Preparations containing proteases that act on mucin

Abstract

Microspheres for delivery to target regions within a patient's body are disclosed. The microspheres of the present invention are configured to encapsulate a mucin-active protease and release the protease continuously upon exposure to physiological conditions. Pharmaceutical compositions containing the microspheres of the present invention and methods of treatment using the microspheres of the present invention are also disclosed.

Description

Technical Field

[0001] The present invention relates to microspheres that internally carry a protease that acts on mucin. In one aspect, the present invention relates to microspheres containing a protease (such as bromelain) that acts on mucin for use in the treatment of mucin-producing cancers and other diseases involving mucin.

Background Art

[0002] Mucin is a family of highly glycosylated high molecular weight proteins produced by epithelial tissues present in the digestive tract, lungs, kidneys, ovaries, breasts, pancreas, etc. Mucin is responsible for protecting epithelial tissues under normal physiological conditions. On the other hand, mucin may be involved in various diseases. For example, overexpression of certain types of mucin (such as MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC16, etc.) is associated with certain types of cancer. The synthesis of mucin on the surface of epithelial cells is usually highly regulated, but in tumors, mucin production increases, such as by an increase in the expression of human mucin. In epithelial-derived cancers, the expression and composition of mucin change, and in patients suffering from epithelial-derived cancers, it is known that mucus production is a negative prognostic factor.

[0003] Abnormal accumulation of mucin can have an adverse effect on the health of patients and may cause non-cancerous diseases such as cystic fibrosis and chronic obstructive pulmonary disease.

[0004] Therefore, diseases involving mucin need to be treated, and it is necessary to provide a better outcome for patients suffering from diseases involving mucin. Mucin-related diseases can be treated, for example, with mucolytics, which act on mucin proteins (such as by degrading or destroying mucin proteins) to reduce their viscosity, promote the removal of mucin by the body, or facilitate the penetration of cytotoxic agents (such as when tumors are covered with mucin).

[0005] Mucin-acting proteases are a type of mucolytic agent and are proteolytic enzymes that cause the breakdown of mucin proteins. However, mucin-acting proteases generally have complex properties and carry the risk of side effects, making it difficult to effectively deliver them to patients. Furthermore, mucin-acting proteases also have stability issues under physiological conditions.

[0006] For example, bromelain is a protease that acts on mucin. Bromelain is an extract from the pineapple (Ananas Comosus), a type of plant, and contains various thiol proteases. Bromelain has proteolytic activity in vitro and in vivo, and possesses anti-edema activity, anti-inflammatory activity, antithrombotic activity, and fibrinolytic activity, so it can be used to treat diseases such as deep vein thrombosis and blood coagulation disorders. In addition, bromelain exhibits anti-cancer properties against certain types of cancer, both alone and in combination with other chemotherapy agents, in in vitro and in vivo models.

[0007] Therefore, bromelain has been proposed as a therapeutic agent for treating certain types of cancer and other mucin-related diseases. However, clinical trials on systemic administration of therapeutically effective doses of bromelain have not been conducted because (as seen in past animal studies) systemic administration of bromelain carries risks (particularly fibrinolytic and hemorrhagic effects).

[0008] It would be advantageous to deliver a therapeutically effective dose of a mucin-acting protease (such as bromelain) to the patient in a way that minimizes the possibility of side effects. [Overview of the project] [Means for solving the problem]

[0009] In a first embodiment, the present invention provides microspheres for delivery to a target area within a patient's body. These microspheres contain a mucin-acting protease and are configured to continuously elute the mucin-acting protease when exposed to physiological conditions.

[0010] The practice of encapsulating specific drugs within microspheres for local delivery into a patient's body is well-known, for example, in a technique known as transarterial chemoembolization (TACE). Microspheres marketed by Biocompatibles UK Ltd under the trademark name DC Bead® are, for example, indicated for the intra-arterial delivery of doxorubicin and irinotecan, anticancer drugs used to treat primary and secondary liver cancer. However, the drugs intended to be encapsulated within DC Beads for sustained release are all relatively small (approximately 600 Da) positively charged molecules, and it is believed that drugs other than doxorubicin and irinotecan cannot be properly retained within these microspheres. In fact, even if a drug could be encapsulated within these microspheres, many drugs would be released almost instantaneously under physiological conditions (commonly referred to as "burst release"). Other microspheres (some of which will be discussed later) are similarly indicated only for use with small molecules such as doxorubicin and irinotecan.

[0011] In contrast to such low molecular weight molecules, mucin-acting proteases are enzymes (or enzyme mixtures) with high molecular weights. In the case of bromelain, for example, some enzymes have been reported to have a molecular weight of approximately 33,000 Da. Therefore, according to prior art teachings, it is considered impossible to support mucin-acting proteases such as bromelain within microspheres such as DC beads, and even if it were possible to support mucin-acting proteases within such microspheres, it is considered unlikely that these proteases could be continuously released from the microspheres in an active form under physiological conditions. In fact, past studies by the present inventors and other researchers have failed in attempts to support bromelain within microspheres. In some of these attempts, for example, the support of bromelain within the microspheres itself failed. In other attempts, it was found that bromelain breaks down microspheres, and that bromelain itself decomposes under ambient conditions, or that bromelain exposed to physiological conditions is "burst-released" (having the same effect as when delivered systemically).

[0012] Given the prior art teachings described herein, it was generally believed that microspheres like those described herein were not useful for the sustained delivery of off-label molecules, much less for the sustained delivery of large, active enzymes or enzyme mixtures containing large enzymes.

[0013] The inventors unexpectedly discovered that bromelain can actually be supported within microspheres (such as DC beads) as described herein (it was later found that papain can also be supported), and that microspheres supporting bromelain can be locally delivered into a patient's body. Based on this discovery, the inventors completed the present invention. Furthermore, surprisingly and unexpectedly, it was found that bromelain supported on microspheres is continuously eluted from the microspheres in an active state when exposed to physiological conditions. Given that bromelain is normally unstable under ambient conditions, the inventors were surprised that bromelain could maintain its activity for such a long period. Thus, contrary to conventional knowledge, the inventors discovered that bromelain can be supported within microspheres and configured to be continuously released. Subsequent experiments by the inventors revealed that papain, another protease that acts on mucin, also exhibits comparable support and elution characteristics to bromelain. Therefore, the inventors reasonably predict that other proteases that act on mucin will also be useful in the present invention. For example, papain and ficin are similar in structure and function.

[0014] The inventors have found that these discoveries make it possible to provide a topical delivery medium for therapeutically effective doses of bromelain or other mucin-acting proteases that minimizes the potential for side effects. The significant advantages of these features will be apparent to those skilled in the art and will be discussed later.

[0015] In a second embodiment, the present invention provides a pharmaceutical composition comprising a microsphere for delivery to a target area in a patient's body and a pharmaceutically acceptable carrier, wherein the microsphere is configured to encapsulate a mucin-acting protease and to continuously elute the mucin-acting protease upon exposure to physiological conditions.

[0016] In a third aspect, the present invention provides a pharmaceutical composition comprising microspheres according to the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0017] In a fourth embodiment, the present invention provides a method for immobilizing a mucin-acting protease within microspheres. This method comprises the steps of: adding microspheres to a solution having an acidic pH (for example, a low pH such as pH 2 or pH 2.5); mixing the solution containing the microspheres with a solution containing a mucin-acting protease; and shaking the mixture for a sufficient time to allow the mucin-acting protease to be immobilized within the microspheres. The solution to which the microspheres are added may optionally have an ionic strength equivalent to that of a target area in the patient's body.

[0018] In a fifth embodiment, the present invention provides a method for treating mucin-producing cancer, pseudomyxoma peritonei, cystic fibrosis, or chronic obstructive pulmonary disease. The method comprises administering to a patient a therapeutically effective amount of microspheres containing a mucin-acting protease, wherein the microspheres are configured to continuously elute the mucin-acting protease after administration.

[0019] As mentioned earlier, in a method called transarterial chemoembolization (TACE) for the treatment of unresectable cancers such as hepatocellular carcinoma, microspheres loaded with doxorubicin or irinotecan are used. These microspheres are injected into an artery upstream of the tumor and form an embolism in areas where the artery diameter is small. Subsequently, doxorubicin or irinotecan is eluted from the microspheres, which are located very close to the blood vessel leading to the tumor, and delivered directly to this blood vessel, allowing for a high local concentration of the drug. This precisely targeted drug delivery can virtually eliminate drug-related adverse events.

[0020] The inventors believe that even when the microspheres according to the present invention are intra-arterially delivered to a patient, the protease that acts on mucin can be delivered locally equally effectively. Further, the inventors predict that the intralesional delivery, intraperitoneal delivery or intracavitary delivery (for example, delivery to the abdominal cavity or pleural cavity) of the microspheres is equally effective for the treatment of other related diseases, which will be described later.

[0021] In a sixth aspect, the present invention provides a method for treating mucin-producing cancer, pseudomyxoma peritonei, cystic fibrosis or chronic obstructive pulmonary disease, the method comprising administering a therapeutically effective amount of the microspheres according to the first aspect of the present invention or the pharmaceutical composition according to the second or third aspect of the present invention to a patient in need of treatment of said disease.

[0022] In a seventh aspect, the present invention provides the use of the microspheres according to the first aspect of the present invention for the manufacture of a medicament for the treatment of mucin-producing cancer, pseudomyxoma peritonei, cystic fibrosis or chronic obstructive pulmonary disease.

[0023] In an eighth aspect, the present invention provides the use of the microspheres according to the first aspect of the present invention for the treatment of mucin-producing cancer, pseudomyxoma peritonei, cystic fibrosis or chronic obstructive pulmonary disease.

[0024] In a ninth aspect, the present invention provides the microspheres according to the first aspect of the present invention for use as a medicament.

[0025] In a tenth aspect, the present invention provides the microspheres according to the first aspect of the present invention for use in the treatment of mucin-producing cancer, pseudomyxoma peritonei, cystic fibrosis or chronic obstructive pulmonary disease.

[0026] In the eleventh aspect, the present invention provides a composition comprising microspheres carrying internally a protease that acts on mucin, wherein the microspheres are configured to continuously elute the protease that acts on mucin when exposed to physiological conditions.

[0027] In the twelfth aspect, the present invention provides an injectable composition comprising microspheres carrying internally a protease that acts on mucin, wherein the microspheres are configured to continuously elute the protease that acts on mucin when exposed to physiological conditions.

[0028] In the thirteenth aspect, the present invention provides a sustained release formulation comprising microspheres carrying internally a protease that acts on mucin, wherein the microspheres are configured to continuously elute the protease that acts on mucin when exposed to physiological conditions.

[0029] Other aspects, features and advantages of the present invention will be described below.

Mode for Carrying Out the Invention

[0030] As described above, the present invention provides microspheres for delivery to a target region in a patient's body. The microspheres of the present invention carry internally one (or more) type(s) of protease that acts on mucin, and are configured to continuously elute this protease when exposed to physiological conditions.

[0031] Intra-arterial delivery of microspheres is a relatively well-established field, and biocompatible microspheres containing chemotherapeutic agents, intended for local delivery to tumors, are used to treat certain types of tumors. For example, polyvinyl alcohol (PVA) hydrogel microspheres containing the chemotherapeutic agents doxorubicin or irinotecan (both positively charged low-molecular-weight substances) are marketed under the brand name DC Beads by Biocompatibles UK Ltd., and DC Beads are used to treat primary or secondary liver cancer by local delivery to tumors via a technique known as transarterial chemoembolization (TACE). In addition, PVA hydrogel beads that elute such drugs are used with radiolabeling and are also used, for example, in selective internal radiotherapy (SIRT).

[0032] After the inventors made the surprising and unexpected discovery that bromelain could be supported within microspheres such as DC Beads (and other microspheres described later) (it was later found that papain could also be supported in microspheres), they conducted experiments (described later) and, even more surprisingly, found that bromelain was continuously eluted from the microspheres in an active form under in vitro physiological conditions. From these preliminary experimental data, the inventors reasonably predicted that it would be possible to support, contain, and elute bromelain (and other proteases that act on mucin) in the same manner in other types of microspheres. Microspheres made from materials that are biocompatible with the patient's body and do not cause unfavorable interactions with the proteases contained within may be useful in the present invention, and routine tests and experiments can be conducted to confirm whether specific types of microspheres are suitable for the continuous elution of the proteases contained within.

[0033] Proteases that act on mucin As mentioned above, mucin-acting proteases are a type of protease that can impart therapeutic effects by causing the proteolysis of mucin proteins. In this specification, "acting on mucin" means acting on mucin in a therapeutic manner, such as by liquefying or degrading mucin (i.e., reducing its viscosity and promoting its elimination by the body) or destroying mucin. Proteases having such effects may be useful in the treatment of mucin-producing cancers (which may include mucin-secreting cancers, mucin-containing cancers and / or mucin-producing cancers (as defined later)) and other diseases involving mucin (for example, diseases described below). In our view, any mucin-acting protease may be used in the present invention, and to determine whether a particular mucin-acting protease is suitable, only the necessary routine tests and experiments (in light of the teachings described herein) should be performed.

[0034] The present invention will be described below primarily in relation to bromelain and papain, both plant-derived protease enzymes that act on mucin. However, those skilled in the art will understand, through routine tests and experiments, that the teachings described herein are also applicable to other proteases that act on mucin.

[0035] The protease acting on mucin may be selected from, for example, one (or more) of the group consisting of plant-derived proteases acting on mucin, fungal proteases acting on mucin, and bacterial proteases acting on mucin.

[0036] Some plant-derived proteolytic enzymes exhibit the same properties as bromelain, and the inventors anticipate that any plant-derived protease enzyme that has a therapeutic effect on mucin (e.g., mucin production) can be used in the present invention. The suitability of a particular plant-derived protease enzyme can be confirmed by routine experiments. For example, in some embodiments, the plant-derived protease enzyme may be selected from one or more of the group consisting of bromelain, papain (a protease extracted from papaya), fikain (a protease extracted from fig), actinidine (a protease extracted from fruits such as kiwi fruit, pineapple, mango, banana, and papaya), zingipain (a protease extracted from ginger), and Fastuosain (a cysteine ​​proteinase extracted from Bromelia fastuosa). Other proteases derived from asparagus, mango, kiwi fruit, papaya, etc., may also be used.

[0037] Furthermore, the inventors believe that both fungal proteases acting on mucin and bacterial proteases acting on mucin may be equally useful in the present invention. Suitable fungal proteases include proteases derived from Aspergillus, serine proteases (subtilisin family), aspartate proteases (pepsin family), and metalloproteases (some of which are known to have anticancer activity targeting epithelial cell walls). Suitable bacterial proteases include proteases derived from silkworm peptizymes.

[0038] In this specification, “bromelain” encompasses one or more therapeutically active substances found in extracts of the plant pineapple (Ananas Comosus) that act on mucin and may have other therapeutic effects. Bromelain is a mixture of various substances (various thiol endopeptidases and other components (such as phosphatases, glucosidases, peroxidases, cellulases, esterases, and several protease inhibitors)), and it is considered unnecessary to load all substances present in the extract onto microspheres if some of these substances loaded onto microspheres can act on mucin (for example, by causing proteolysis of mucin proteins). The bromelain used in the experiments described herein was a commercially available product from Challenge Bioproducts Co Ltd.

[0039] Adaptation The microspheres of the present invention, containing a mucin-acting protease, may be delivered to a target area in a patient's body to treat any disease or condition in which the mucin-acting protease is effective. The microspheres of the present invention, which contain a mucin-acting protease and are configured to continuously elute the protease when exposed to physiological conditions, can be used to treat any mucin-related disease, and in particular, to treat diseases in which systemic delivery of the mucin-acting protease may cause problems.

[0040] For example, as mentioned above, bromelain is known to have proteolytic activity in vitro and in vivo. Bromelain also possesses anti-edema, anti-inflammatory, antithrombotic, and fibrinolytic activity, and is expected to be a promising anticancer agent. However, systemic administration of bromelain carries risks due to its fibrinolytic and hemorrhagic effects, and for this reason, bromelain has not yet been used in the clinical treatment of cancer. On the other hand, according to the present invention, bromelain can be continuously released locally, making it possible to increase the local concentration of bromelain in the target area of ​​the patient's body without the risk of systemic toxicity. The present invention can also improve the penetration of drugs into cancer and produce a synergistic effect against the cytotoxicity of certain chemotherapeutic agents.

[0041] The present invention may be provided, for example, for the treatment of mucin-producing cancers, pseudomyxoma peritonei, cystic fibrosis, and chronic obstructive pulmonary disease. If a protease acting on mucin has further therapeutic activity, the present invention may be provided for the treatment of another condition. For example, in the case of bromelain, the present invention may be provided for the treatment of deep vein thrombosis and blood coagulation disorders.

[0042] Proteases that act on mucin cause proteolysis of mucin proteins, and therefore, when delivered to a target region in the patient's body, they act on the mucin in that target region (e.g., by degradation). Therefore, by delivering the microspheres of the present invention to a target region in the patient's body (e.g., a mucin-producing tumor), it is possible to act on the mucin in that region (e.g., around the tumor) at a minimum, resulting in some therapeutic effect (e.g., reduction of mucin mass in the target region, improved blood flow, or improved digestive capacity). Furthermore, co-administered therapeutic agents (e.g., those described later) can penetrate into the target region (e.g., the tumor) more effectively than if the mucin were unaffected. As those skilled in the art will understand, this method yields extremely useful therapeutic effects, significantly enhances the effectiveness of existing treatment plans, and allows for a reduction in the dose of co-administered therapeutic agents.

[0043] The present invention may be used in the treatment of mucin-producing cancers. In this specification, “mucin-producing” cancer means both cancers containing mucin and cancers that produce mucin. Examples of cancers containing mucin include signet ring cell carcinoma and goblet cell carcinoma. Mucin can also be present in the cytoplasm of cells that are not identified as signet ring cells or goblet cells. Examples of cancers that produce mucin include mucin-secreting cancers such as pseudomyxoma, cancers that overexpress mucin, and cancers that secrete mucin around cells (cell membranes). The secreted mucin acts as a barrier to the penetration of chemotherapeutic agents and also hinders the recognition of immune cells.

[0044] Cancers that produce mucin include, for example, lung cancer, liver cancer, pancreatic cancer, thyroid cancer, stomach cancer, appendiceal cancer, peritoneal cancer, hepatocellular carcinoma, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, mesothelioma, neuroblastoma, small intestine cancer, lymphoma, and leukemia. Many of these cancers are difficult to treat with conventional therapies. The treatment of hepatocellular carcinoma (primary liver cancer), liver metastases (secondary liver cancer), and pancreatic cancer are particularly preferred applications of the present invention. Furthermore, the microspheres of the present invention can also be used in the treatment of adenocarcinoma. In particular, the adenocarcinoma may be signet ring cell carcinoma. The microspheres of the present invention may also be used in the treatment of pseudomyxoma peritonei.

[0045] Hepatocellular carcinoma (primary liver cancer) is generally caused by hepatitis B or C infection, cirrhosis due to alcohol, non-alcoholic steatohepatitis (NASH), and other less common causes. Current treatments include liver transplantation, resection, and thermal ablation, but only a small number of patients are treatable with these effective methods. The majority of patients are treated with transcatheter arterial chemotherapy (TACE) and doxorubicin microsphere delivery, but the response rate is not very high, and survival rates are not very high for many patients.

[0046] Liver metastases (secondary tumors) can arise from various cancers, including colorectal cancer, gastric cancer, and pancreatic cancer, as well as adenocarcinomas and tumors originating from the abdomen and other parts of the body. Hepatectomy is the optimal treatment, but in certain cases, thermal ablation can now achieve comparable results. Systemic chemotherapy is widely used, but the outcomes are not very good. Delivery of irinotecan-loaded microspheres is used as palliative therapy for liver metastases originating from colorectal cancer.

[0047] By delivering the microspheres of the present invention into the patient's tumor via a nutrient artery (for example, the hepatic artery in the treatment of liver cancer), the effect of sustained release of mucin-acting proteases can be maximized, and the risk of side effects associated with systemic delivery can be reduced. Since such delivery of microspheres allows for the delivery of mucin-acting proteases to the target site, liver tumors or pancreatic tumors can be treated in a highly minimally invasive manner.

[0048] Furthermore, some proteases that act on mucin may possess anticancer activity themselves. For example, bromelain has been shown to have anticancer activity against numerous cancers, such as pancreatic cancer, hepatocellular carcinoma, prostate cancer, breast cancer, colorectal cancer, thyroid cancer, gastric cancer, appendiceal cancer, peritoneal cancer, hepatocellular carcinoma, mesothelioma, pseudomyxoma peritonei and other peritoneal cancers, ovarian cancer, lung cancer, and small intestine cancer. Papain may also be used to treat cancers such as lung cancer, pancreatic cancer, liver cancer, ovarian cancer, neuroblastoma, lymphoma, leukemia, and other solid tumors. Therefore, by delivering the microspheres of the present invention containing bromelain or papain to a mucin-producing tumor, it is possible to act on the mucin surrounding the tumor (for example, by destroying or degrading it), thereby allowing bromelain or papain to penetrate into the tumor more effectively and improving the efficacy of the anticancer activity of bromelain or papain (particularly because it can be delivered continuously over a certain period of time).

[0049] Pseudomyxoma peritonei (PMP) is a type of tumor characterized by the secretion of mucin by tumor cells, which accumulates excessively in the abdominal cavity. These tumor cells primarily originate from the appendix, but disseminated cancers of the colon, rectum, stomach, gallbladder, small intestine, bladder, lung, breast, pancreas, and ovaries can also contribute to this disease. The secreted mucin accumulates in the abdominal cavity in clumps, increasing the internal pressure in the digestive tract. This results in malnutrition, worsening the condition, and ultimately leading to death.

[0050] Traditionally, the preferred treatment for PMP patients has been open surgery to remove mucinous masses and tumor cells, followed by hyperthermic intraperitoneal chemotherapy (HIPEC). However, because this disease is progressive, the condition can worsen and lead to death, and several types of treatment may be necessary during the course of the disease.

[0051] By delivering the microspheres of the present invention into the patient's peritoneal cavity, the effects of the sustained release of mucin-acting proteases can be maximized, and the risk of side effects associated with systemic delivery can be reduced. Furthermore, because the mucin-acting proteases liquefy accumulated mucin (i.e., making it easier to eliminate mucin from the body or to aspirate the liquefied mucin from the peritoneal cavity), mucin masses can be removed in a very non-invasive manner, and cancer can also be treated (for example, by the anticancer activity of the protease or the anticancer activity of co-administered chemotherapy agents).

[0052] The present invention may be used to treat cystic fibrosis and chronic obstructive pulmonary disease. Cystic fibrosis is a disease that damages the lungs and digestive system. This disease causes lesions in cells that produce mucus, sweat, and digestive fluids, increasing the viscosity of these fluids and making them sticky, which can obstruct lumens, ducts, and pathways. Chronic obstructive pulmonary disease (COPD) is a group of lung diseases (including emphysema and chronic bronchitis) in which breathing becomes difficult due to obstruction of airflow. It is expected that an effective treatment plan can be achieved by continuously delivering mucin-acting proteases to the patient's lungs.

[0053] In some embodiments, certain proteases that act on mucin may have other therapeutic applications in addition to their properties of acting on mucin. Examples of such embodiments are described below.

[0054] Thrombus formation is a cause of various serious diseases, including myocardial infarction, coronary artery disease, stroke, extensive pulmonary embolism, and acute limb ischemia. Patients with stents may also have an increased risk of developing thrombosis. Thrombosis may be treated with anticoagulants (such as heparin or warfarin). However, such anticoagulants are only effective in inhibiting thrombus formation or the growth of existing thrombi. There is some evidence that administering proteases to patients generally reduces blood coagulation. Such therapeutic effects have been well reported, for example, with proteases such as bromelain. Therefore, in embodiments of the present invention that include bromelain, the microspheres of the present invention may be useful in treating diseases such as (at least) deep vein thrombosis, blood coagulation disorders, hemophilia, myocardial infarction, coronary artery disease, stroke, extensive pulmonary embolism, acute limb ischemia, stent thrombosis, and hematologic thromboembolism. As mentioned above, local delivery of bromelain to the appropriate area within the patient's body is far more effective and has fewer side effects than systemic delivery of bromelain.

[0055] Furthermore, when a protease that acts on mucin is used in combination with another therapeutically effective drug, a synergistic effect may be obtained. For example, when bromelain is used in combination with another mucolytic agent (details of which will be described later), the effectiveness of the microspheres of the present invention may be improved in the treatment of other mucin-related diseases such as gluer's ear infection, sputum retention, thoracic infections, and mucus retention and cellular debris associated with the placement of biliary stents / pancreatic stents.

[0056] As described herein, by combining a mucin-acting protease, such as bromelain, with another or multiple chemotherapeutic agents, a synergistic effect can be obtained in which bromelain promotes the penetration of the chemotherapeutic agent into the tumor (and deeper into it). As those skilled in the art will understand, such a mechanism may increase the effectiveness of the chemotherapeutic agent and reduce the dosage.

[0057] Microspheres The microspheres of the present invention may take any suitable form and may be made from any suitable biocompatible material or combination thereof, provided that they are capable of carrying a mucin-acting protease internally (and retaining it for a therapeutically important period without significantly adversely affecting its activity), can be delivered to a target region in the patient's body, and can sustainably elute the protease upon exposure to physiological conditions (i.e., upon reaching the target region).

[0058] Microspheres may utilize any suitable mechanism to retain mucin-acting proteases within them. In some embodiments, for example, the protease is retained by the chemical charge or functional groups of the microsphere. Alternatively (or in addition to the foregoing), the protease is retained within the microsphere by steric effects (e.g., pore size) until exposed to physiological conditions. Similarly, the elution of mucin-acting proteases from microspheres may utilize any suitable mechanism. In some embodiments, for example, the protease may leach out of the pores of the microsphere under physiological conditions. In some embodiments, the microspheres themselves may biodegrade under physiological conditions, and the protease may be continuously released as the microspheres degrade. In some embodiments, when the microspheres are exposed to physiological conditions, chemical factors (e.g., the chemical charge or functional groups of the microspheres) may change, thereby preventing the microspheres from retaining the protease and causing continuous release of the protease. In some embodiments, when microspheres are exposed to physiological conditions, the pores of the microspheres may enlarge, leading to the sustained release of proteases.

[0059] Based on the factors described in the preceding paragraph and the teachings described herein, the inventors believe it is reasonably predictable whether a particular microsphere is useful in the present invention using a specific protease that acts on mucin. To confirm this prediction, the conventional experiments described below (adapted as appropriate depending on the circumstances) can be performed.

[0060] The microspheres may contain (or be defined as) a matrix capable of supporting the aforementioned protease enzyme. Under appropriate conditions (for example, as described later), when a mucin-acting protease and microspheres are brought into contact with each other, the protease is incorporated into the matrix and supported within the microspheres.

[0061] The inventors envision that microspheres are typically purchased as commercially available products (approved for therapeutic use in humans), but it is also possible to prepare microspheres from compositions containing proteases. In such embodiments, a matrix is ​​formed around the enzyme, and in the microspheres thus formed, the enzyme is more homogeneously dispersed throughout the microsphere, allowing for sustained release of the enzyme from the microspheres at the target site over a longer period of time.

[0062] For example, the microspheres may contain (or be defined as) a hydrogel capable of encapsulating a mucin-acting protease. A suitable hydrogel is polyvinyl alcohol (PVA) hydrogel. Of the microspheres formed from PVA hydrogels, those tested by the inventors are commercially available biocompatible polyvinyl alcohol (PVA) hydrogel microspheres sold under the brand name DC Beads by Biocompatibles UK Ltd. These microspheres are manufactured from a sulfonate-modified polyvinyl alcohol (PVA) hydrogel and have been previously used for the controlled encapsulation and delivery of the chemotherapeutic agents doxorubicin or irinotecan, and are also used in transarterial chemoembolization (TACE). Another type of commercially available DC Beads is described, for example, in WO2001 / 68722 under the name "Hydrogel biomedical articles" (the contents of this document are incorporated herein by reference). DC Beads are available in a variety of sizes, including 70-150 μm, 100-300 μm, 300-500 μm, and 500-700 μm.

[0063] Another suitable hydrogel is poly(vinyl alcohol-co-sodium acrylate) hydrogel. Among the microspheres formed from poly(vinyl alcohol-co-sodium acrylate) hydrogel, those tested by the inventors are called HepaSphere. TM These are commercially available microspheres sold under the brand name "Microsphere." Hepasphere microspheres are manufactured from vinyl acetate and methyl acrylate in an acidic environment. Anticancer drugs such as doxorubicin can be loaded into Hepasphere microspheres, and these microspheres are suitable for delivery to patients using the TACE method described above. Hepasphere microspheres are available in various sizes, including 30-60 μm, 50-100 μm, 100-150 μm, and 150-200 μm.

[0064] Another suitable hydrogel is one comprising a hydrogel core made of polysodium methacrylate and an outer shell of poly(bis[trifluoroethoxy]phosphazene). Of the microspheres formed from such hydrogels, those tested by the inventors include Embozene TANDEM from Boston Scientific. TM These are commercially available microspheres sold under the trademark name [brand name]. Similar to the aforementioned microspheres, doxorubicin-HCl or irinotecan-HCl can be loaded into Embozene TANDEM microspheres for use in the TACE method. Embozene TANDEM is available in sizes of 40±10 μm, 75±15 μm, or 100±25 μm.

[0065] However, as mentioned above, the sustained-release molecules that are said to be able to be supported inside DC Beads, Hepasphere microspheres, and Embozene TANDEM microspheres are all positively charged, relatively small (approximately 600 Da) molecules, and it is believed that other drugs cannot be properly retained within the microspheres. Therefore, the fact that proteolytic enzymes such as bromelain and papain can be supported inside the microspheres, stably retained within these microspheres, and then sustainably eluted from these microspheres is truly astonishing.

[0066] Another commercially available microsphere that the inventors are aware of and believe would be suitable for use in the present invention is the microsphere sold by Terumo Europe NV under the trademark name LifePearl. This microsphere consists of a hydrogel network of poly(ethylene glycol) and 3-sulfopropyl acrylate. The inventors also believe that microspheres formed from a poly(lactic acid-co-glycolic acid) (PLGA) hydrogel network and microspheres formed from a polylactic acid (PLLA) hydrogel network are also suitable for use in the present invention.

[0067] In some embodiments, the microspheres of the present invention may include an outer coating, which imparts advantageous properties to the microspheres. For example, it may be advantageous to coat the microspheres with a coating that dissolves (or is removed) before the protease enzyme begins to elute. Thus, for example, after delivery of the microspheres, time may be required for them to reach the tumor site and for enzyme elution to begin. Alternatively, it may be advantageous to coat the microspheres with a coating that protects them after delivery until appropriate physiological conditions (e.g., pH and ion concentration in the target region) are reached.

[0068] For example, the inventors have found that microspheres having an outer coating made of alginate can delay the onset of sustained release of protease enzymes after exposure to physiological conditions. Other coating agents, such as those containing chitosan, may also be useful in the present invention.

[0069] The inventors anticipate that glass microspheres, resin microspheres, and ceramic microspheres will also be useful in the present invention. For example, glass microspheres sold under the trademark name TheraSphere® are used in radiotherapy for hepatocellular carcinoma (HCC) in several countries. These glass microspheres (20-30 μm in diameter) are radioactive and, when injected into the arteries supplying nutrients to liver tumors, cause embolisms in the hepatic capillaries, delivering high doses of radiation from yttrium-90 to the malignant tumors. The inventors believe that TheraSphere glass microspheres can be configured to carry mucin-acting proteases for use as taught in the present invention. Similarly, ceramic microspheres sold under the trademark name Ceramispheres, for example, or resin microspheres sold under the trademark name SIR-spheres®, for example, are also considered to be able to carry mucin-acting proteases for use as taught in the present invention.

[0070] In some embodiments, it is conceivable that different types of microspheres may be combined and co-administered to the patient. In different types of microspheres, the mucin-acting proteases may be the same or different, and other active agents, as described later, may be contained in the microspheres. Different types of microspheres may differ in terms of size, particle size distribution and / or composition.

[0071] For the microspheres to be useful in this invention, it is desirable that they are generally spherical and on the order of micrometers in size. Spherical microspheres are suitable for embolization, for example, because they offer low resistance to blood flow when delivered through blood vessels. Furthermore, spherical particles of a specific size can be dense at a particular volume.

[0072] Microspheres can be of any size that defines them as microspheres (when measured by diameter), and the size of microspheres useful for a particular application depends on several factors, including the characteristics and amount of mucin-acting proteases to be carried within the microspheres (for example, the more proteases carried, the more microspheres are needed), and the delivery route of the microspheres to the patient (for example, in embolization therapy, the size of the blood vessels to which the embolization is induced determines the size of the microspheres required). As will be understood by those skilled in the art, each batch of microspheres is always specified within a range of diameters.

[0073] In some embodiments, for example, the diameter of the microspheres may be about 30 to about 700 μm, and for intraperitoneal delivery and application in the peritoneal cavity, a diameter slightly less than 1000 μm may be appropriate. In some embodiments, for example, the diameter of the microspheres may be about 30 to about 500 μm, about 50 to about 400 μm, about 60 to about 300 μm, about 80 to about 200 μm, about 60 to about 100 μm, about 50 to about 100 μm, about 40 to about 80 μm, about 30 to about 60 μm, about 30 to about 50 μm, or about 40 to about 100 μm. In some embodiments, for example, the diameter of the microsphere may be about 700 μm, about 600 μm, about 500 μm, about 400 μm, about 300 μm, about 200 μm, about 100 μm, about 80 μm, about 70 μm, about 60 μm, about 50 μm, about 40 μm, or about 30 μm.

[0074] Generally, larger microspheres are more useful for delivery via intracavitary routes (e.g., intraperitoneal delivery for the treatment of pseudomyxoma peritonei (PMP) and other peritoneal cancers), and a greater amount of mucin-acting proteases (and optionally other active agents) is beneficial. Also, generally, smaller microspheres are more useful for intra-arterial delivery routes, where microspheres travel through the arteries and form embolisms in the target area.

[0075] The microspheres of the present invention are configured to continuously elute a mucin-acting protease upon delivery to a target region. The mechanism by which the protease is eluted is not important, as long as continuous elution is achieved. As mentioned above, the microspheres may have, for example, numerous pores capable of eluting the protease. In some embodiments, the supported protease may be exposed by the degradation of the microspheres themselves under physiological conditions.

[0076] In this specification, “sustained” means that one or more proteases acting on mucin, contained within the microsphere, are eluted over a therapeutically beneficial period of time. When microspheres carrying mucin-acting proteases are first exposed to physiological conditions, a “burst release” often occurs, in which a certain proportion of the proteases are rapidly eluted. Subsequently, the rate of protease elution decreases, and the remaining proteases within the microsphere elute over several hours, days, or even weeks. The rate of protease release does not have to be constant throughout the entire elution period.

[0077] The rate at which proteases elute from microspheres, and the time it takes, can vary depending on the specific application. However, it is generally desirable that the protease be released for at least the time required for cell replication in the target region. Thus, proteases (and other active agents contained in the microspheres) are thought to inhibit cell replication and induce cell death.

[0078] Typically, the rate at which proteases are delivered to and eluted from the target region must be taken into consideration. For example, when delivered to a region with relatively high blood flow, the protease is expected to be eliminated more rapidly than when delivered to a region with relatively low blood flow (however, it should be noted that blood flow may be significantly impaired by embolization). The release rate of proteases from microspheres should be adjusted to take these factors into account.

[0079] In a particular embodiment, for example, a mucin-acting protease may be continuously released from the microspheres delivered to the target region for up to about 120 hours or more. In some embodiments, for example, the mucin-acting protease may be released from the microspheres for about 10 to about 120 hours, about 20 to about 100 hours, about 30 to about 80 hours, about 10 to about 50 hours, about 15 to about 40 hours, about 10 to about 30 hours, or about 10 to about 20 hours.

[0080] In the microspheres of the present invention (which are size-constrained and carry a protease), the amount of mucin-acting protease may be any amount that can produce a therapeutic effect on the relevant disease. The amount of protease that can be carried in a particular microsphere usually needs to be determined on a case-by-case basis based on empirical rules as well as release characteristics. The amount of protease carried in the microsphere and subsequently delivered into the patient's body is determined depending on various factors such as the characteristics of the disease being treated, the sustained release rate of the protease, and the time required for protease release.

[0081] In some embodiments, it may be necessary to deliver a relatively large amount of microspheres to obtain specific protease release characteristics and / or to deliver a specific amount of protease. When delivering bromelain using microspheres with a diameter of 300-500 μm, for example, approximately 1800 μg of bromelain may be loaded onto 60 μL of microspheres. For example, when treating a tumor localized to a specific site with bromelain, the characteristics and size of the tumor are the main factors that influence the required amount of bromelain-loaded microspheres.

[0082] The microspheres of the present invention can be delivered to a target area within a patient's body. A delivery method may be used in the present invention that allows the microspheres to reach the target area substantially intact and retain as little of the mucin-acting protease (or other drug) they contain as possible.

[0083] Typically, microspheres are configured for local delivery to a target area (determined primarily on the basis of the disease being treated). Such local delivery maximizes the number of microspheres delivered to the area of ​​the body where delivery is needed (and consequently maximizes the amount of proteases acting on mucin), thereby maximizing therapeutic efficacy and minimizing the potential for side effects. For example, microspheres may be configured for intra-arterial, intra-lesional, intraperitoneal, or intracavitary delivery to the patient (e.g., delivery to the patient's peritoneal or pleural cavity). Other intracavitary delivery routes include intranasal and intrabronchial delivery routes (considered useful for treating conditions such as cystic fibrosis), intravesical, or intraductal delivery routes (considered useful for conditions such as cholangiocarcinoma).

[0084] The target area within the patient's body may be a tumor. The tumor may be located, for example, in the patient's abdomen (e.g., the patient's pancreas, liver, colon, ovaries, or prostate). The tumor may also be located, for example, in the patient's lungs. Similar to the transarterial chemoembolization (TACE) described above, by administering microspheres to the blood vessels supplying such a tumor, the local concentration of mucin-acting proteases can be increased during the sustained administration period. If a longer-term sustained release is beneficial for treatment, additional doses of microspheres may be delivered.

[0085] In an alternative method for treating pseudomyxoma peritonei or other peritoneal cancers, the microspheres of the present invention may be delivered by intraperitoneal injection. As described above, larger microspheres can be delivered to body cavities such as the patient's peritoneal cavity, which means that high doses of protease (or other drugs) can be administered.

[0086] In an alternative method, when treating thrombosis such as deep vein thrombosis, the microspheres of the present invention may be delivered to the thrombus site by injection.

[0087] The microspheres of the present invention are configured to continuously elute proteases when exposed to physiological conditions. As those skilled in the art will understand, by using various administration methods, the microspheres can be delivered to various parts of a patient's body (e.g., intraarteries or body cavities), in which case the microspheres will be exposed to various physiological conditions. For example, while a patient's body temperature is considered to be nearly constant throughout the body, (e.g.) pH and electrolyte concentrations may differ between arteries and body cavities. Therefore, those skilled in the art will understand that the microspheres of the present invention can be adapted to physiological conditions by evaluating these parameters (by conducting prior tests as necessary).

[0088] Further drugs The proteases contained in the microspheres of the present invention that act on mucin are effective on their own (i.e., as described above, they are effective against diseases involving mucin), but they may be used in combination with further agents. Specific examples of such further agents will be described later. If necessary (or if beneficial), the amount of such further agents may be determined by conducting very routine tests and experiments as needed.

[0089] In addition to the mucin-acting protease, further agents may be incorporated into the microspheres themselves (i.e., co-carried with the protease and further agents). Alternatively, further agents may be delivered to the patient (i.e., the target area) in combination with the microspheres (the microspheres and further agents may be administered together via the same or different routes, or separately (e.g., sequentially in any order)). The further agents may, for example, be present in the carrier of the microspheres or chemically bound to the surface of the microspheres. In other ways, or in addition to these, further agents may be incorporated into microspheres separate from the microspheres containing the mucin-acting protease, and these microspheres may be delivered to the patient in combination (either may be administered first, second, or simultaneously). Local delivery of microspheres containing a mucin-acting protease (such as bromelain) to the tumor may be combined, for example, with a systemic chemotherapy regimen (i.e., oral or intravenous delivery of chemotherapeutic agents). In some embodiments, additional drugs may be delivered systemically (for example, by oral or intravenous administration) before, during, or after delivery of the microspheres.

[0090] Further agents may be selected from, for example, one or more of the group consisting of chemotherapeutic agents, radiotherapy agents, other mucolytic agents, and contrast agents. Each of these further agents is described in detail below.

[0091] Chemotherapy agents are pharmacological agents used in the treatment of cancer. Examples of chemotherapeutic agents considered useful in the present invention are described in WO2014 / 094041 (the contents of this document are incorporated herein by reference). Specific examples of chemotherapeutic agents that may be used in the present invention include, for example, gemcitabine, paclitaxel, docetaxel, doxorubicin, irinotecan, mitomycin C, oxaliplatin, carboplatin, 5-fluorouracil (or its analogues), and / or cisplatin. The inventors have previously reported that desirable synergistic effects are observed when some of these chemotherapeutic agents are co-administered with bromelain or another mucolytic agent, and such synergistic effects are assumed to be available in the present invention. In particular, doxorubicin, gemcitabine, 5-fluorouracil, mitomycin C, paclitaxel, taxol, oxaliplatin, and cisplatin have all been observed by the inventors to exhibit synergistic effects with bromelain.

[0092] For example, as mentioned above, regardless of whether one or more chemotherapeutic agents are delivered systemically, co-supported on the same microsphere, or supported on separate microspheres (simultaneous or sequential administration), it is expected that the efficacy of chemotherapy will be improved by intra-arterial administration of bromelain supported on microspheres. The inventors believe that delivery of bromelain supported on microspheres to target regions can serve as an alternative treatment for hepatocellular carcinoma, i.e., primary liver cancer, liver metastases, and pancreatic cancer, and can enhance the antitumor effects of chemotherapeutic agents such as doxorubicin.

[0093] For example, radiotherapy agents may be co-delivered with microspheres containing mucin-acting proteases to specify the delivery site and / or improve the effectiveness of the mucin-acting protease. For example, bromelain is a known PARP inhibitor and, when co-administered with a radioactive material, may interfere with the repair of radiation-damaged DNA, leading to local cell death.

[0094] Theoretically, radiotherapy agents can be co-loaded within microspheres containing proteases, but it is necessary to develop a method that ensures the proteases are not damaged and the therapeutic activity is not affected by co-loading the radiotherapy agent. Alternatively, the radiotherapy agent may be delivered to the patient separately from the protease-containing microspheres to minimize radiation damage to the proteases that act on mucin.

[0095] For example, the radiotherapy agent may be provided separately by loading it onto glass spheres, resin spheres, or ceramic spheres, such as microspheres marketed under the trademark names QuiremSpheres® or SIR-spheres® Y-90 resin microspheres. Alternatively (or in addition to the foregoing), the radiotherapy agent may be co-delivered by external beam radiation therapy or brachytherapy, which can enhance tumor sensitivity.

[0096] As mentioned above, mucolytics act on mucus (for example, by disruption or dissolution) and are currently used to alleviate respiratory distress. Mucin-acting proteases are a type of mucolytic, but the mucolytics described herein are defined in the context of this invention as not being enzymatic agents and, in this respect, differ from mucin-acting proteases. Combining such mucolytics with mucin-acting proteases may be advantageous, and some such embodiments are described herein.

[0097] In WO2014 / 094041, some of the inventors described the beneficial effects of bromelain when administered together with a mucolytic agent (such as N-acetylcysteine) or a chemotherapeutic agent. The combination of bromelain and a mucolytic agent has been found to significantly enhance the efficacy and cytotoxicity of chemotherapeutic agents against mucin-producing cancer cells, exert direct antitumor and inhibitory effects on the viability and proliferation of cancer cells, significantly influence mucin production by tumors, and be highly effective in liquefying mucin produced by tumors. The advantages of bromelain include improved penetration of chemotherapeutic agents into cancer cells, improved penetration of chemotherapeutic agents into the tumor stroma, and synergistic effects with certain chemotherapeutic agents. In addition, bromelain has the advantage of facilitating entry into tumors, particularly in tumors with a fibrous capsule or tumors surrounded by adhesions.

[0098] In WO2017 / 063023 (the contents of which are incorporated herein by reference) by some of the inventors, a surprising and unexpected synergistic effect of bromelain is observed when bromelain is administered together with the mucolytic agent cysteamine (or its metabolites, pharmaceutically acceptable salts, solvates, or prodrugs). This combination has proven to be highly effective in the treatment of solid or hard tumors.

[0099] When the microspheres of the present invention contain a mucolytic agent, the microspheres of the present invention are thought to be even more effective in treating mucin-related diseases such as mucin-producing cancers (as described above), pseudomyxoma peritonei, gluyer's ear infection, cystic fibrosis, sputum retention, thoracic infections, mucus retention and cellular debris associated with biliary / pancreatic stent placement; as well as thrombosis-related diseases such as hemophilia, myocardial infarction, coronary artery disease, stroke, extensive pulmonary embolism, acute limb ischemia, stent thrombosis, or hematologic thromboembolism. These diseases can also be treated with mucin-acting proteases alone, but the effectiveness of treatment may be improved by co-administration of additional mucolytic agents.

[0100] The mucolytic agent may be, for example, a thiol-containing mucolytic agent that reduces or breaks the disulfide bonds of mucin. Specific examples of mucolytic agents include N-acetylcysteine ​​("NAC"), cysteamine, nasistrine, mercaptoethanesulfonate, carbocysteine, N-acystelyn, erdosteine, dorunase α, gelzolin, thymosin P4, dextran, and heparin. NAC is also an antioxidant and antigenotoxic agent, and its long-term safety at high doses has been well established in human patients, mainly in respiratory diseases. Other mucolytic agents are described in WO2014 / 094041 and WO2017 / 063023 (these references are incorporated herein by reference).

[0101] Microspheres may be encapsulated with a contrast agent if it offers advantages such as enabling detection of the location of delivered microspheres or precise identification of the administration site. The fluorescence of the contrast agent is thought to facilitate visual confirmation of the precise site and easier verification of the dose distribution.

[0102] Method for forming microspheres The present invention further provides a method for immobilizing a mucin-acting protease within microspheres. This method comprises the steps of: adding microspheres to a solution having an acidic pH and optionally an ionic strength equivalent to that of a target region in the patient's body; mixing the solution containing the microspheres with a solution containing a mucin-acting protease; and shaking the mixture for a sufficient time for the mucin-acting protease to be immobilized within the microspheres.

[0103] The inventors have discovered that the pH at which mucin-acting proteases are loaded into microspheres can affect the amount of protease that can be loaded, and thus affect the release rate of the protease when subsequently exposed to physiological conditions. In the case of bromelain, for example, it was found that lowering the pH increased the amount that could be loaded into the microspheres, and slowed the release rate after delivery to the target region. Although not bound by any particular theory, the inventors hypothesize that these effects are due to the increase in the net charge of bromelain at lower pH, and / or that lowering the pH affects the pore size, thereby influencing the release pattern of the microspheres.

[0104] In this regard, preliminary experiments by the inventors have shown that when the protease acting on mucin is bromelain, it may be advantageous to support the protease at a low pH such as pH 2 or pH 2.5.

[0105] Similarly, the inventors discovered that the solvent used to support mucin-acting proteases within microspheres can affect the release rate of the proteases when subsequently exposed to physiological conditions.

[0106] The inventors have found that by using a loading solvent having an ion concentration and acidic pH that is typically expected to be equivalent to that of a target region in the patient's body, proteases can be successfully loaded into microspheres, and subsequently, the proteases can be sustainedly released. Specific examples of loading bromelain or papain into particular microspheres are described in more detail in the examples.

[0107] Pharmaceutical composition The present invention further provides a pharmaceutical composition comprising a microsphere (e.g., the aforementioned microsphere) for delivery to a target area in a patient's body and a pharmaceutically acceptable carrier, wherein the microsphere is configured to encapsulate a mucin-acting protease and to continuously elute the mucin-acting protease upon exposure to physiological conditions.

[0108] The pharmaceutically acceptable carrier used in the pharmaceutical composition of the present invention is determined according to the route of administration of the pharmaceutical composition. Examples of liquid formulations include liquids, suspensions, and emulsions, and examples include aqueous solutions or aqueous propylene glycol solutions for parenteral injection, intraperitoneal administration, or intraperitoneal injection. Suitable pharmaceutically acceptable carriers used in the pharmaceutical composition of the present invention include physiological saline, dextrose solution, and Ringer's solution.

[0109] The liquid and aerosol formulations containing microspheres of the present invention are considered useful, for example, for intranasal administration in the treatment of cystic fibrosis. Examples of aerosol formulations suitable for inhalation include liquid formulations and powdered solids, which may be combined with a pharmaceutically acceptable carrier such as a compressed inert gas (e.g., nitrogen gas).

[0110] Pharmaceutical compositions suitable for delivery to patients may be prepared immediately before delivery into the patient's body, or they may be prepared in advance and stored appropriately until delivery.

[0111] The pharmaceutical compositions and pharmaceuticals of the present invention may contain pharmaceutically acceptable carriers, adjuvants, excipients and / or diluents. These carriers, diluents, excipients and adjuvants must be "acceptable" in terms of compatibility with the other components of the pharmaceutical composition or pharmaceutical of the present invention and their delivery method, and are generally harmless to the recipient. Examples of pharmaceutically acceptable carriers or diluents considered suitable for use in some embodiments include: deionized water or distilled water; physiological saline; vegetable oils such as peanut oil, safflower oil, olive oil, cottonseed oil, and corn oil; sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil, peanut oil, or coconut oil; silicone oils containing polysiloxanes such as methylpolysiloxane, phenylpolysiloxane, and methylphenylpolysiloxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin, and squalane; methylcellulose, ethylcellulose, carboxymethylcellulose, and calcamine. Examples of carriers include, but are not limited to, cellulose derivatives such as sodium methylcellulose and hydroxypropyl methylcellulose; lower alkanols such as ethanol and isopropanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols such as polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol, and glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate, and ethyl oleate; polyvinylpyrrolidone; agar; tragacanth gum or acacia gum; and petrolatum. Typically, one or more carriers constitute about 10% to about 99.9% by weight of the pharmaceutical composition or drug of the present invention.

[0112] Some of the components in the microspheres or pharmaceutical compositions of the present invention may be provided in the form of their metabolites, pharmaceutically acceptable salts, solvates, or prodrugs, if it is appropriate to do so.

[0113] In this invention, the term "metabolites" in the components of the microspheres refers to metabolic intermediates and metabolic products.

[0114] In the context of pharmaceutically acceptable carriers and excipients, "pharmaceutically acceptable" means that the specific compound is pharmacologically acceptable and is not substantially toxic to the target recipient.

[0115] "Pharmacologically acceptable salts" refer to conventional acid-addition or base-addition salts that retain the biological efficacy and properties of the original component and are formed from appropriate and non-toxic organic acids, inorganic acids, organic bases, or inorganic bases. Examples of acid-addition salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, and salts derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, and fumaric acid. Examples of base-addition salts include salts derived from ammonium, potassium, sodium, and quaternary ammonium hydroxides (such as tetramethylammonium hydroxide). The technique of chemically modifying pharmaceutical compounds (i.e., drugs) to convert them into salt forms to improve the physical stability, chemical stability, hygroscopicity, fluidity, and solubility of compounds is well known to pharmacists. For example, see pp. 196 and pp. 1456-1457 of H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) (this reference is incorporated herein by reference).

[0116] It is also conceivable that some of the components in the microspheres or pharmaceutical compositions of the present invention may be “prodrugs” or “solvates.” A “prodrug” means a compound (e.g., a drug precursor) that is converted in vivo to produce the compound desired by the present invention, or its metabolites, pharmaceutically acceptable salts, or solvates. This conversion can occur through various mechanisms (e.g., metabolism or chemical processes). Considerations regarding the use of prodrugs are discussed in T. Higuchi and W. Stella, “Prodrugs as Novel Delivery Systems,” Vol. 14, ACS Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0117] Treatment method The present invention further provides methods for treating mucin-related diseases and conditions in which the activity of mucin-acting proteases is therapeutically effective. For example, bromelain has therapeutically effective activity for treating mucin-producing cancers, pseudomyxoma peritonei, cystic fibrosis, chronic obstructive pulmonary disease, deep vein thrombosis, and blood coagulation disorders. Furthermore, co-administration of bromelain with another chemotherapeutic agent further enhances its efficacy because it facilitates the penetration of the chemotherapeutic agent into the tumor. Papain has therapeutically effective activity for treating several mucin-producing cancers and other conditions. Other mucin-acting proteases are expected to have similar activity, and benefits can be obtained by sustained local delivery of such proteases as described herein (for example, problems associated with systemic delivery can be solved or improved).

[0118] The present invention provides a method for treating patients with mucin-producing cancers, pseudomyxoma peritonei, cystic fibrosis, and chronic obstructive pulmonary disease (including other diseases or conditions that can be treated depending on the type of protease carried on the microspheres, as described above). The method comprises administering to a patient a therapeutically effective amount of microspheres (e.g., the aforementioned microspheres) that carry a mucin-acting protease inside, the microspheres configured to continuously release the protease after administration.

[0119] As mentioned above, bromelain has various therapeutic benefits, including anticancer activity, but it has not been able to enter clinical trials due to side effects when administered systemically. However, microspheres containing bromelain can specifically target areas of the body that require treatment, allowing for local delivery of a relatively small amount of bromelain (compared to the amount required for systemic administration), and thus significantly reducing side effects. Cancers for which microspheres containing bromelain are effective include, as mentioned above, cancers with a large blood supply, such as hepatocellular carcinoma, pancreatic cancer, and colorectal cancer.

[0120] The aforementioned method may include intra-arterial delivery of microspheres, in which a catheter is pre-positioned as close as possible to the tumor's supplying vessels (to avoid occluding vessels leading to other sites), and the microspheres are injected through this catheter. In this way, the microspheres are delivered directly into (or very close to) the tumor, forming an embolism at the delivery site and continuously releasing bromelain (or other proteases acting on mucin). Such a method is similar to the method currently used in transarterial chemoembolization (TACE) described above.

[0121] Direct injection of microspheres carrying mucin-acting proteases into the tumor (intra-lesional injection) is also considered a useful delivery method. In this way, mucin-acting proteases can be delivered directly to the tumor in relatively large doses at therapeutically effective levels, maximizing their efficacy while minimizing the risk of side effects associated with delivery methods that are largely untargeted.

[0122] This method may include intracavitary delivery of microspheres into the patient's body cavity (e.g., the abdominal cavity or pleural cavity). As described above, such a method is considered particularly useful for the treatment of pseudomyxoma peritonei and other peritoneal cancers, or cancers associated with the lung or pleura. The microspheres or pharmaceutical composition of the present invention may be administered to the recipient by routes such as intrathecal injection, subcutaneous injection, or intramuscular injection.

[0123] In this specification, “therapeutic dose” means an amount of the drug or composition used in the present invention that is sufficient to produce the desired therapeutic effect without exhibiting toxicity. The exact dose required will vary from subject to subject depending on various factors, such as the animal species being treated, the age and general condition of the subject, the severity of the disease being treated, the specific drug being administered, and the method of administration. Therefore, it is impossible to define an exact “effective dose” that is applicable to all embodiments. However, in any case, an appropriate “effective dose” can be determined by a person skilled in the art simply by performing routine experiments.

[0124] Typically, the microspheres and pharmaceutical compositions of the present invention can be administered in a manner that is appropriate to the route of administration and the recipient's physical characteristics (such as health condition), and that induces the desired effect. For example, the appropriate dosage may be determined based on various factors, including (but not limited to) the physical characteristics of the subject (e.g., age, weight, sex), the use of the drug, composition, or pharmaceutical as monotherapy or adjunct therapy, the progression of the disease or condition being treated (i.e., the state of the illness), and other factors readily apparent to those skilled in the art. Various general considerations when determining the appropriate dosage of drugs, compositions, and pharmaceuticals are described, for example, in Gennaro et al. (Eds), (1990), “Remington's Pharmaceutical Sciences”, Mack Publishing Co., Easton, Pennsylvania, USA, and Gilman et al., (Eds), (1990), “Goodman And Gilman's: The Pharmacological Bases of Therapeutics” Pergamon Press.

[0125] The microspheres of the present invention may typically be administered in an amount effective to achieve the desired objective. More specifically, the microspheres of the present invention may be administered in a therapeutically effective dose, meaning an amount effective in preventing the onset of the target disease or condition, or an amount effective in alleviating existing symptoms of the target disease or condition. Determining the effective dose is readily possible for those skilled in the art. For example, the therapeutically effective dose of a particular microsphere can be estimated first using a cell culture assay. For instance, a specific dose can be formulated for an animal model so that a blood concentration range, including the IC50 determined by cell culture, is achieved. Using such information, a more accurate determination of a useful dose for mammalian subjects, such as humans, can be made.

[0126] Typically, the microspheres of the present invention may be administered to a patient in a specific amount that yields a therapeutic effect. The characteristics of the therapeutic effect depend on factors such as the mucin-related disease or condition being treated, and the proteases that act on the administered mucin. For example, when treating a tumor, the inventors believe it is more appropriate to consider factors such as tumor volume rather than body weight when determining the appropriate dosage. For example, the average size of a pancreatic tumor is approximately 20 cm². 3 ±16cm 3 It is presumed that the concentration of bromelain (measured in vitro) required to exert a sufficient cytotoxic effect on pancreatic cells must be greater than 20 μg / mL, therefore 20 cm 3 For tumors of this size, it is necessary to deliver a sufficient amount of bromelain from the microsphere to locally deliver more than 400 μg of bromelain (note that this is the amount when bromelain is used alone, and the required amount may be less when used in combination with chemotherapeutic agents). Also, as mentioned above, the clearance rate of protease from the target region must be taken into consideration when calculating the amount and rate of protease delivery to the tumor.

[0127] Typically, in therapeutic applications, treatment may be administered during the duration of the disease or condition. Furthermore, those skilled in the art will readily understand that the optimal dosage and interval between doses can be determined according to the characteristics and severity of the disease or condition being treated; the form of administration, route of administration, and site of administration; and the characteristics of the specific target being treated. The optimal dosage can be determined using prior art. Those skilled in the art will also readily understand that conventional tests for determining treatment plans can be used to verify whether the administration plan is optimal.

[0128] When administering two or more substances (for example, drugs or pharmaceuticals) "together" to a subject, they may be administered simultaneously as a single composition, simultaneously as separate compositions, or at different times as separate compositions. If administered at different times, one may be administered first, and the other may be administered later.

[0129] For example, certain embodiments of the present invention may involve administering the microspheres or pharmaceutical composition in multiple doses. Therefore, the therapeutic methods described herein encompass administering multiple doses to a subject over a predetermined period. In some embodiments, the methods of the present invention may include administering a priming dose, followed by a booster dose. In some embodiments, the microspheres or pharmaceutical composition of the present invention may be administered at least once, twice, three times, or more times.

[0130] The therapeutically effective dose refers to the amount of microspheres (and mucin-acting proteases) required to alleviate the symptoms of the target during treatment and / or to prolong its survival. The toxicity and therapeutic efficacy of enzymes and other compounds can be measured by standard pharmaceutical assays in cell culture and / or by experimental animals (for example, by determining the LD50 (the dose at which 50% of the population dies) and ED50 (the dose at which 50% of the population is therapeutically effective)). The ratio of the toxic dose to the therapeutic dose is a therapeutic index that can be expressed as the ratio of LD50 to ED50. Drugs, compositions, and pharmaceuticals exhibiting a high therapeutic index are preferred. Data obtained from cell culture assays and / or animal studies may be used to determine the dose range for use in humans or other mammals. The dose of such compounds is preferably within a blood concentration range that includes the ED50 and is non-toxic or has little toxicity. The dose may vary within the above range depending on the dosage form and route of administration used. The precise prescription, route of administration, and dosage can be easily selected by the attending physician, taking into account the patient's condition (see, for example, the description by Fingl et al. (1975) in “The Pharmacological Basis of Therapeutics”, Ch. 1 p.1 (this document is incorporated herein by reference)).

[0131] The present invention may be used to treat appropriate patients or subjects. In some embodiments, the patient is a mammalian subject. Typically, the patient is a human patient, but other subjects may also benefit from the present invention. For example, the subject may be a mouse, rat, dog, cat, cow, sheep, horse, or other mammal that is socially, economically, or researchly important. [Examples]

[0132] Experimental results The inventors' experiments, which successfully eluted mucin-acting proteases in the form of bromelain or papain by supporting them in commercially available microspheres and then exposing them to physiological conditions, are described below.

[0133] Example 1 - Supporting bromelain or papain in DC beads, which are microspheres made of polyvinyl alcohol (PVA) hydrogel. The following experiments were conducted using PVA hydrogel microspheres, commercially available under the trademark name DC Beads®, in two sizes: 100-300 μm and 300-500 μm. The loading of bromelain into these beads and its subsequent release were investigated.

[0134] Experiment 1: 300-500 μm PVA beads (DC Beads) Three solutions containing bromelain at concentrations of 3 mg / ml, 5 mg / ml, or 10 mg / ml were each added to 100 μl of PVA hydrogel beads (300-500 μm DC beads), and incubated at room temperature (23°C) for 24 hours with vigorous shaking. After 24 hours of incubation, the amount of bromelain remaining in each solution was analyzed to determine how much bromelain was supported within the beads.

[0135] Following this analysis, the beads from each batch supporting bromelain were gently washed with distilled water, and then the bromelain was released in 5 ml of distilled water at 37°C. 250 μl samples were taken from each solution at regular intervals, and the same amount of fresh distilled water was added to each solution. The solutions taken at each time point were analyzed by azocasein assay to measure the amount of bromelain released from the beads. The results of this analysis are shown in Table 1 and Graph 1 below. [Table 1] JPEG0007862932000002.jpg77149

[0136] As can be seen from the graph, after the initial burst release, the remaining bromelain was observed to be continuously released from the microspheres, and this trend was particularly noticeable in microspheres loaded with low concentrations of bromelain. From these results, the amount of bromelain that can be loaded into DC beads was determined, and it was found that the more bromelain loaded, the greater the burst release.

[0137] Experiment 2: 100-300 μm PVA beads (DC Beads) In the same manner as in Experiment 1, 200 μl of bromelain solution containing 1.0 mg / ml or 3.0 mg / ml of bromelain in distilled water was added to 60 μl of PVA hydrogel beads (100-300 μm DC beads), and incubated at room temperature (23°C) for 24 hours with shaking.

[0138] Beads were collected from 200 μl of bromelain solution, washed, and then added to 5 ml of pH 7.0 distilled water to initiate elution. At regular intervals, 250 μl was taken from the solution, and the same amount of fresh distilled water was added to each solution. Bromelain was analyzed using the solutions taken at each time point, in the same manner as in Experiment 1. The results of this analysis are shown in Table 2 and Graph 2 below.

[0139] The bromelain release rate is calculated from the linear increase after the first 30 minutes in the bromelain plotted against elapsed time (graph). In this experiment, the burst release was found to be relatively small, which is probably due to washing the beads before releasing the bromelain in distilled water. [Table 2] JPEG0007862932000004.jpg102155

[0140] The experimental results described in Experiments 1 and 2 demonstrated that a relatively large amount of bromelain could be loaded into DC beads of various sizes, and that the loaded bromelain was released continuously (although an initial burst release was observed).

[0141] Experiment 3: Measurement of the durability and proteolytic activity of bromelain eluted from DC beads (300-500 μm) made of PVA hydrogel. Bromelain was immobilized within DC beads (300-500 μm) using the following method. After washing 80 μl of PVA hydrogel beads with 1.0 ml of distilled water, the beads were immersed in 200 μl of bromelain solution (1.0 mg / ml, pH 3.7) and incubated at 23°C for 24 hours with vigorous shaking. 100 μl of this bromelain solution was taken, and the proteolytic activity of bromelain was analyzed using an azocasein assay. This analysis indicated that a total of 197 μg (almost 100%) of bromelain was immobilized within the microspheres.

[0142] Next, the bromelain supported in this manner was eluted from DC beads, which are PVA beads, as follows. The beads supporting the bromelain were carefully collected and immersed in 5.0 ml of distilled water (pH 7.0) in a 50 ml centrifuge tube. The centrifuge tube containing the beads was placed in a 37°C water bath and shaken continuously. At intervals of 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, and 8 hours, 250 μL samples were taken from the solution in the centrifuge tube, and the same amount of distilled water adjusted to pH 7.0 was replenished with each sample. The bromelain was analyzed using the solutions taken at each time point. The results are shown in Graph 3 below. JPEG0007862932000005.jpg78150

[0143] As can be seen from Graph 3 above, analysis using the azocasein assay revealed that bromelain was released from the beads for approximately 77 hours, after which no proteolytic activity was observed. This suggests that the remaining bromelain was either trapped within the hydrogel beads or continued to be released from the beads, but lost its proteolytic activity, likely due to being maintained at 37°C for more than 76 hours or due to the effects of shaking. However, the fact that bromelain, which is relatively sensitive to heat, maintained its proteolytic activity at 37°C for approximately 80 hours was a surprising finding for the inventors.

[0144] Within the time period described above, approximately 37% (66 μg) of the bromelain supported within the microspheres was released in an active form (i.e., a form with proteolytic activity).

[0145] The same procedure was performed using a 3.0 mg / ml bromelain solution, and 60 μl of DC beads (300-500 μm) were immersed in 200 μl of this bromelain solution. The results of this experiment are shown in Graph 4 below. JPEG0007862932000006.jpg77146

[0146] As can be seen from the graph, when 60 μl of DC beads (300-500 μm) were immersed in 200 μl of 3.0 mg / ml bromelain solution for 24 hours, 288 μg (48%) of bromelain could be loaded onto the beads. Furthermore, when an elution test was performed in the same manner as in Experiment 3, it was shown that active bromelain (evaluated by azocasein assay) was eluted from the microspheres over 108 hours, indicating that approximately 55% (158 μg) of bromelain diffused from the microspheres over the aforementioned period.

[0147] Experiment 4 - Loading of papain into DC beads (300-500 μm) made of polyvinyl alcohol hydrogel 80 μL of DC beads (300-500 μm) were placed in a 1.5 mL centrifuge tube and washed twice with distilled water. 200 μL of 5 mg / mL papain solution was added, and the mixture was incubated at room temperature for 6 hours with gentle shaking. Next, the beads were washed twice with distilled water and suspended in 5 mL of pH 6.5 PBS. As the first sample, 250 μL was taken after 30 minutes, and samples were taken every hour for the following 16 hours. 250 μL of PBS was replenished after each sample collection. The papain concentration in the samples was measured by an azocasein assay, and the cumulative amount of papain released from the DC beads as a function of time is shown in the graph below. JPEG0007862932000007.jpg88157

[0148] As can be seen from Graph 5, sustained release of papain was achieved and continued for more than 16 hours.

[0149] Next, an experiment was conducted to measure the effectiveness of papain released from the beads. In this experiment, HT29 cells (human colorectal cancer cell line) were seeded in a 24-well plate. After 24 hours, beads loaded with 5 mg / ml of papain (500 μg per 80 μl of beads) were placed in a transwell chamber, and the plate seeded with HT29 cells was treated with these beads. The transwell chamber containing the beads was moved to a new well every 3 hours. This procedure was continued for a maximum of 3 days. This experiment showed that the papain released from DC beads maintained its proteolytic activity and killed all cells in the transwell chamber after 3 hours.

[0150] Experiment 5 - Measurement of the effect of released solvent volume and sample volume on bromelain release from 100-300 μm DC beads. The initial drug concentration delivered in vivo to the tumor is primarily determined by the size of the tumor, while the subsequent drug concentration in body fluids is determined by the body's volume (body weight). The drug clearance rate from various target areas within the patient's body is determined by the different perfusion rates (blood supply) for each organ. The models described below (Graphs 6.0, 6.1, 6.2, 6.3, and Table 3) illustrate the initial release of bromelain into body fluids and the subsequent clearance rate. JPEG0007862932000008.jpg100148

[0151] In the same manner as described above, 81.0 μg of bromelain was loaded onto 60 μl of PVA beads (100-300 μm). Next, the beads were added to 20 mL of PBS (pH 7.4, 37°C), and the amount of eluted bromelain was measured as a function of time. As in the previous experiment, 250 μl samples were taken at various time points, and the same amount of PBS was added to maintain the volume of elution solvent. The bromelain content was analyzed using the samples taken at each time point. As can be seen from the graph above (Graph 6.0), the release of bromelain into 20 ml of PBS (pH 7.4) showed a burst release of 30 μg in the first 30 minutes, followed by a sustained release (dx / dt) of 1.78 μg / hour for the next 28 hours. JPEG0007862932000009.jpg106156

[0152] The same beads loaded with bromelain were added to 5 mL of PBS (pH 7.4, 37°C), and the amount of eluted bromelain was measured as a function of time. The results are shown in Graph 6.1. As can be seen from the graph, 8 μg was released in the first 30 minutes (burst release), and then a sustained release was observed for the following 37 hours (the straight line portion of the graph). dx / dt = 58 / 37 = 1.57 μg / hour.

[0153] In this model (i.e., the model shown in Graph 6.1), the amount of release solvent (PBS at pH 7.4) was only 5.0 ml. Compared to the previous model (Graph 7.0) which used four times the amount, the initial burst release was significantly smaller. This result indicates that reducing the amount of release solvent reduces the amount of bromelain eluted during burst release.

[0154] The same beads loaded with bromelain were added to 20 mL of PBS (pH 7.4, 37°C), and samples were taken. The amount of eluted bromelain was measured as a function of time. In this experiment, the sample volume was 500 μL instead of 250 μL to mimic the target area in the body of a patient with a higher blood flow rate or a higher drug clearance rate than in the previous experiment. As can be seen from Graph 6.2 (below), in this experiment, dx / dt = 2.45 μg / hour, and the entire amount of 81 μg of loaded bromelain was released from 60 μL of PVA beads within 23 hours. JPEG0007862932000010.jpg102149JPEG0007862932000011.jpg109152

[0155] At the end of this series of experiments, the same beads loaded with bromelain were added to 20 mL of PBS (pH 7.4, 37°C), samples were taken, and the amount of eluted bromelain was measured as a function of time. In this experiment, the sample volume was 1 mL instead of 500 μL or 250 μL to mimic the target area in the body of a patient with even higher blood flow or a higher drug clearance rate. The results of this experiment are shown in Graph 6.3 above. As can be seen from the graph, compared to the other release models used, the sample volume was the largest (indicating high blood flow or clearance), and the entire amount of loaded 81 μg of bromelain was released within 17 hours (dx / dt = 4.5 μg / hour). [Table 3] JPEG0007862932000013.jpg79149

[0156] The selection of release rate and volume in vivo depends on various factors such as the body's volume, metabolic rate, perfusion flow rate in organs, pH, and the bead delivery site. The in vivo clearance rate when mucin-acting proteases are eluted is an important factor in determining an appropriate administration plan. The experiments described so far were conducted to simulate increased blood flow in the target region and showed that the degree of the initial burst varied depending on the selection of the release solvent, and the subsequent release rate varied depending on the sample volume (i.e., the in vivo clearance rate) (see Graph 7).

[0157] Experiment 6 - Measurement of the effect of pH of bromelain-supported solution on the release characteristics of microspheres In the following experiment, bromelain was loaded onto 300-500 μm DC beads using bromelain solutions at various pH levels, such as pH 2.5, pH 3.4, or pH 4.0. Next, the bromelain-loaded DC beads were added to 5.0 ml of PBS (pH 6.5). At regular intervals, 250 μl samples were taken from each solution, and the same amount of fresh PBS was added to each solution. The results of this experiment are shown in Graph 8 and Table 4. JPEG0007862932000014.jpg79155 [Table 4]

[0158] This experiment demonstrated that the pH of the loading solution affects the burst release and subsequent bromelain release rates. While the loading amounts were very similar at these three pH levels, the release rates were significantly slower at a loading solution pH of 2.5, and very similar at pH 3.4 and pH 4.0.

[0159] Experiment 7: Measurement of the effect of pH on the loading of bromelain onto DC beads (100-300 μm) made of PVA hydrogel and the release of bromelain from the DC beads. 200 μl of bromelain solution (3.0 mg / ml) prepared in water (pH 2.8, pH 3.0, or pH 3.2) or PBS (pH 2.77) was added to 100-300 μm DC beads (60 μl), and the mixture was incubated at ambient temperature (25°C) for 24 hours while shaking on a shaker to load the DC beads with bromelain. Next, the bromelain solution was carefully collected using a pipette, and the remaining bromelain was analyzed to determine the total amount loaded onto the beads. The results of this experiment are summarized in Table 5 below.

[0160] Next, the PVA beads were added to 10 ml of PBS (pH 6.5) and gently shaken in a 37°C water bath. To measure the burst release of bromelain, 500 μl of the solution was taken after 30 minutes and analyzed (500 μl of fresh PBS was added to maintain a constant solution volume). Subsequently, the same procedure was performed every hour to analyze the bromelain. The results of this experiment are shown in Graph 9 and Table 5 below. JPEG0007862932000016.jpg104152 [Table 5]

[0161] When bromelain dissolved in PBS at pH 2.77 was used, the loading rate (%) was almost 100%, indicating that this method is a good method for efficiently loading bromelain. Furthermore, the bromelain aqueous solution at the pH investigated in this study also allowed for very efficient loading of bromelain, with a loading efficiency in the range of 86-89%.

[0162] Compared to loading in water at other pH levels or in PBS (pH 2.77), bromelain loading in water at pH 2.8 resulted in the least burst release. However, when comparing burst release as a percentage of the total loading, loading in water at pH 2.8 and loading in PBS at pH 2.77 appear to yield similar results.

[0163] Experiment 8 - Measurement of the effect of pH of the PBS solution used for loading bromelain onto DC beads (100-300 μm) made of PVA hydrogel and on the release of bromelain from the DC beads. Bromelain solutions (3.0 mg / ml) were prepared as separate samples in PBS at pH 2.0, pH 2.2, pH 2.4, or pH 2.6. 60 μl of DC beads (100-300 μm) were added to 200 μl of each bromelain solution, and the mixture was shaken on a stirrer at ambient room temperature (23°C) for 24 hours. 200 μl of the supernatant was carefully collected from each sample, and the amount of bromelain adsorbed onto the beads was quantified by analyzing the bromelain content remaining in the sample using an azocasein assay.

[0164] Each bead was added to 10 ml of PBS (pH 6.5), and 500 μl of each solution was collected after 30 minutes, and then at 1, 2, and 3 hours (simultaneously replenishing with the same amount of fresh PBS), and burst release was evaluated. Bromelain eluted into 500 μl of PBS was quantified over time until no more bromelain release was detected (protein degradation activity was measured by azocasein assay). The results of this experiment are shown in the following graphs and tables (Graph 10, Table 6.0, Table 6.1, Table 6.2). It appears that the higher the acidity of the support solution, the longer and more sustained the release (compare with Graph 8).

[0165] The release rate of bromelain after burst release was divided into 12-hour intervals, and the bromelain elution rate was calculated from the linear portion of the graph plotted against the elapsed time. JPEG0007862932000018.jpg108150 [Table 6] TIFF0007862932000020.tif48155TIFF0007862932000021.tif37158

[0166] As can be seen from the graph, the pH during bromelain loading within microspheres affects the amount of bromelain that can be loaded, burst release, and the subsequent elution rate of the loaded bromelain. Therefore, the elution characteristics of the microspheres may be adjusted by changing the pH of the loading solvent to suit specific patients and treatment plans.

[0167] For example, the average size of a pancreatic tumor is about 20 cm. 3 ±16cm 3 It is estimated that... Using 60 μl of 100-300 μm DC beads loaded with bromelain (3.0 mg / ml) at pH 2.6, if 120 μg is released in a burst, the bromelain concentration in the tumor will effectively be 120 / 20 = 6 μg / ml. However, when bromelain is administered alone, a concentration of bromelain exceeding 20 μg / ml is required to exert a sufficient cytotoxic effect as a monotherapy against tumor cells (in in vitro studies, the IC50 value for PANC-1 cells was 18 μg / ml and the IC75 value was 50 μg / ml). Therefore, 20 cm 3 To increase the bromelain concentration to 60 μg / ml for a tumor of this size, it is estimated that 600 μl of bromelain-laden beads would be required.

[0168] After a burst release, 5.33 μg / hour of bromelain is released from 60 μl of 100-300 μm DC beads. If the amount of beads is increased tenfold (600 μl), it is estimated that a total of 53.3 μg / hour of bromelain will be released in the first 12 hours. Assuming a clearance of 53.3 μg / hour, a steady state of 60 μg / ml of bromelain can then be maintained for at least 12 hours, after which the release rate decreases by approximately 0.007% per hour.

[0169] A lean patient weighing 80 kg is thought to have a blood volume of approximately 6 L. If bromelain is released at a rate of 53 μg / hour, it will be diluted to approximately 8.83 ng / ml, the majority of which will bind to albumin, antitrypsin, and macroglobulin. Toxicity to blood coagulation parameters may be a concern, but this issue is related to blood flow and is not seen in upstream vascular embolization models used for liver cancer and pancreatic cancer. Low-dose bromelain exposure can also be used in synergistic effect models combined with chemotherapy.

[0170] In this tumor model, a similar treatment to the previous example may be simulated by using PVA beads loaded with bromelain at pH 2.4, perhaps in 20 times the amount.

[0171] Example 2: 30-60 μm hepaspheres made of sodium acrylate-alcohol copolymer Experiment 9 - Loading of bromelain (3.0 mg / ml) into HEPASphere microspheres (30-60 μm) and release of bromelain from HEPASpheres using bromelain solutions (3.0 mg / ml) in PBS at various pH levels. 300 μl of bromelain solution (3 mg / ml) in PBS at various pH levels (pH 2.0, pH 2.2, pH 2.4, or pH 2.6) was added to a HEPA microsphere (40 μl) and treated for 24 hours with continuous shaking. Next, the tube containing the beads and bromelain solution was centrifuged, and the supernatant (300 μl) was aspirated and collected. The amount of bromelain loaded was quantified by analyzing the remaining bromelain content.

[0172] Next, HEPA beads were added to 10 ml of PBS (pH 6.5) in a 50 ml centrifuge tube, and the tube was continuously shaken in a 37°C water bath. After 30 minutes, 500 μl of this solution was taken, and thereafter, samples were taken every hour. After each sample collection, the same amount of PBS as the collected solution (500 μl) was replenished. Next, the bromelain content in the collected solutions was analyzed using an azocasein assay. The results of this experiment are shown in Graph 11 and Tables 7, 7.1, and 7.2 below. JPEG0007862932000022.jpg104163 [Table 7] TIFF0007862932000024.tif43147

[0173] The release rate (dx / dt) every 12 hours was calculated using the straight line portion of the graph, excluding the amount of bromelain released in bursts. [Table 8]

[0174] Beads (40 μl) were exposed to each bromelain solution (300 μl) containing 900 μg of bromelain. There was only a slight difference in load-bearing capacity due to differences in pH. The load-bearing capacity was similar at pH 2.2 and pH 2.0, and slightly higher than at pH 2.6 and pH 2.4. In another experiment (not described), it was found that exposure to 1200 μg of bromelain at pH 3.4 resulted in a very high load-bearing capacity (87%).

[0175] When bromelain was supported at pH 2.6, the burst release was highest (125 μg: 19.2% of the total supported amount), and when bromelain was supported at pH 2.0, the burst release was lowest. Since pH is known to affect the pore size of microspheres, it is thought that pH affected the burst release.

[0176] When the release rate was calculated every 12 hours, it was again shown that the release rate was highest at pH 2.6, suggesting a possible relationship between the pH at the time of loading and the pore size of the beads at a specific pH. After 52 hours, HEPA microspheres loaded with bromelain at pH 2.6 eluted 86% of the loaded bromelain, compared to 48% when loaded at pH 2.4, 26% when loaded at pH 2.2, and 13% when loaded at pH 2.0. These results suggest that the pH at the time of loading may play an important role in the release pattern and the total amount released at a specific time point.

[0177] HEPA microspheres are a copolymer of polyvinyl alcohol and sodium acrylate. In an acidic environment, the positively charged protonated amine groups in bromelain form bonds with the negatively charged acetate groups, thereby tethering the bromelain to the beads. When the bromelain-supported beads are added to the elution solvent, the ionic bonds are easily broken, releasing the bromelain. Bromelain is released until equilibrium is reached between the bromelain bound to the beads and the free bromelain in pH 6.5 PBS. When the sample is taken and when fresh PBS is added to the solution, the bromelain concentration decreases, resulting in further release of bromelain from the beads until equilibrium is reached. In this experimental model, 10 ml of PBS was used and 500 μl of sample was taken; this amount corresponds to a 5% change in blood flow or a 5% clearance.

[0178] The average size of liver tumors has been reported to be 21.8 cc (Dachman et al Tumor size on Computed Tomography Scans. Cancer, 2001; 91(3):555-560), and treatment with bromelain-carrying microspheres that can deliver a concentration of approximately 20 μg / ml is necessary for unresectable tumors. A burst release of 125 μg of bromelain from a 40 μl microsphere loaded with bromelain at pH 2.6 corresponds to a dose of 5.73 μg / cc in the tumor. When 160 μl of microspheres are delivered to the tumor site, the bromelain concentration effectively increases to 22.9 μg / cc. Assuming a clearance of approximately 10% / hour, 2.3 μg / hour is eliminated. The release rate per hour during the first 24 hours was approximately 8 μg / hour (×4 = 32 μg / hour), and this release rate offsets the bromelain eliminated at the tumor site.

[0179] Example 3 - 75 μm TANDEM microspheres Experiment: Bromelain loading onto 10-75 μm TANDEM microspheres and release of bromelain from TANDEM microspheres. 40 μL of TANDEM beads (75 μm) were placed in a 1.5 mL centrifuge tube. 200 μL of 5 mg / mL bromelain in distilled water was added. The tube was gently shaken at room temperature for 24 hours. The next day, the beads were washed twice with distilled water and resuspended in 5 mL of distilled water. First, a 250 μL sample was taken after 30 minutes, and then the next sample was taken every hour for the following 32 hours (250 μL of fresh water was added after each sample was taken). The bromelain content in each sample was measured by azocasein assay, and the bromelain release characteristics were calculated using the measured bromelain content. The results are shown in Graphs 12.1 and 12.2. JPEG0007862932000026.jpg106145JPEG0007862932000027.jpg106148

[0180] After 32 hours, no bromelain was released. This experiment demonstrated that bromelain can be immobilized within other forms of commercially available microspheres and subsequently eluted continuously in its active form. The bromelain release pattern from TANDEM microspheres may be modified by adjusting the microsphere size, pH during immobilization, load amount, coating, and other techniques described above.

[0181] Example 4 - Coated DC beads (300-500 μm) (PVA hydrogel microspheres) Experiment 11 - Loading onto DC beads (PVA) coated with alginate and release from coated DC beads Bromelain (5 mg / mL) was added to DC beads (300 μm to 500 μm) and allowed to be loaded at room temperature for 24 hours. The amount of bromelain loaded onto the beads was calculated to be 900 μg. Next, the beads were washed twice with distilled water, immersed in a 2% alginate solution, and then immersed in a 2% CaCl solution for 15 minutes to form an alginate coating on the outermost surface of the microspheres.

[0182] Next, beads loaded with bromelain and coated with alginate were added to 10 mL of water, and the release of bromelain over the next 30 hours was measured in the same manner as described above. The results are shown in Graph 13.2. 300-500 μm DC beads loaded with bromelain (uncoated) were used as a control (Graph 13.1). JPEG0007862932000028.jpg97142JPEG0007862932000029.jpg105150

[0183] As can be seen from the graph above, the bromelain contained in the alginate-coated DC beads eluted at a very slow rate. In about 10 hours, only about 10% of the total bromelain was eluted from the coated beads, compared to about 66% from the uncoated microspheres. Furthermore, burst release was suppressed in the alginate-coated beads.

[0184] Example 5 - DC Beads Microspheres Co-supported with Bromelain and Doxorubicin Three batches of DC beads (300 μm to 500 μm) were prepared in the same manner as described above. Bromelain (1 mg / mL) was loaded onto the DC beads of the first batch alone. 0.25 mg / mL of doxorubicin was loaded onto the DC beads of the second batch alone. For the third batch of DC beads, 1 mg / mL of bromelain was first loaded over 24 hours, and then 0.25 mg / mL of doxorubicin was loaded over 6 hours the following day.

[0185] CFPAC-1 cells (human pancreatic cancer cell line) were seeded in 96-well plates. Beads were serially diluted and dispensed into the wells in four-compartment incubators, and incubated for 72 hours. After incubation, an SRB assay was performed, and the number of beads per well was counted. The results are summarized in the following graphs (Graphs 14.1, 14.2, and 14.3). JPEG0007862932000030.jpg133152

[0186] Graph 14.1 shows that inhibition of growth was observed when 40 beads and 22 beads were placed per well, demonstrating a dose-dependent effect (bead dilution). However, no inhibition of growth was observed when 9 beads were placed per well. JPEG0007862932000031.jpg116153JPEG0007862932000032.jpg108160

[0187] As can be clearly seen from the experimental results above (Graphs 14.1, 14.2, and 14.3), the number of cells killed increased when beads co-loaded with bromelain and doxorubicin were used compared to when beads loaded with bromelain alone or when beads loaded with doxorubicin alone. These experimental data demonstrate a synergistic effect of doxorubicin and bromelain compared to doxorubicin alone or bromelain alone.

[0188] Example 6 - Efficacy of 100-300 μm DC bead microspheres containing bromelain In the same manner as described above, bromelain (400 μg / mL) was loaded onto DC beads (100-300 μm). Next, the microspheres were serially diluted, and their efficacy against CFPAC-1 cells was measured in the same manner as in Example 5. The results of this experiment are shown in Graphs 15.1 and 15.2 below. JPEG0007862932000033.jpg97157

[0189] These data (Graph 15.1 above) showed that the inhibition of proliferation in the pancreatic cancer cell line CFPAC-1 decreased when the number of beads per well fell below 62, with a loading of 0.149 μg per bead. 130 loaded beads were required to kill 90% of the cells.

[0190] When the amount of bromelain loaded onto the beads was significantly increased (Graph 15.2 below), the inhibition of proliferation against the pancreatic cancer cell line CFPAC-1 decreased when the number of beads per well fell below 22. Graph 15.1 showed that when the amount of bromelain loaded onto the beads was not significantly increased (0.149 μg per bead), 130 beads were required to kill 90% of the cells. However, when the amount of bromelain loaded onto the beads was significantly increased (0.403 μg per bead), only 43 beads were required to achieve almost the same efficacy as in Graph 15.2. JPEG0007862932000034.jpg101165

[0191] This experiment tested the inhibitory effects of beads loaded with different concentrations of bromelain (0.149 μg or 0.403 μg per bead). The experimental data showed that the higher the bromelain concentration per DC bead, the fewer beads were needed to inhibit cell proliferation.

[0192] Example 7 - Tests at various points in time to evaluate the required exposure time Human ovarian cancer cell line OVCAR-3 cells were seeded in 96-well plates. After 24 hours, the plates were treated with doxorubicin (50 nM), N-acetylcysteine ​​(2.5 mM), and various concentrations of bromelain (not supported on microspheres). At 1, 3, 6, 18, 24, and 48 hours, each drug and medium was removed, and the plates were washed with PBS. Treatment with doxorubicin was restarted in appropriate wells, and drug-free medium was added to the remaining wells. All plates were treated for a further 72 hours. The results of this experiment are shown in Graphs 16.1, 16.2, and 16.3 below.

[0193] This study aimed to measure the time required for bromelain to elute from microspheres and exert a synergistic effect with concomitantly administered chemotherapeutic agents. The results showed that 24 hours of exposure to bromelain, preferably 48 hours, resulted in a good synergistic effect with 50 nM doxorubicin. With even higher concentrations of doxorubicin, such as 100 nM (results not shown), a synergistic killing effect against cancer cells was observed even with just 3 hours of exposure to bromelain. JPEG0007862932000035.jpg97152JPEG0007862932000036.jpg90148JPEG0007862932000037.jpg89149

[0194] Example 8 - Preclinical animal study using DC beads loaded with bromelain and doxorubicin Bromelain was supported on DC beads (100-300 μm) in the same manner as described above.

[0195] In this safety study, New Zealand rabbits were treated with DC beads loaded with a total of 5 mg or 10 mg of bromelain, in the same manner as described above. The bead suspension was injected directly into the common hepatic artery (i.e., via an intra-arterial route), allowing the DC beads to be carried through the bloodstream, forming an embolism and releasing bromelain over time.

[0196] The rabbits were euthanized 1 hour, 3 hours, 6 hours, 24 hours, or 7 days after the procedure. After euthanasia, the internal organs were observed comparatively, and bromelain concentrations in the plasma and liver were measured. The observation results are shown in the following graphs 17.1-17.6. JPEG0007862932000038.jpg79151JPEG0007862932000039.jpg81162JPEG0007862932000040.jpg81157 JPEG0007862932000041.jpg85162JPEG0007862932000042.jpg89154JPEG0007862932000043.jpg94164

[0197] As can be seen from the graphs above, the bromelain contained in the DC Beads was eluted in the liver over approximately 24 hours after injection into the common hepatic artery (Graphs 17.1, 17.2, and 17.5). The amount of bromelain in the bloodstream was lowest after 6 hours (Graphs 17.3, 17.4, and 17.6). Macroscopic evaluation of the liver showed that the beads were distributed in the target liver lobe. Recovery of normal tissue was observed 7 days after the procedure (results not shown). In short, the results of this study demonstrated the safety of DC Beads loaded with bromelain.

[0198] Example 9 - Cytotoxic activity of bromelain-supported DC beads (100-300 μm) against cancer cells (ASPC-1 cells and HT-29 cells) ASPC-1 and HT-29 cell lines were seeded into 96-well plates. After 24 hours, the plates were treated with 5 mg / ml bromelain supported in DC beads (100-300 μm) in the same manner as described above (see Example 5). The microspheres were then serially diluted. After 48 hours, the drug and medium were removed, and cell proliferation was tested using an SRB assay. The results of this experiment are shown in Graphs 18.1 and 18.2 below. JPEG0007862932000044.jpg79146JPEG0007862932000045.jpg70145

[0199] In this experiment, the inhibitory effects of serially diluted bromelain-laden beads (50, 30, or 10 beads per well) were tested. The experimental data showed that serially diluted bromelain-laden DC beads were more effective against the colorectal cancer cell line HT-29 than against the pancreatic cancer cell line ASPC-1 (Graph 18.1).

[0200] Example 10 - Duration of activity and cytotoxic activity of bromelain-supported DC beads (300-500 μm) against pancreatic cancer cells (CFPAC-1) Pancreatic cancer cell line CFPAC-1 cells were seeded in a 24-transwell plate. After 24 hours, the plate was treated with bromelain supported in DC beads (300-500 μm) using a transwell chamber insert. The transwell insert containing the beads was transferred to a new well every 3 hours. This procedure was continued for 3 days. Cell proliferation was tested using an SRB assay. The results of this experiment are shown in Graph 19.1 below. JPEG0007862932000046.jpg109156

[0201] The results of this experiment showed that an effective amount of cytotoxic bromelain was released from the beads for up to 17 hours.

[0202] Example 11 - Methods usable for cancer treatment The following examples describe the inventors' considerations on how to treat cancerous tumors (e.g., primary or secondary liver cancer) using microspheres according to embodiments of the present invention. This treatment method is similar to the TACE method described above, in which the microspheres of the present invention are injected into the arteries supplying nutrients to the cancerous tumor. The microspheres are transported through the patient's arteries and physically occlude the arteries before reaching the bed of arteries. In this way, the microspheres block or restrict the blood supply to the tumor, and continuously deliver bromelain (or another protease acting on mucin) and any further drugs co-carried or co-administered to the site.

[0203] As described above, the present invention provides a novel delivery medium capable of delivering an effective amount of bromelain (or other proteases that act on mucin and are therapeutically indicated) to a patient in a manner that minimizes the potential for side effects. Embodiments of the present invention offer many advantages over existing treatments, some of which are summarized below. The microspheres of the present invention provide a method for locally delivering a mucin-acting protease, thereby achieving sustained release of the mucin-acting protease (and optionally other active substances), and consequently improving the effect of the protease while minimizing the possibility of side effects. The present invention makes it possible to increase the local concentration of proteases that act on mucin in the target region, resulting in benefits without the risks associated with systemic toxicity. The present invention may improve the penetration of drugs into cancer (particularly tumors having a fibrous capsule or tumors surrounded by adhesions), and may provide a synergistic effect when used in combination with other chemotherapeutic agents. To ensure cell death, the protease acting on mucin can be recombined so that it is continuously released beyond the regeneration time of the disease-involved cells.

[0204] Those skilled in the art who have referenced the present invention will understand that various modifications may be made without departing from the spirit and scope of the invention. Any such modifications are included within the scope of the following claims.

[0205] The above description relates to specific forms of the microspheres, pharmaceutical compositions, and therapeutic methods of the present invention. Such detailed descriptions are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[0206] It is understood that none of the prior art documents cited herein constitute part of the common technical knowledge in this field.

[0207] In the claims described later and in the prior descriptions of the present invention, unless the context requires a non-inclusive meaning through explicit terminology or necessary implication, the term “comprise” or its variations such as “comprises” or “comprising” is used in an inclusive sense, that is, to identify the presence of the described features in various embodiments of the present invention, but not to exclude the presence or addition of further features.

Claims

1. The use of microspheres for manufacturing therapeutic drugs for mucin-producing cancer, pseudomyxoma peritonei, cystic fibrosis, or chronic obstructive pulmonary disease, The aforementioned pharmaceutical product is characterized by being used to be delivered to the patient via intra-arterial, intraperitoneal, or intracavitary delivery, The microspheres have a diameter of 30 to 700 μm, carry a mucin-acting protease inside, and are configured to continuously elute the mucin-acting protease over a period of 5 to 120 hours after administration. The microspheres include a hydrogel on which a protease acting on the mucin is supported.

2. The use according to claim 1, wherein the protease acting on the mucin is selected from one or more of the group consisting of plant-derived proteases, fungal proteases, and bacterial proteases.

3. The use according to claim 2, wherein the plant-derived protease is selected from one or more of the group consisting of bromelain, papain, ficain, actinidine, zingipain, and fastuosain.

4. The use according to any one of claims 1 to 3, characterized in that the pharmaceutical product is used in such a way that a therapeutically effective amount of further therapeutically effective drugs is co-delivered.

5. The use according to claim 4, wherein an additional agent effective in the aforementioned treatment is selected from one or more of the group consisting of chemotherapeutic agents, radiotherapy agents, mucolytic agents, and contrast agents.

6. The use according to claim 4 or 5, wherein an additional agent effective in the aforementioned treatment is carried on within the microsphere containing a protease that acts on the mucin and co-delivered.

7. The use according to claim 4 or 5, wherein an additional agent effective in the aforementioned treatment is co-delivered in a form separate from the microsphere containing the protease acting on the mucin.

8. The use according to claim 7, wherein an additional agent effective for the aforementioned treatment is co-delivered simultaneously with or subsequently to the microsphere containing the protease acting on the mucin, and if the additional agent is subsequently delivered to the microsphere, the delivery of the additional agent takes place before or after the delivery of the microsphere.

9. The use according to any one of claims 1 to 8, wherein the mucin-producing cancer is selected from the group consisting of liver cancer (primary or secondary liver cancer), pancreatic cancer, lung cancer, thyroid cancer, stomach cancer, appendiceal cancer, peritoneal cancer, hepatocellular carcinoma, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, mesothelioma, neuroblastoma, small intestine cancer, lymphoma, and leukemia.

10. The use of microspheres for manufacturing therapeutic drugs for mucin-producing cancers, The aforementioned pharmaceutical product is characterized by containing a therapeutically effective amount of the microspheres, each containing a protease that acts on mucin, and being used to be delivered intra-arterial to the patient. The microspheres have a diameter of 60 to 120 μm and are configured to continuously elute the protease that acts on the mucin over a period of 5 to 120 hours after administration. The microspheres include a hydrogel on which a protease acting on the mucin is supported.

11. The use according to claim 10, wherein the mucin-producing cancer is primary liver cancer or secondary liver cancer.

12. The use of microspheres for manufacturing a drug for the treatment of pseudomyxoma peritonei, The aforementioned pharmaceutical product contains a therapeutically effective amount of the microspheres, each containing a protease that acts on mucin, and is used to be delivered to the patient intraperitoneally or intracavitarially. The microspheres have a diameter of 30 to 700 μm and are configured to continuously elute the protease that acts on the mucin over a period of 5 to 120 hours after administration. The microspheres include a hydrogel on which a protease acting on the mucin is supported.

13. A drug for the treatment of mucin-producing cancer, pseudomyxoma peritonei, cystic fibrosis, or chronic obstructive pulmonary disease, The aforementioned pharmaceutical product is characterized by comprising microspheres and being used to be delivered to a patient via intra-arterial, intraperitoneal, or intracavitary delivery, A pharmaceutical product comprising a hydrogel containing a mucin-acting protease, wherein the microspheres have a diameter of 30 to 700 μm and are configured to continuously elute the mucin-acting protease over a period of 5 to 120 hours after administration.

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