusate from DTX-TIPS became smaller over time. (b) PC3 cells cultured in 2D in tissue culture wells were treated in the same manner and showed morphological characteristics of cytotoxicity. (c) At day 12, cell viability of spheroids incubated with perfusate was analyzed using Live / Dead® staining (calcein AM-live cells and ethidium homodimer-1 (EthD-1) dead cells), scale bar 200 μm. (d) PC3 cells in spheroids incubated with perfusate from DTX-TIPS microspheres and transferred to tissue culture plates on day 12 migrated out of the spheroids and appeared non-viable, whereas cells from spheroids incubated with control microspheres survived and migrated out of the spheroids, Scale bar 200 μm. Data represent mean ± standard deviation from n=20. ***p<0.001 between control and DTX-treated samples. [Figure 11] FIG. 11 shows that DTX-TIPS inhibits tumor growth in vivo without systemic toxicity. (a) Mice receiving IV DTX showed progressive and significant weight loss indicative of systemic toxicity. (b) Tumor volume steadily increased over the study time in mice receiving control TIPS microspheres or IV saline. (c) In mice receiving IV DTX on day 14, there was no significant difference in tumor size increase between mice receiving DTX IV and DTX-TIPS, indicating that DTX-TIPS is equally effective in preventing tumor growth. [Figure 12]Figure 12 shows the measurement of DTX in plasma collected from non-tumor-bearing BALB / cAnNCrl mice. Low levels of DTX were detectable in plasma only 1 and 24 hours after delivery of DTX-TIPS microspheres. Higher levels of DTX at 1 and 24 hours were detected after IV DTX administration, with detectable levels of DTX maintained at 48 and 72 hours, as well as on days 10 (240 hours) and 15 (360 hours). [Figure 13] Figure 13 shows histology of excised PC3 xenograft tumor (T). DTX-TIPS microspheres (*) implanted around the tumor remained at the delivery site at day 35. The microspheres are surrounded by loose connective tissue. Higher magnification (inset) reveals that the microspheres remain intact after implantation. [Figure 14] FIG. 14 shows that (a) the greatest increase in tumor volume was observed in the groups receiving IV saline or control TIPS microspheres. No increase in tumor volume was observed in the groups receiving IV DTX. The increase in tumor volume was attenuated in the group receiving DTX-TIPS. (b) Macroscopic images of representative resected PC3 prostate cancer xenograft tumors illustrating the differences in tumor volume associated with the different treatment groups. (c) Body weight change in NSG™ immunodeficient mice after treatment. Progressive weight loss was observed in mice receiving IV DTX. No significant weight change was observed in mice receiving DTX-TIPS. (****p<0.0001). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description of the Invention According to a first aspect of the present invention, there is provided a method for attaching an insoluble active pharmaceutical ingredient (API) to microspheres produced by thermally induced phase separation (TIPS), comprising the steps of: i) mixing the microspheres with an aqueous solution to form a first composition; ii) dissolving the insoluble API in a first solvent, and subsequently adding the insoluble API dissolved in the first solvent to the first composition to form a second composition; and iii) mixing a second composition; The present invention provides a method comprising:

[0017] The method allows for the loading of insoluble APIs into TIPS microspheres. As outlined above, the approach avoids exposure of the API to solvents during the fabrication process, and therefore achieves high drug loading efficiency. The term "added" as used herein includes any form of addition, but preferably refers to passive addition. Preferably, the insoluble API is deposited on the surface of the microsphere. The insoluble API may be attached to the microsphere by physical adsorption. Preferably, the insoluble API is deposited on the surface of the microsphere in crystalline form. By "insoluble" it is meant that the API is not soluble in an aqueous solution.

[0018] The term API, as used herein, means a substance that can be used in an ultimate pharmaceutical product and is intended to provide pharmacological activity or otherwise act directly in the diagnosis, cure, mitigation, treatment or prevention of disease, or to restore, correct or modify human physiological function.

[0019] The term "microsphere" refers to a preparation of homogeneous, substantially spherical particles. The term is well known in the art. Microspheres can contain numerous radial pores. This means that the pores extend from the central portion of the microsphere to the surface, preferably substantially parallel to the radius of the microsphere. The pores are preferably tubular and interconnected. The radial pores provide the microsphere with a level of mechanical strength.

[0020] The structure is produced by a thermally induced phase separation method. In particular, the structure can be produced by any of the methods disclosed in WO2008 / 155558, the disclosure of which is incorporated by reference in its entirety.

[0021] The method includes mixing the microspheres with an aqueous solution to form a first composition. Preferably, the aqueous solution includes water, saline, Ringer's solution, or a mixture thereof. When water is used, the water may further include sodium chloride and / or dextrose. Mixing may be performed by any suitable means, such as, for example, shaking, rotating, rolling, or vortexing. Preferably, mixing is performed by vortexing for a suitable period of time, such as, for example, 2, 5, 10, 15, or several seconds. Preferably, vortexing is performed for about 10 seconds.

[0022] In one embodiment, the microspheres are mixed with an aqueous solution to form a first composition in a container. The container can be any suitable container known to those skilled in the art, such as, for example, a beaker, a flask, a bottle, a jar, a test tube, a cylinder, a centrifuge tube, a microtube, or a vial. Preferably, the container is a vial. The container can be made of any suitable material in which the API dose does not adsorb, such as, for example, plastic or glass. Preferably, the container is made of glass, such as, for example, borosilicate glass. Preferably, the container is a glass vial, a lyophilization vial, or a siliconized vial, more preferably a borosilicate glass vial.

[0023] The volume of the aqueous solution may be any volume suitable for the container utilized. For example, when the container is a 20 ml vial, the amount of water may be 0.5 to 5 ml, preferably 1.5 to 4.5 ml, and more preferably 3 to 4.5 ml. Preferably, the container is sealed during mixing by any suitable means known to one of skill in the art, including lids, caps, bungs, stoppers, or parafilm. Preferably, the container is sealed with a stopper. An example of a preferred stopper is a butyl injection stopper.

[0024] The insoluble API is dissolved in a first solvent. The first solvent can be selected from the Q3C-Tables and List Guidance for Industry (http: / / academy.gmp-compliance.org / guidemgr / files / UCM073395.PDF) known to those skilled in the art. Preferably, the solvent is selected from Class 3 solvents.

[0025] The first solvent may be selected from acetic acid, acetone, nitromethane, dioxane, tetrahydrofuran, pyridine, methyl ethyl ketone, DMSO, methyl acetate, halogenated hydrocarbons, glycerin, toluene, formamide, propylene glycol, polyethylene glycol, lower alcohols, and mixtures thereof. Halogenated hydrocarbons include, but are not limited to, dichloromethane, chloroform, tetrachloroethane, and trichloroethane. Lower alcohols include, but are not limited to, isopropyl alcohol, methanol, and ethanol.

[0026] Preferably, the solvent is a lower alcohol, and most preferably, the solvent is ethanol. Preferably, the ethanol is 45% to 100% ethanol, more preferably 95% or 100% ethanol. Preferably, the first solvent is added to the insoluble API in a volume to result in a final concentration in the second composition of 1-30% v / v, preferably 10-30% v / v, more preferably 10-20% v / v, and most preferably 10% v / v. At concentrations above 30% v / v in the second composition, the microspheres may aggregate together.

[0027] Following dissolution of the insoluble API in the first solvent, the insoluble API dissolved in the first solvent is added to the first composition to form a second composition. Preferably, during this step, the container containing the microspheres and the aqueous solution is inverted to mix, resulting in the microspheres moving away from the rubber stopper sealing the opening of the vial. Diluting the first solvent with the aqueous solution present in the first composition prevents the microspheres from coming into direct contact with the first solvent, which may result in agglomeration of the microspheres.

[0028] Preferably, the addition of the insoluble API dissolved in the first solvent to the first composition is via a needle. Preferably, a syringe with an attached needle is used to pierce the rubber stopper sealing that encloses the vial while the vial is inverted.

[0029] The second composition is then mixed. Mixing can be performed by any suitable means, such as, for example, shaking, rotating, rolling or vortexing. Preferably, mixing is performed by vortexing for a suitable time, such as, for example, 2, 5, 10, 15 or several seconds. Preferably, vortexing is performed for about 10 seconds. Preferably, vortexing is then followed by a period of rotation or rolling, such as, for example, 5 minutes to 24 hours, preferably 30 minutes to 60 minutes.

[0030] The thermally induced phase separation method preferably comprises the following: i) dissolving the polymer in a second solvent to form a solution; ii) quenching the droplets of the solution with a quenching fluid; and iii) freeze-drying the resulting spheres; preferably wherein the solution is introduced into the quench using a syringe, vibrating needle or atomizer, and more preferably further comprising the step of sonicating the solution.

[0031] Any hydrophobic polymer can be used, but it is preferred that the polymer is medicamentously acceptable and completely soluble in the solvent. The polymer can be degradable or non-degradable. It can be synthetic or non-synthetic. A combination of polymers can be used, for example a synthetic polymer is used in combination with a non-synthetic polymer. Examples of polymers include poly(lactide-co-glycolide) (PLGA), poly(α-hydroxy ester), polyanhydride, polyorthoester, polyphosphazine, polypropylene fumarate, poly(propylene-fumarate-co-ethylene glycol), polyethylene oxide, polyhydroxybutyrate (PHB) and polyhydroxyvalerate (PHV). Co-polymers of two or more polymers can also be used, particularly PHB and PHV. Others include poly(α-hydroxy ester)-co-PEG copolymers, or copolymers containing PEGylated drugs. Natural polymers that can be used include fibrin. Preferably the polymer is not chitosan.

[0032] The type of polymer (e.g., permanent or degradable, natural or synthetic), porosity, mechanical strength and size may be selected depending on the application of the microsphere or the selected delivery site. For example, degradable materials may be preferred when tissue from the delivery site will replace the temporary scaffolding function of the structure. Most preferably, the polymer is poly(lactide-co-glycolide) (PLGA).

[0033] The second solvent can be selected to have a relatively high freezing temperature, higher than the temperature of the quenching fluid. Examples of solvents include dimethyl carbonate, chloroform, acetone, dimethyl chloride, tetrahydrofuran, and supercritical carbon dioxide. Preferably, the second solvent is dimethyl carbonate.

[0034] Preferably, the method further comprises the step of sonicating the solution. The quenching fluid used to form the microspheres can be a liquid or a gas. Examples of quenching fluids include liquid nitrogen, liquid oxygen, liquid CO2, etc. 2 , Freon, water, ethanol, methanol. Preferably, the quenching fluid is liquid nitrogen.

[0035] The solution can be introduced into the quenching fluid using any suitable method, for example, droplets can be produced using a syringe or vibrating needle, or the solution can be sprayed through an atomizer, for example, using an aerosolized or pumped system, or drawn into the quenching fluid using electrostatic forces or a coaxial air stream.

[0036] Preferably, the insoluble API is a chemotherapeutic agent. By "chemotherapeutic agent" it is meant that the agent is selectively destructive to malignant cells and tissues. Non-exhaustive examples of chemotherapeutic agents include alkylating agents such as chlorambucil, melphalan, dacarbazine, and temozolomide; anthracyclines such as idarubicin and valrubicin; histone deacetylase inhibitors such as vorinostat and romidepsin; topoisomerase inhibitors such as etoposide, teniposide, and tafluposide; kinase inhibitors such as bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, and vismodegib; and cyclosporine inhibitors such as cyclosporine. For example, nucleotide analogues and precursor analogues such as azacitidine, azathioprine, fluorouracil, mercaptopurine, methotrexate, and thioguanine; platinum-based agents such as carboplatin, cisplatin, and oxaliplatin; retinoids such as tretinoin, alitretinoin, and bexarotene; vinca alkaloids and derivatives such as vinblastine and vindesine, and taxanes such as cabazitaxel, paclitaxel, and docetaxel.

[0037] Preferably, the chemotherapeutic agent is a taxane, more preferably cabazitaxel, paclitaxel or docetaxel, even more preferably docetaxel.

[0038] Any insoluble API not bound to the microspheres is removed from the second composition by washing with an aqueous solution. Preferably, the aqueous solution comprises water, saline, Ringer's solution or a mixture thereof. When water is used, it may further comprise sodium chloride and / or dextrose. Non-limiting examples of washing procedures may include: (i) removing the API dissolved in the first solvent; (ii) adding the same volume of aqueous solution; and (iii) washing by any suitable means, such as shaking, rotating, rolling or vortexing. Preferably, washing is performed by vortexing for a suitable time, such as for example 10 seconds to 5 minutes. Preferably, vortexing is performed for about 2 minutes. After this step, the aqueous solution may be removed. Preferably, removal is performed by aspiration.

[0039] According to a second aspect of the present invention, there is provided microspheres with an insoluble API bound to their surface, produced by a thermally induced phase separation process. Preferably, the insoluble API is bound to the microspheres by the methods described above. Preferably, the thermally induced phase separation method is preferably one of the following: i) dissolving the polymer in a second solvent to form a solution; ii) quenching the droplets of the solution with a quenching fluid; and iii) freeze-drying the resulting spheres; preferably wherein the solution is introduced into the quench using a syringe, vibrating needle or atomizer, and more preferably further comprising the step of sonicating the solution.

[0040] The solvent in this embodiment of the invention is the second solvent described in the method above. Preferably, the polymer is PLGA, the solvent is dimethyl carbonate, and / or the quenching fluid is liquid nitrogen.

[0041] The term "microsphere" has been defined above and as used herein may encompass approximately spherical particles that are of a suitable size for attachment of an insoluble API. Preferably, the microspheres are about 10-900 μm in diameter as characterized by electron microscopy, such as scanning electron microscopy. Preferably, the microspheres may be about 50-450 μm in diameter, more preferably about 100-400 μm in diameter, and even more preferably about 250-350 μm in diameter.

[0042] The desired size range of 250-350 μm is such that the microspheres can be easily delivered to a patient via a minimally invasive approach through a suitable gauge needle or delivery port while providing wide enough gaps between the packed microspheres to increase surface area for drug release and allowing tissue infiltration between the tightly packed microspheres.

[0043] The pore size of the microsphere may also be selected according to the intended use and the required mechanical strength, and may also be selected according to the diameter of the microsphere. Furthermore, the pores are preferably of uniform size, i.e., the pores are preferably of substantially the same diameter, i.e., the diameters of the pores preferably differ by no more than 10%. Porous microspheres have good mechanical strength due to the nature of the pores.

[0044] According to a third aspect, the present invention relates to a composition comprising the microspheres of the present invention for use in therapy. According to a fourth aspect of the invention there is provided a composition comprising the microspheres of the invention for use in the treatment of cancer.

[0045] When cancer is "treated", this means that one or more clinical signs of cancer are improved. It does not mean that the cancer symptoms are completely corrected, such that they are no longer present in the patient, although in some ways they may be. "Treatment" results in one or more cancer symptoms being less severe than before treatment. For example, a tumor may be reduced in size or completely eradicated.

[0046] The cancer may be any type of cancer, preferably carcinoma, sarcoma or lymphoma.Preferably, the cancer is selected from prostate cancer, breast cancer, bladder cancer, colorectal cancer, thyroid cancer, oral cancer, lung cancer, kidney cancer, uterine cancer or ovarian cancer.More preferably, the cancer is prostate cancer.In a particularly preferred embodiment, the patient suffering from prostate cancer has undergone radical prostatectomy.

[0047] The composition comprising the microspheres of the present invention is administered directly into the tissue cavity created after tumor resection or, for example, radical prostatectomy. In addition to delivering chemotherapy directly to the required site, TIPS microspheres can act as a conformable tissue scaffold after surgery. The microspheres can be easily delivered through a suitable gauge needle or delivery port by a minimally invasive approach, while providing sufficiently wide gaps between closely packed microspheres to increase surface area for drug release and allowing tissue infiltration between closely packed microspheres.

[0048] According to a fifth aspect of the present invention there is provided the use of a composition comprising the microspheres of the present invention in the manufacture of a medicament for the treatment of cancer. According to a sixth aspect of the present invention, there is provided a method of treating cancer comprising administering to a subject in need of such treatment a composition comprising the microspheres of the present invention.

[0049] According to a seventh aspect of the present invention, there is provided a kit for treating cancer, comprising: i) TIPS microspheres provided in a sealed container; ii) an insoluble API dissolved in a first solvent; and iii) a carrier vehicle; A kit comprising:

[0050] The container can be any suitable container known to those skilled in the art, such as, for example, a beaker, a flask, a bottle, a jar, a test tube, a cylinder, a centrifuge tube, a microtube or a vial. Preferably, the container is a vial. The container can be made of any suitable material in which the API dose does not adsorb, such as, for example, plastic or glass. Preferably, the container is made of glass, such as, for example, borosilicate glass. Preferably, the container is a glass vial, a lyophilization vial or a siliconized vial, more preferably a borosilicate glass vial.

[0051] Preferably, the container is sealed under vacuum. Dry TIPS microspheres under such conditions have a shelf life of more than six years. The insoluble API and the first solvent are as described above.

[0052] The carrier vehicle may comprise one or more suitable excipients. Excipients acceptable for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985). The vehicle may comprise or be added to any suitable binder, lubricant, suspending agent, coating agent or solubilizing agent that is an excipient. In one embodiment, the carrier vehicle may comprise a hydrogel.

[0053] The kit may further comprise an aqueous solution suitable for delivery in injectable form. The aqueous solution may comprise water, saline, Ringer's solution, or a mixture thereof. When the aqueous solution is water, it may further comprise sodium chloride and / or dextrose. The kit may further comprise a syringe, needle, and / or cannula of a suitable gauge, eg, 16G-30G, for delivery of the microspheres in the carrier vehicle to a patient.

[0054] Those skilled in the art will appreciate that all aspects of the present invention are equally applicable to all of the other aspects of the present invention, whether they relate to, for example, attachment methods, microspheres, uses, or treatment methods. In particular, for example, attachment method aspects may have been described in more detail than other aspects of the present invention, such as, for example, the use of microspheres. However, those skilled in the art will appreciate that when more detailed information is given to a particular aspect of the present invention, this information is generally equally applicable to the other aspects of the present invention. The invention will now be described in more detail, by way of example only, with reference to the drawings in which: EXAMPLES

[0055] Example 1 Materials and Methods Fabrication of TIPS microspheres TIPS microspheres composed of poly(D,L-lactide-co-glycolide) (PLGA) were prepared as previously described (Ahmadi R, Mordan N, Forbes A, Day RM. Enhanced attachment, growth and migration of smooth muscle cells on microcarriers produced using thermally induced phase separation. Acta Biomater. 2011 Apr;7(4):1542-1549). PLGA PURASORB7507 (75:25) polymer (Corbion, Amsterdam, Netherlands) was dissolved in dimethyl carbonate (Sigma Aldrich, Dorset, UK) overnight using a magnetic stirrer to produce a 10% (w / v) aqueous solution of the polymer. The polymer aqueous solution was then pumped into a Nisco Encapsulator Unit (Nisco Engineering, Zurich, Switzerland; Frequency: 2.75 kHz, Amplitude: 70%) at a constant flow rate of 2 mL / min by a syringe pump (Harvard Apparatus, Kent, UK). Polymer droplets were formed using a 100 μm sapphire nozzle and collected in liquid nitrogen. Residual solvent was removed from the frozen polymer droplets by lyophilization for 48 hours. Dry PLGA TIPS microspheres were sieved into a particle size range of 250-350 μm and stored in rubber-stoppered glass vials at room temperature under reduced pressure.

[0056] Addition of Taxanes using TIPS microspheres A simple method was developed to load DTX and other taxanes, e.g., paclitaxel and cabazitaxel, onto TIPS microspheres by poor solvent precipitation (Figure 1). 5 mg of PLGA TIPS microspheres were transferred to a 20 ml clear Type 1B borosilicate glass vial and sealed with a butyl injection stopper. 4.5 ml of ultrapure water was added to the vial and vortexed for 10 seconds. 0.5 ml of 0.1% (w / v) taxane in ethanol, or Taxotere® or Taxol® formulations corresponding to docetaxel or paclitaxel, was added using a 1 ml syringe with a 25 G needle through the rubber stopper when the vial was inverted. The vials were then vortexed for 10 seconds and placed on a roller mixer (IKA® Roller 6 Digital; 60 rpm) at room temperature for the scheduled times (5, 15, 30, 60, 120 minutes).

[0057] The drug loading efficiency (DLE) of taxanes, such as DTX, onto TIPS microspheres at each time point was calculated according to Equation 1. The amount of free taxane remaining in solution was measured by UV spectroscopy at a wavelength of 229 nm using a Nanodrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA).

[0058] [ka]

[0059] Scanning electron microscopy was used to investigate the morphological changes at the surface of TIPS microspheres upon the loading of taxanes. Unbound taxanes were removed from the microspheres by washing three times with 5 ml of ultrapure water, followed by drying under reduced pressure. Samples of the dried particles were coated with gold for 60 seconds using a Q150R ES gold coater (Quorum Technologies, Oxford, UK). Samples were imaged using a Hitachi S3400N scanning electron microscope. X-ray photoelectron spectroscopy (XPS) was performed using a Thermo Scientific Kα photoelectron spectrometer using monochromatic Alkα radiation. Higher resolution scans were recorded for the first peaks of N(1), C(1) at a pass energy of 50 eV.

[0060] Comparison of methods used to load API onto PLGA TIPS microspheres 1. Dry TIPS PLGA microspheres (250-350 μm size range): Docetaxel was loaded onto 5 mg of PLGA TIPS microspheres as described immediately above. Dry-loaded TIPS microspheres were prepared by placing washed microspheres in a desiccator for at least 7 days or by lyophilization to remove excess water ("freeze drying").

[0061] 2. "Pre-wet" TIPS PLGA microspheres: 5 mg of PLGA TIPS microspheres were mixed with complete medium (Ham's F12-K medium (Kaighn modification) (Invitrogen, 21127-022) supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% antibiotics, followed by the addition of ethanol to a concentration of 7% (v / v).

[0062] 3. 5 mg of PLGA TIPS microspheres were transferred to a 20 ml clear Type 1B borosilicate glass vial and sealed with a butyl injection stopper. 4.5 ml of ultrapure water was added to the vial and vortexed for 10 seconds. 0.5 ml of 0.1% (w / v) docetaxel in ethanol was added using a 1 ml syringe with a 25G needle through the rubber stopper when the vial was inverted. The vial was then vortexed for 10 seconds and placed on a roller mixer (IKA® Roller 6 Digital; 60 rpm) for the scheduled times (5, 15, 30, 60, 120 min) at room temperature.

[0063] 4. The drug loading efficiency (DLE) of docetaxel (DTX) onto TIPS microspheres at each time point was calculated according to Equation 1. The amount of free docetaxel remaining in the solution was measured by UV spectroscopy at a wavelength of 229 nm using a Nanodrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA).

[0064] Docetaxel release from DTX-TIPS microsphere combination products The release profile of DTX from TIPS microspheres was investigated using a dynamic perfusion system to simulate the tissue perfusion of the drug-device combination in a physiological environment when used clinically after radical prostatectomy. Thus, the perfusion system was placed in a 37°C incubator, and a physiologically simulated medium (phosphate buffered saline (PBS); pH 7.4) was used as the perfusate. The DTX-loaded microspheres were mixed with 100 μl of 70% (v / v) GranuGel® (Convatec, UK) diluted with ultrapure water, and the mixture was placed between two 25 mm circular filter papers (Whatman® qualitative cellulose filter paper, Grade 1), where their position was held by a Swin-Lok™ plastic membrane filter holder. A hypodermic needle (18G×40 mm) connected to the outlet of the filter holder was inserted through the lid of a 50 ml polypropylene container to collect the perfusate. PBS was pumped through the filter holder at a flow rate of 0.01 mL / min by a peristaltic pump (Harvard Apparatus). The conditioned perfusate was sampled at specified intervals and used for further experiments.

[0065] The amount of DTX released into the perfusate was determined using a predetermined calibration curve. At each measurement, the concentration of DTX in the release medium collected in the polypropylene container was measured by UV spectroscopy at 229 nm as previously described, and the cumulative DTX release was calculated according to Equation 2.

[0066] [ka]

[0067] In vitro efficacy of docetaxel released from DTX-TIPS microsphere combination products Human prostate cancer cells (PC3, American Type Culture Collection) were used to test the activity of docetaxel released from DTX-TIPS microspheres. PC3 cells were maintained in Ham's F12-K medium (Kaighn's modification) (Invitrogen, 21127-022) supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% antibiotics (hereafter referred to as complete medium). Cells were incubated at 37°C in a humidified incubator with 5% CO 2 PC3 cells were cultured in 6-well plates under atmospheric conditions. PC3 cells cultured in 6-well plates were incubated for 48 hours in 2 ml of perfusate (conditioned complete medium) collected from DTX-TIPS microspheres added to the perfusion system outlined above. Colony formation assays were performed as previously described (Franken NAP, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat Protoc. 2006;1(5):2315-2319). PC3 cells exposed to the perfusate were detached from 6-well plates using trypsin-EDTA solution (0.5 g / l porcine trypsin and 0.2 g / l EDTA.4Na in Hank's Balanced Salt Solution with phenol red; Sigma-Aldrich), washed with fresh complete medium, single cell suspensions were made, resuspended in fresh complete medium, and replated in 9 cm diameter Petri dishes at a density of 400 cells per dish. After plating, the dishes were incubated at 37°C in a humidified incubator with 5% CO for 2 weeks. 2Colonies were allowed to form under ambient conditions and incubated. Colonies were fixed with methanol for 20 min and stained with 0.5% crystal violet (Sigma Aldrich, Dorset, UK) in distilled water for 2 h. Colonies were counted by ImageJ using the "Colony Counter" plugin (processing parameters: size 100-30,000; circularity 0.5-1) (https: / / imagej.nih.gov / ij / plugins / colony-counter.html).

[0068] Plating efficiency (PE) was estimated by dividing the number of colonies counted by the number of cells initially plated, and this number was used for normalization when calculating survival fraction (SF).

[0069] [ka]

[0070] Morphological detection of apoptosis in 2D cultures Morphological changes to PC3 cells indicative of apoptosis, including shrinkage and fragmentation into membrane-bound apoptotic bodies, were assessed following exposure to perfusate-conditioned complete medium. Cells were incubated for 24 hours in perfusate-conditioned complete medium, collected at 24-hour intervals over 12 days. Images of cell morphology were obtained using phase-contrast microscopy using a Zeiss Primovert microscope, and the number of cells showing nuclear fragmentation in each image was calculated, with at least 100 cells analyzed in each group.

[0071] Efficacy studies using tumor spheroids 3D spheroids of PC3 cells were generated by using methylcellulose as a scaffold as previously described (Korff T. (2004) Three-Dimensional Spheroid Culture of Endothelial Cells. In: Augustin HG (eds) Methods in Endothelial Cell Biology. Springer Lab Manuals. Springer, Berlin, Heidelberg). PC3 were grown at 2 × 10 4 Cells were seeded at a concentration of 200 μL of complete medium containing 20% ​​wt methylcellulose. Cells were incubated at 37°C in a humidified incubator with 5% CO until spheroids formed. 2 The cells were incubated at room temperature for 2 days. The medium was replaced with 200 μL of perfusate-conditioned complete medium, which was replaced daily with medium withdrawn from the perfusion system at the corresponding time points over 12 or 14 days. Images of the spheroids were taken daily during culture. The dimensions of the imaged spheroids were measured using Image J, and the average diameter of the spheroids was estimated using the Feret diameter and plotted against time (GraphPad Prism software).

[0072] Prostate tumor formation in vivo Five million human prostate cancer cells (PC3, American Type Culture Collection) were injected subcutaneously into the flank of female NSG™ (NOD scid γ) immunodeficient mice (6-7 weeks old). After 2 weeks, ∼20 mm 3 Palpable tumors measuring 0.01 mm were formed. The size of each tumor and the weight of each animal were recorded three times a week.

[0073] Tumor treatment Mice were assigned to one of four treatment groups (DTX-TIPS microspheres n=7; control TIPS microspheres n=7; native DTX n=7; saline n=4) and received the following treatment regimens:

[0074] 1. Peritumoral delivery of DTX-TIPS microspheres: 5 mg of PLGA TIPS microspheres loaded with 500 μg of DTX solution (described in Example 1) was mixed with 100 μl of GranuGel. 2. Peritumoral delivery of control TIPS microspheres: 5 mg of PLGA TIPS microspheres were mixed with 100 μl of GranuGel.

[0075] 3. Tail vein injection of 10 mg of DTX dissolved in ethanol and Tween® 80 to make a 10 mg / ml solution, followed by dilution to 1 mg / ml in saline. Mice were given 10 mg / kg; once a week for 3 weeks. 4. Tail vein injection of saline (10 μl / g); once a week for 3 weeks. At the end of the life phase, animals were humanely sacrificed. Tumors were excised and weighed prior to histological analysis.

[0076] Measurement of DTX released in vivo from DTX-TIPS microspheres Docetaxel-loaded TIPS microspheres were prepared by antisolvent precipitation as outlined above. To measure the concentration of DTX present in the circulation after treatment, DTX-TIPS microspheres were mixed into a homogenous suspension in 100 μl of 70% (v / v) GranuGel and implanted subcutaneously in non-tumor-bearing BALB / cAnNCrl mice (n=5) (7-8 weeks, 17-20 g, Charles River) with 1×100 μl depot using a 1 mL syringe and 16G needle. Control animals received either control TIPS microspheres (n=5) or IV DTX 10 mg / kg once a week for 3 weeks via tail vein delivery (n=5). For measurement of circulating DTX in plasma, blood samples were collected via the tail vein on days 0 (pre-dose), and days 1, 2, 3, 7, 10, and then every 5 days until just before the end of the life phase on day 35. Blood samples were collected in 10 μl capillary tubes containing sodium heparin (Hirschmann, Eberstadt, Germany), placed into wells of a 2 ml deep-well plate, and stored at −80° C. until extraction.

[0077] On the day of extraction, the standard curve and sample plates were thawed. 125 μl of 70:30 water:acetonitrile containing 0.4% blood was added to blanks, standard curves, and QCs. 125 μl of 70:30 water:acetonitrile containing 0.4% DMSO was added to all samples. 40 μl of each blood:water:acetonitrile sample was taken and proteins were precipitated with 120 μl acetonitrile containing DTX-D9 (25 nM). Samples were mixed and centrifuged. Supernatants were taken and diluted 40:60 with 0.5% sodium acetate (20 μM) in ammonium acetate (10 mM, pH 5).

[0078] Liquid chromatography-mass spectrometry was performed on a Waters Acquity HSS PFP column (1.8 μm, 50 mm × 2.1 mm id) with a gradient consisting of 10 mM ammonium acetate (pH 5.0) and acetonitrile mobile phase using a Waters (Milford, MA) H-class Acquity solvent and sample manager. The flow rate was 0.6 ml / min, and the run time was 5.6 min. Analytes and internal standards were ionized using electrospray ionization in positive ion mode. Detection of analytes was by tandem mass spectrometry (MS / MS) using a Waters Xevo TQ-S mass spectrometer in multiple reaction monitoring (MRM) mode. Transition states m / z 830.4-248.1 / 304.2 and m / z 839.4-313.0 were observed for DTX and DTX-D9 (IS), respectively. The calibration curve was linear over the concentration range of 1 to 10,000 nM.

[0079] In vivo tumor growth inhibition activity Female NSG™ immunodeficient mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ; Charles River), 6-7 weeks old and weighing 18-20 g, were acclimated for 1 week prior to injection of tumor cells. PC3 cells (5.0 × 10 in 100 μl phosphate-buffered saline [PBS]) were 6 Cells) were injected subcutaneously into the right flank of each mouse. Tumor dimensions were measured three times a week using digital calipers, and tumor volumes were calculated using the following formula:

[0080] [ka]

[0081] Fourteen days after cell injection, mice were randomized into four groups: Group 1, intravenous (IV) DTX (10 mg / kg delivered via tail vein once weekly for 3 weeks; n=7); Group 2, peritumoral injection of DTX-TIPS microspheres (n=7); Group 3, peritumoral injection of control TIPS microspheres (n=7); Group 4, IV saline (10 μl / g body weight; n=4). Mice in Groups 2 and 3 received TIPS microspheres (+ / - DTX) after mixing the microspheres into a uniform suspension in 100 μl of 70% (v / v) GranuGel. The suspension of TIPS microspheres in GranuGel was delivered subcutaneously in a 1×100 μl depot around the periphery of the tumor using a 1 mL syringe and a 16G needle. Mice were observed for signs of toxicity (weight loss, body condition, and uncoordinated movement) over the course of the study. Mice were euthanized if they lost more than 15% of their starting weight or showed excessive signs of toxicity.

[0082] At the end of the life phase (days 1, 10, and 35 after treatment), mice were treated with an overdose of CO 2Mice were euthanized at 37 °C followed by cervical dislocation and organs (heart, liver, kidneys, lungs and spleen) were harvested, weighed and frozen in liquid nitrogen immediately prior to storage at -80 °C until further analysis. Assessment of docetaxel concentration was performed by tissue liquid chromatography tandem mass spectrometry following tissue homogenization in 3 ml / g (spleen 5 ml / g) of 10 mM PBS using a Precellys24 homogenizer (Bertin technologies, Montigny-le-Bretonneux, France). 45 μl of tissue homogenate was spiked with 5 μl of DMSO, samples were mixed and proteins were precipitated with 150 μl of acetonitrile containing DTX-D9 as an internal standard (25 nM). Samples were mixed and centrifuged. Supernatants were collected and diluted 40:60 with 0.5% sodium acetate (20 μM) in ammonium acetate (10 mM, pH 5). Blanks, calibration curves, and QCs were prepared as described above using tissues obtained from the same mouse strain (NSG) spiked with DMSO, working calibration standards, and working QCs, respectively. Blanks consisted of proteins precipitated with acetonitrile alone, while blanks+, standards, and QCs were proteins precipitated with acetonitrile containing DTX-D9.

[0083] Tumors were explanted and processed for histology. Tissues were fixed in 10% formalin, dehydrated, and embedded in low melting point wax (Paraplast X-TRAR, Sigma). Histological sections cut from the wax-embedded tissues were stained with hematoxylin and eosin.

[0084] statistical methods Data were tested for Gaussian distribution and analyzed for statistical significance using GraphPad Prism software. For data sets that were Gaussian distributed, statistical evaluation was performed by Two-Way ANOVA with Dunnett's test for multiple comparisons unless otherwise stated in the figure legend. For data sets with non-Gaussian distribution, statistical evaluation was performed with the Friedman test.

[0085] Example 2 Immobilization of DTX on PLGA TIPS microspheres The second composition of PLGA TIPS microspheres initially floated on top of the DTX solution, but the solution was constantly mixed by rotation of the glass vial throughout the loading phase. The amount of DTX loaded onto the microspheres was indirectly quantified by calculating the amount of DTX remaining in solution at various time points during the loading phase. Approximately 80% of the DTX was loaded onto the microspheres from the solution within 60 min of the start of mixing (Figure 2a). No further increase in loading was detected beyond 60 min during the loading phase. The rapid loading of TIPS microspheres within 1 h is likely enhanced by their unusually high surface electrostatic charge. Such rapid loading is beneficial when using biodegradable devices. Scanning electron microscopy of the microspheres revealed the presence of crystalline material on the surface of the microspheres incubated in the DTX solution compared to unloaded control TIPS microspheres (Figures 2b and c). The crystalline material was visible on the surface of the microspheres within 1 min of incubation and gradually increased with the duration of the incubation time (Figure 2d). DTX(C 43 H 53 NO 14 The molecular composition of the DTX enabled X-ray photoelectron spectroscopy (XPS) analysis of nitrogen to be used to confirm the elemental composition of the crystalline material on the microsphere surface (FIG. 2e). A strong nitrogen signal was detected on the DTX-loaded microspheres, which was absent in the unloaded control microspheres. Similarly, a faster rate of drug loading is achieved with dry PLGA TIPS microspheres compared to solid microspheres composed of certain PLGA polymers or to "pre-wet" TIPS microspheres followed by loading of PLGA TIPS microspheres (see comparison of the method used to load API onto PLGA TIPS microspheres in Example 1, where the microspheres are immersed in medium prior to the addition of ethanol) (FIG. 2f).

[0086] Scanning electron microscopy of the TIPS microspheres also revealed the presence of crystalline material on the surface of TIPS microspheres incubated in paclitaxel or cabazitaxel solutions compared to no-addition control TIPS microspheres (FIG. 2g).

[0087] The presence of taxane crystals on the surface of TIPS microspheres indicates that the taxane was loaded onto the surface instead of by incorporation into the particles. The taxane crystals observed on the TIPS particles were likely formed by anti-solvent precipitation and grew during the drying process as a result of solvent evaporation. The hydrophobic and porous nature of the PLGA TIPS microspheres provided a favorable surface for taxane precipitation as it facilitated drug nucleation, thereby promoting crystallization on the surface, explaining the high drug loading efficiency of ∼80%. Maximum loading of DTX onto TIPS particles occurred within 60 min without the use of heating and cooling steps, making it a feasible time frame for preparing drug-device combinations at the point of care. However, it was noted that the rate of anti-solvent precipitation loading of TIPS microspheres differed for various taxanes. Docetaxel showed a faster loading rate onto TIPS microspheres compared to cabazitaxel. Approximately 80% of docetaxel was loaded from solution to the microspheres within 60 min after mixing began (Figure 2h). At the same time point, approximately 60% of cabazitaxel was loaded from solution to the microspheres. No further increase in docetaxel loading was detected during the entire loading phase up to 60 min.

[0088] Example 3 Excipients in injectable formulations of taxanes interfere with antisolvent precipitation drug loading onto TIPS microspheres Injectable docetaxel (e.g., Taxotere®) is formulated in a vehicle consisting of a 1:1 mixture of Tween® 80 and ethanol, which is further diluted with saline prior to administration. Injectable paclitaxel (e.g., Taxol®) is formulated in a vehicle consisting of a 1:1 mixture of Cremophor EL and ethanol, which is diluted 5-20 times with saline prior to administration. The use of injectable formulations of taxanes containing excipients has not been compatible with the loading of taxanes into TIPS microspheres.

[0089] Scanning electron microscopy of the microspheres revealed the presence of less crystalline material on the surface of the microspheres incubated in the injectable formulations of docetaxel and paclitaxel compared to the TIPS microspheres incubated with the pure formulations of docetaxel and paclitaxel without the addition of excipients (Figure 3).

[0090] The corresponding Taxotere® or Taxol® formulations of docetaxel or paclitaxel showed much lower drug loading onto the microspheres compared to the pure formulations without added excipients (Figure 4). After 3 hours of mixing, approximately <10% of docetaxel was loaded onto the microspheres from solution. After 4 hours of mixing, approximately <10% of paclitaxel was loaded onto the microspheres from solution.

[0091] Example 4 Freeze-drying of TIPS microspheres loaded with drugs by antisolvent precipitation does not affect taxane release from TIPS microspheres Following antisolvent precipitation of the drug onto the TIPS microspheres, the microspheres can be used immediately after a final washing step ("wet") or can be subsequently dried to allow for long-term dry storage of the microspheres. Dry storage is important because the microspheres are composed of PLGA, a polymer that undergoes hydrolytic degradation when exposed to water. Therefore, any residual water present in the stored finished product will result in degradation of the microspheres. This will result in loss of microsphere structure and release of the drug loaded onto the surface of the microspheres.

[0092] Scanning electron microscopy of the microspheres revealed a similar distribution of crystalline material on the surface of the "wet" and "dry" microspheres (Figure 5). The cumulative release of docetaxel showed similar release rate trends for the "wet" and "dry" docetaxel-loaded TIPS microspheres over the first 72 hours (Figure 6).

[0093] The diameter of the spheroids calculated from the acquired images showed a significant reduction in size compared to both the starting size of the spheroids and the control spheroids measured at the same time points, producing a similar effect to the perfusate collected from "wet" and "dry" docetaxel-loaded TIPS microspheres (Figure 7).

[0094] Example 5 Taxane-loaded TIPS microspheres are stable when stored dry at a variety of temperatures. The long-term stability of docetaxel-loaded TIPS microspheres allows the product to be stored prior to clinical use. It is important to determine the climatic conditions under which the drug-loaded microspheres are stable. SEM imaging confirmed comparable amounts of docetaxel present on the surface of TIPS microspheres after one month of storage (Figure 8).

[0095] Example 6 Cytotoxic activity of sustained DTX release against prostate cancer cells Sustained drug release was observed from the microspheres when placed in a tissue perfusion simulating dynamic system. The release profile of DTX from TIPS microspheres was investigated using a dynamic perfusion system designed to simulate in vivo tissue perfusion during the intended clinical use of the drug-device combination. DTX-containing perfusate from the TIPS microspheres was withdrawn at regular intervals and the amount of DTX was measured using UV absorbance. A plot of cumulative release of DTX showed that approximately 95% of the total amount of DTX loaded onto the TIPS microspheres was released over a 5-day period, with approximately one-third released during the first 24 hours (FIG. 9a). The amount of DTX released beyond the 5-day period was below the detection threshold of the system used for UV absorbance. Therefore, a two-cell-based assay using PC3 prostate cancer cells was used to assess whether there was sustained cytotoxic activity of the DTX released from the TIPS microspheres in the perfusate. Colony formation assays revealed that DTX released into the perfusate from TIPS microspheres harvested from all time points up to day 10 continued to have an inhibitory effect on PC3 cell colony formation (Figure 9b). DTX present in the perfusate harvested from days 1-4 completely inhibited the formation of all colonies. Colony formation after incubation in perfusate harvested between days 5-8 was less than 25% of the number of colonies established in the control group containing no DTX. The number of colonies formed at day 10 was approximately 50% of the number of colonies established in the control group containing no DTX. Further confirmation of the sustained cytotoxic effect was revealed by morphological analysis of cells first incubated in DTX-TIPS perfusate for 48 h, followed by incubation in fresh complete medium for 1, 5 or 10 days (Figure 9c). Phase contrast microscopy revealed characteristic morphological features of apoptosis, including nuclear fragmentation, cell rounding with shrinkage and cytoplasmic condensation (signs of early apoptosis), and apoptotic bodies (signs of late apoptosis).

[0096] Since DTX was added to the surface of the microspheres, the release of DTX was mainly controlled by DTX crystal dissolution, not by the degradation of the PLGA polymer. The DTX crystals slowly dissolved over time and maintained a concentration gradient for diffusion, resulting in sustained release over 5 days. This is beneficial because the drug-device combination is implanted at the tumor resection site, where its long-term localized release helps eradicate residual or sloughed tumor cells released during surgery, minimizing the chance of recurrence. The amount of DTX released into the perfusate during the first 5 days of the DTX release assay, simulating release and localized activity in vivo, was sufficient to achieve 100% toxicity to PC3 cells in the colony formation assay and represented approximately 95% of the DTX released from the microspheres. DTX release from the microspheres continued until the amount of DTX present in the perfusate collected every other day for up to 10 days fell below the detection threshold of the UV specification, indicating mitotic arrest, beyond 5 days. PC3 prostate cancer cells incubated in perfusate harvested at later time points were reduced in number and displayed phenotypic characteristics typical of apoptosis.

[0097] Example 7 Cytotoxic activity of sustained DTX release against prostate cancer spheroids Drug released into the perfusate from microspheres harvested at defined time intervals inhibited colony formation and demonstrated sustained cytotoxicity against PC3 prostate cancer cells over 10 days. The cytotoxicity of DTX released from the DTX-TIPS combination in a more physiological 3D culture system was evaluated as follows: 3D spheroids of PC3 cells were incubated with perfusate harvested daily for 12 days. The medium containing the spheroids was replaced daily with perfusate harvested from the perfusion system on the anniversary day. The diameter and volume of the spheroids were calculated from images obtained throughout the experiment and showed a significant reduction in size compared to both the starting size of the spheroids and the control spheroids measured at the same time points (Figure 10a). PC3 cells cultured in 2D in tissue culture wells exposed to daily harvested perfusate for up to 12 days showed morphological features of cytotoxicity similar to those observed in the colony formation assay (Figure 10b). Live / Dead staining with Calcein AM (an enzymatic fluorescent dye that passively enters and stains all metabolically active cells) and Ethidium homodimer-1 (EthD-1; a fluorescent dye that only stains dead cells by binding to nucleic acids after penetrating their damaged membrane) was performed on spheroids incubated with perfusate collected over time on day 12 (FIG. 10c). In both DTX-treated and control spheroids, dead cells were visible towards the center of the spheroid, indicating the presence of a necrotic core due to oxygen and nutrient diffusion limitation. Exposure of spheroids to DTX in the perfusate on day 12 resulted in a size reduction in spheroid diameter. PC3 cells in the remaining spheroid state stained positively with Calcein AM and EthD-1, whereas the percentage of EthD-1 positive cells at the periphery of spheroids exposed to DTX was increased compared to the control group. The cytotoxic effect of DTX released from DTX-TIPS particles on 3D spheroid cultures was confirmed by transferring the spheroids to tissue culture plates on day 12. No viable cells were found to migrate out of the spheroids in the DTX-treated spheroids, whereas cells were found to migrate out of the spheroids in the control group.

[0098] Example 8 DTX-TIPS inhibits tumor growth in vivo without systemic toxicity Mice receiving IV DTX (as described above in Example 1, In Vivo Prostate Tumor Formation and Tumor Treatment) showed progressive and significant weight loss and demonstrated systemic toxicity (FIG. 11a). No significant weight change was observed in mice receiving DTX-TIPS compared to mice receiving IV saline or control TIPS microspheres, suggesting no systemic toxicity.

[0099] Tumor volume increased steadily throughout the study period in mice receiving control TIPS microspheres or IV saline (Figure 11b). Tumor volume in mice receiving DTX-TIPS was substantially reduced compared to the saline or control microsphere groups. Tumor growth inhibition was most evident in mice receiving IV DTX.

[0100] At day 14, there was no significant difference in tumor size increase between mice receiving DTX IV and DTX-TIPS, indicating that DTX-TIPS was equally effective at preventing tumor growth (Figure 11c). The increase in tumor size was significantly lower in mice receiving DTX-TIPS compared to mice receiving IV saline and mice receiving control TIPS microspheres.

[0101] Example 9 Docetaxel-loaded TIPS microspheres prepared by antisolvent precipitation provide localized and sustained release of non-toxic therapeutic doses of DTX that effectively arrest tumor growth in vivo The efficacy of docetaxel-loaded TIPS microspheres was verified in vivo after peritumoral injection in a PC3 xenograft tumor model, where the level of tumor-destroying inhibition was comparable to that achieved with intravenous delivery of DTX. However, unlike intravenous delivery of DTX, implantation of DTX-TIPS microspheres was not associated with toxicity or elevated systemic levels of DTX in organs, tissues, or plasma. Analysis of the in vivo DTX release profile was investigated in plasma collected from non-tumor-bearing BALB / cAnNCrl mice. Detectable levels of DTX released from TIPS microspheres were observed 1 and 24 hours after administration. Thereafter, DTX was not detectable in plasma. Plasma samples collected from the group that received IV DTX once a week for 3 weeks contained significantly higher levels of DTX at 1 hour (102-fold increase) and 24 hours (8-fold increase) after administration compared to the DTX-TIPS group (p<0.01) (Figure 12). Administration of IV DTX resulted in detectable levels of DTX remaining in plasma for 48 and 72 hours, and on days 10 and 15, corresponding to the second and third doses of IV DTX delivered on days 7 and 14.

[0102] The antineoplastic activity and systemic toxicity of DTX-TIPS microspheres were investigated in vivo using a human prostate tumor xenograft model in NSG mice. PC3 cells were subcutaneously implanted into immunodeficient mice. Palpable tumors were formed 14 days after cell injection, measuring 0.03–0.05 cm. 3 Peritumoral delivery of TIPS microspheres (+ / - DTX) and retention at the implantation site was confirmed by histology with the microspheres remaining in situ throughout the study (Figure 13).

[0103] Tumor volume measurements in mice receiving IV DTX did not increase over the course of the study (Figure 14a). Mice receiving DTX-TIPS microspheres did not have a significant increase in tumor volume compared to mice receiving IV DTX through day 21 (p<0.05). At this time point, the increase in tumor volume in the group treated with DTX-TIPS microspheres was approximately six-fold compared to the group treated with saline only at the same time point. In mice receiving either TIPS microspheres only or IV saline, tumor volume was significantly increased at day 16 (p<0.05) and day 14 (p<0.05), respectively, compared to mice receiving IV DTX. Qualitative macroscopic evaluation of excised tumors at the end of the study revealed visibly smaller tumors recovered from the DTX-treated group (Figure 14b).

[0104] Significant toxicity was associated with IV delivery of DTX, as indicated by progressive weight loss beginning on day 7, compared to starting weight, and culminating in -7.0% ± 2.3% at day 28 and -12. 7% ± 3.5% at day 35 (p<0.0001; weight loss exceeded >15% at day 35 in 3 / 7 mice). In contrast, no significant weight loss was observed in groups receiving DTX-TIPS microspheres, TIPS microspheres only, or IV saline (Figure 14c).

[0105] Tissue levels of DTX in explanted organs (heart, kidney, lung, spleen, liver) were analyzed at 1, 10, and 35 days post-dosing as shown in Table 1 below. High levels of DTX were detected in all organs at all time points after IV DTX administration compared to DTX-TIPS administration, except for liver and spleen on day 35, where DTX was undetectable for either treatment group.

[0106] [Table 1] Table 1 - DTX concentrations measured in tissues from explanted organs at days 1, 10, and 35 after administration of DTX-TIPS microspheres or IV DTX (n=5-6 samples per time point for each group).

[0107] All cited references are incorporated herein in their entirety.

Claims

1. 1. A method for attaching an insoluble active pharmaceutical ingredient (API) to microspheres produced by thermally induced phase separation (TIPS), comprising: i) mixing the microspheres with an aqueous solution to form a first composition; ii) dissolving an insoluble API in a first solvent, and subsequently adding the insoluble API dissolved in the first solvent to the first composition to form a second composition; and iii) mixing a second composition; The method includes:

2. 13. The method of claim 1, wherein the microspheres are mixed with the aqueous solution in a container and the container is tumbled during formation of a second composition, preferably the addition of the insoluble API dissolved in the first solvent to the first composition is via a needle.

3. The thermally induced phase separation method comprises the steps of: i) dissolving the polymer in a second solvent to form a solution; ii) quenching the droplets of the solution with a quenching fluid; and iii) freeze-drying the resulting spheres; 3. The method of claim 1 or 2, comprising the steps of: preferably wherein the solution is introduced during quenching using a syringe, vibrating needle or atomizer; and more preferably further comprising the step of sonicating the solution.

4. i) the polymer is PLGA; ii) the second solvent is dimethyl carbonate; and / or iii) the quenching fluid is liquid nitrogen; The method of claim 3.

5. 3. The method of claim 1 or claim 2, wherein the microspheres comprise PLGA.

6. The method of any one of claims 1 to 5, wherein the insoluble API is a chemotherapeutic drug, preferably, the chemotherapeutic drug is a taxane, and more preferably, cabazitaxel, paclitaxel, or docetaxel.

7. 7. The method of any one of claims 1 to 6, wherein the insoluble API is deposited on the surface of the microspheres in crystalline form.

8. 8. The method according to any one of claims 1 to 7, wherein the first solvent is an alcohol, preferably ethanol, optionally at a final concentration in the second composition of 10% to 30% v / v.

9. In addition: a. removing any unbound insoluble API from said second composition by washing with an aqueous solution; and / or b. mixing of said second composition is performed by rotation; The method according to any one of claims 1 to 8, comprising:

10. A microsphere produced by a thermally induced phase separation process having an insoluble API bound to its surface, the insoluble API being a chemotherapeutic taxane, the microsphere comprising poly(lactide-co-glycolide) (PLGA).

11. A microsphere produced by a thermally induced phase separation process having an insoluble API bound to its surface, wherein the insoluble API is bound to the microsphere by the method of any one of claims 1 to 9.

12. The thermally induced phase separation method comprises the steps of: i) dissolving a polymer in a solvent to form a solution; ii) quenching the droplets of the solution with a quenching fluid; and iii) freeze-drying the resulting spheres; 12. The microspheres of claim 10 or 11, preferably wherein the solution is introduced during quenching using a syringe, vibrating needle or atomizer, and more preferably further comprising the step of sonicating the solution.

13. i) the polymer is PLGA; ii) the solvent is dimethyl carbonate; and / or iii) the quenching fluid is liquid nitrogen; 13. The microsphere of claim 12.

14. The microsphere of claim 10 or 11, wherein the microsphere comprises PLGA.

15. The microsphere of any one of claims 10 to 14, wherein the insoluble API is a chemotherapeutic drug, preferably, the chemotherapeutic drug is a taxane, and more preferably, cabazitaxel, paclitaxel or docetaxel.

16. The microsphere of any one of claims 10 to 15, wherein the insoluble API is deposited on the surface of the microsphere in crystalline form.

17. 17. Microspheres according to any one of claims 10 to 16, wherein the microspheres have a diameter of 10 to 900 μm, preferably 50 to 450 μm, more preferably 100 to 400 μm and even more preferably 250 to 350 μm.

18. A composition comprising a microsphere according to any one of claims 10 to 17 for use in therapy.

19. A composition comprising a microsphere according to any one of claims 10 to 17 for use in cancer therapy.

20. The composition of claim 19, wherein the cancer is selected from prostate cancer, breast cancer, bladder cancer, colorectal cancer, thyroid cancer, oral cancer, lung cancer, renal cancer, uterine cancer and ovarian cancer.

21. 21. The composition for use according to claim 19 or 20, wherein the cancer is prostate cancer, preferably wherein the patient suffering from prostate cancer has undergone radical prostatectomy.

22. Use of a composition comprising the microspheres of any one of claims 10 to 17 in the manufacture of a medicament for the treatment of cancer.

23. The use of claim 22, wherein the cancer is selected from prostate cancer, breast cancer, bladder cancer, colorectal cancer, thyroid cancer, oral cancer, lung cancer, renal cancer, uterine cancer and ovarian cancer.

24. 24. Use of a composition according to claim 22 or 23, wherein the cancer is prostate cancer, preferably wherein the patient suffering from prostate cancer has undergone radical prostatectomy.

25. A pharmaceutical composition for treating cancer, comprising a microsphere according to any one of claims 10 to 17.

26. The pharmaceutical composition of claim 25, wherein the cancer is selected from prostate cancer, breast cancer, bladder cancer, colorectal cancer, thyroid cancer, oral cancer, lung cancer, renal cancer, uterine cancer and ovarian cancer.

27. 27. The pharmaceutical composition of claim 25 or 26, wherein the cancer is prostate cancer, preferably wherein the subject suffering from prostate cancer has undergone radical prostatectomy.

28. 1. A kit for treating cancer, comprising: i) TIPS microspheres provided in a sealed container; ii) an insoluble API dissolved in a first solvent; and iii) a carrier vehicle; Kit including:

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