Encapsulated pancreatic islets

Abstract

The present invention provides an improved encapsulated islet. This encapsulated islet is a preparation in which a core is coated with five layers of films, the core includes an islet, the first, third, and fifth films from the inside among the five layers of films include alginic acid, and the second and fourth films from the inside include polyornithine.

Description

[Technical Field]

[0001] Techniques for encapsulating pancreatic islets are disclosed. [Background technology]

[0002] Pancreatic islet transplantation is a diabetes treatment method that places a relatively low burden on the human body, but strong immunosuppression is essential for the long-term survival of the transplanted islets, and therefore, in medical practice, transplant patients are administered immunosuppressants. However, because administering immunosuppressants carries the risk of side effects, a method has been investigated in which a large number of pancreatic islets are transplanted into the abdominal cavity after immunoisolation using minute microencapsulation. In this case, immunosuppression is unnecessary or the dosage can be reduced, but satisfactory therapeutic results have not yet been obtained. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2001 / 052871 [Non-patent literature]

[0004] [Non-Patent Document 1] METHODS IN ENZYMOLOGY, VOL. 137, 575-580, 1988 [Non-patent document 2] TRANSPLANTATION, Vol. 53, 1180-1183, No. 6, June 1992 [Non-patent document 3] Bioartificial Pancreas, Vol., 98, No. 6, 1996, 1417-1422 [Non-patent document 4] Drug Delivery System, Vol. 12, No. 2, 1997 [Non-patent document 5] JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS, 2013 VOL., 101B, ISSUE 2, 258-268 Summary of the Invention [Problem to be solved by the invention]

[0005] One challenge is to provide improved encapsulated pancreatic islets. [Means for solving the problem]

[0006] As a means for solving such problems, the invention includes the following. Section 1. The core is covered with five layers of membranes. the core comprises a pancreatic islet; The first, third and fifth membranes from the inside of the five-layer membrane contain alginic acid, and the second and fourth membranes from the inside contain polyornithine. formulation. Section 2. Item 1. The formulation according to Item 1, wherein the average diameter of the formulation is 400 μm or more. Section 3. Item 3. The formulation according to Item 1 or 2, wherein the average diameter of the formulation is 400 μm or more and 500 μm or less. Section 4. Item 4. The preparation according to any one of Items 1 to 3, which is used for treating diabetes. Section 5. Item 5. The preparation according to any one of Items 1 to 4, wherein the pancreatic islets are obtained from a 1- to 3-week-old young pig. Section 6. (a) preparing a sodium alginate solution A containing pancreatic islets; (b) adding the sodium alginate solution A dropwise to a divalent cation solution and recovering the gelled particles; (c) adding the particles recovered in step (b) to poly-L-ornithine solution A, stirring for a predetermined time, and then recovering the particles; (d) adding the particles recovered in step (c) to sodium alginate solution B, stirring for a predetermined time, and then recovering the particles; (e) adding the particles recovered in step (d) to poly-L-ornithine solution B, stirring for a predetermined time, and then recovering the particles; (f) adding the particles collected in step (e) to sodium alginate solution C, stirring for a predetermined time, and then collecting the particles; and (g) adding the particles collected in step (f) to a sodium citrate solution, stirring for a predetermined period of time, and then collecting the particles; Item 6. A method for producing the formulation according to any one of Items 1 to 5, comprising: Section 7. Item 7. The method according to Item 6, wherein the poly-L-ornithine concentration of the poly-L-ornithine solution A is 0.05 w / v% or more and less than 1 w / v%. Section 8. Item 8. The method according to Item 6 or 7, wherein in step (d), the particles recovered in step (c) are added to poly-L-ornithine solution A', stirred for a predetermined time, recovered, and then added to sodium alginate solution B. [Effects of the Invention]

[0007] An effective means for treating diabetes is provided. [Brief explanation of the drawings]

[0008] [Figure 1] This shows the results of measuring blood glucose levels in diabetic model mice after administration of encapsulated islets. The black squares represent the results of administration of the triple membrane formulation, the black circles represent the results of administration of the quintuple membrane formulation 1, the black triangles represent the results of administration of the quintuple membrane formulation 2, and the black circles represent the results of administration of the quintuple membrane formulation 3. [Figure 2] The results of measuring the fibrosis rate after administration of encapsulated islets are shown. Black squares indicate the results after administration of the triple membrane preparation, ● indicates the results after administration of the quintuple membrane preparation 1, ▲ indicates the results after administration of the quintuple membrane preparation 2, and × indicates the results after administration of the quintuple membrane preparation 3. DETAILED DESCRIPTION OF THE INVENTION

[0009] Preferably, the preparation has a core covered with five membranes, the core containing pancreatic islets, the first, third, and fifth membranes from the inside of the five membranes containing alginate, and the second and fourth membranes from the inside containing polyornithine. Such a preparation may enable blood glucose levels to be maintained within an appropriate range for a longer period of time. In one embodiment, the preparation may be covered with more than five membranes (e.g., six, seven, eight, nine, or ten membranes). A five-membrane-covered preparation may refer to a preparation prepared by a method including five membrane-forming steps as described below.

[0010] The type of pancreatic islet is not particularly limited and may be any type. Preferably, the pancreatic islet contains insulin-producing β cells, glucagon-containing α cells, somatostatin-secreting delta cells, and pancreatic polypeptide-containing cells (PP cells). Preferably, the majority of the pancreatic islets are insulin-producing β cells. The origin of the pancreatic islets can be selected depending on the purpose, and is preferably human, pig, mouse, rat, monkey, or dog. In one embodiment, the pancreatic islets are preferably derived from pigs, and are preferably pancreatic islets from young pigs (e.g., 3 days to 4 weeks old or 7 days to 3 weeks old). The pancreatic islets can be obtained by any method known in the art. For example, a method using Liberase as a digestive enzyme (TJ Cavanagh et al., Transplantation Proceedings, 30, 367 (1998)) can be preferably used.

[0011] The size of the pancreatic islets is preferably 50 μm to 400 μm, more preferably 50 μm to 350 μm. The size of the pancreatic islets can be measured using a micrometer under a microscope. A preferred characteristic of pancreatic islets is that they contain 10% or more β cells. There is no particular upper limit to the proportion of β cells, but it is, for example, 80%.

[0012] The term "a membrane containing alginic acid" means that the membrane is mainly composed of alginic acid. The term "a membrane containing polyornithine" means that the membrane is mainly composed of polyornithine.

[0013] The preparation preferably contains 1 to 5 pancreatic islets per capsule.

[0014] The type of alginic acid constituting the alginic acid film is not particularly limited, and the type of polyornithine constituting the polyornithine film is not particularly limited.

[0015] From the viewpoint of suppressing fibrosis, the preparation preferably has an average diameter of 400 μm or more, more preferably 420 μm or more. There is no particular upper limit to the average diameter, but it can be set to, for example, 700 μm or less, 600 μm or less, or 500 μm or less. The average diameter can be measured by the measurement method employed in the examples described below.

[0016] The above-mentioned preparation is useful for treating diabetes. The type of diabetes is not particularly limited, and examples thereof include type 1 diabetes. In addition, the above-mentioned preparation is useful for treating patients who have difficulty controlling their blood sugar, regardless of the cause.

[0017] The method for producing pancreatic islets is optional. In one embodiment, the method for producing pancreatic islets preferably comprises the following steps (a) to (g): (a) preparing a sodium alginate solution A containing pancreatic islets; (b) adding the sodium alginate solution A dropwise to a divalent cation solution and recovering the gelled particles; (c) adding the particles recovered in step (b) to poly-L-ornithine solution A, stirring for a predetermined time, and then recovering the particles; (d) adding the particles recovered in step (c) to sodium alginate solution B, stirring for a predetermined time, and then recovering the particles; (e) adding the particles recovered in step (d) to poly-L-ornithine solution B, stirring for a predetermined time, and then recovering the particles; (f) adding the particles collected in step (e) to sodium alginate solution C, stirring for a predetermined time, and then collecting the particles; and (g) adding the particles collected in step (f) to a sodium citrate solution, stirring for a predetermined period of time, and then collecting the particles;

[0018] The sodium alginate concentration of the sodium alginate solution A is not particularly limited, but is preferably, for example, 1 w / v % or more and 3 w / v % or less.

[0019] Sodium alginate solution A preferably contains pancreatic islets at a concentration of 10,000 IEQ / mL or higher, from the viewpoint of improving the glucose responsiveness of the pancreatic islets after encapsulation. Here, IEQ refers to the number of pancreatic islets per 150 μm diameter. The pancreatic islet concentration is preferably 11,000 IEQ / mL or higher, or 12,000 IEQ / mL or higher. There is no particular upper limit to the pancreatic islet concentration, but it can be, for example, 30,000 IEQ / mL or lower, or 25,000 IEQ / mL or lower.

[0020] There are no specific limitations on the method for preparing sodium alginate solution A, as long as it is prepared to contain the pancreatic islets at the above-mentioned concentration. For example, sodium alginate solution A can be obtained by dissolving an appropriate amount of sodium alginate in physiological saline, adding an appropriate amount of pancreatic islets thereto, and stirring as necessary.

[0021] The divalent cation solution used in step (b) is not limited as long as gelling particles (capsules) encapsulating pancreatic islets can be obtained by adding sodium alginate solution A dropwise thereto. Examples of divalent cations constituting such a divalent cation solution include salts that liberate divalent metal ions in aqueous solution (e.g., calcium chloride, calcium lactate, barium chloride, strontium chloride, etc.). In one embodiment, a preferred salt is calcium chloride, and a preferred divalent cation solution is a calcium chloride solution.

[0022] The concentration of the divalent cation in the divalent cation solution is not particularly limited and can be set in the range of, for example, 50 to 500 mM.

[0023] The manner in which sodium alginate solution A is added dropwise to the divalent cation solution is not limited as long as gelling particles containing pancreatic islets are obtained. In one embodiment, sodium alginate solution A is preferably added dropwise to the divalent cation solution through a needle of an appropriate size.

[0024] The gelling particles can be optionally recovered, for example, by leaving the divalent cation solution in which the gelling particles have been formed to stand for a certain period of time and removing the supernatant, or by repeating this procedure.

[0025] The poly-L-ornithine solution A used in step (c) is not particularly limited as long as it can coat the gelling particles formed in step (b). For example, it can be prepared by dissolving poly-L-ornithine in physiological saline.

[0026] The concentration of poly-L-ornithine in poly-L-ornithine solution A is not particularly limited, but is preferably 0.05 w / v% or more and less than 1 w / v% from the viewpoint of controlling the average diameter of the preparation (capsule) to 50 μm or more and 400 μm or less. In one embodiment, the concentration of poly-L-ornithine in poly-L-ornithine solution A is preferably 0.9 w / v% or less or 0.8 w / v% or less.

[0027] The phrase "adding the particles recovered in step (b) to poly-L-ornithine solution A" in step (c) also encompasses an embodiment in which poly-L-ornithine solution A is added to the gelled particles recovered in step (b). The stirring speed and time in step (c) are optional, and are preferably, for example, 1 to 20 minutes.

[0028] The gelling particles can be collected in any manner in step (c), for example, by leaving the poly-L-ornithine solution A to stand for a certain period of time and removing the supernatant. The collected gelling particles preferably have a structure in which the surface of the alginic acid gelling layer is coated with poly-L-ornithine.

[0029] The sodium alginate solution B used in step (d) may have the same concentration as or a different concentration from the sodium alginate solution A used in step (a). In one embodiment, the sodium alginate concentration of sodium alginate solution B is preferably lower than that of sodium alginate solution A, from the viewpoint of ease of coating operation. For example, when the sodium alginate concentration of sodium alginate solution A is 1 w / v% or more and 3 w / v% or less, the sodium alginate concentration of sodium alginate solution B is preferably 0.1 w / v% or more and 0.3 w / v% or less.

[0030] The phrase "adding the particles recovered in step (c) to sodium alginate solution B" in step (d) also encompasses an embodiment in which sodium alginate solution B is added to the gelled particles recovered in step (c). The stirring speed and time in step (d) are optional, and stirring for, for example, 1 to 15 minutes is preferred.

[0031] The gelling particles can be collected in any manner in step (d), for example, by leaving sodium alginate solution B to stand for a certain period of time and removing the supernatant. The collected gelling particles preferably have a gelling layer of alginic acid coated on the surface with poly-L-ornithine, and further have a gelling layer of alginic acid thereon.

[0032] In one embodiment, it is preferable that the particles recovered in step (c) are added to poly-L-ornithine solution A' before being added to sodium alginate solution B, and after stirring for a predetermined time, the particles are recovered and added to sodium alginate solution B in step (d). Here, poly-L-ornithine solution A' may have the same or a different concentration as poly-L-ornithine solution A. In one embodiment, the poly-L-ornithine concentration of poly-L-ornithine solution B is preferably lower than that of poly-L-ornithine solution A. For example, the poly-L-ornithine concentration of poly-L-ornithine solution A' is preferably 0.01 w / v% or more and less than 0.05 w / v%.

[0033] Steps (e) and (f) are essentially repetitions of steps (c) and (d). The concentration of poly-L-ornithine in poly-L-ornithine solution B is not particularly limited, but is preferably 0.01 w / v% or more and less than 0.05 w / v%.

[0034] The phrase "adding the particles recovered in step (d) to poly-L-ornithine solution B" in step (e) also encompasses an embodiment in which poly-L-ornithine solution B is added to the gelled particles recovered in step (d). The stirring speed and time in step (e) are optional, and are preferably, for example, 1 to 20 minutes.

[0035] The gelling particles can be collected in any manner in step (e), for example, by leaving the poly-L-ornithine solution A to stand for a certain period of time and removing the supernatant. The collected gelling particles preferably have a structure in which the surface of the alginic acid gelling layer is coated with poly-L-ornithine, alginic acid, and poly-L-ornithine.

[0036] The sodium alginate solution C used in step (f) may have the same concentration as or a different concentration from the sodium alginate solution B used in step (d). For example, the sodium alginate concentration of sodium alginate solution C is preferably 0.1 w / v% or more and 0.3 w / v% or less.

[0037] The phrase "adding the particles recovered in step (e) to the sodium alginate solution C" in step (f) also encompasses an embodiment in which the sodium alginate solution C is added to the gelled particles recovered in step (e). The stirring speed and time in step (f) are optional, and stirring for, for example, 1 to 15 minutes is preferred.

[0038] The gelling particles can be collected in any manner in step (f), for example, by leaving the sodium alginate solution C to stand for a certain period of time and removing the supernatant. The collected gelling particles preferably have a structure in which the surface of the alginic acid gelling layer is coated with poly-L-ornithine, alginic acid, poly-L-ornithine, and alginic acid.

[0039] The sodium citrate solution used in step (g) preferably contains sodium citrate at a concentration of 0.5 to 3 w / v %. The sodium citrate solution can be added to promote insulin release by chelating the cations in the gelling particles and changing the state of the gel to a liquid state.

[0040] The phrase "adding the particles recovered in step (d) to a sodium citrate solution" in step (g) also encompasses an embodiment in which a sodium citrate solution is added to the gelled particles recovered in step (d). The stirring speed and time in step (e) are optional, and are preferably, for example, 1 to 15 minutes.

[0041] The gelling particles can be collected in any manner in step (e), for example, by leaving the sodium citrate solution to stand for a certain period of time and then removing the supernatant. The collected gelling particles are considered to have an interior that is closer to a liquid state. [Example]

[0042] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0043] 1. Preparation of Triple Membrane Preparation Pancreatic islets isolated from 14-day-old pigs were suspended in 1.7 w / v% sodium alginate solution (Pronova) at a concentration of 16,000 IEQ / mL. The suspension was passed through a needle and dropped into 109 mM calcium chloride solution, using airflow to cut the suspension. The solidified alginate beads were collected from the calcium chloride solution and added to a 0.075 w / v% poly-L-ornithine (PLO) (molecular weight 5,000-15,000) solution and stirred for 10 minutes. The collected beads were then added to a 0.038 w / v% PLO solution and stirred for 6 minutes. Then, they were added to a 0.17 w / v% sodium alginate solution and stirred for 6 minutes. Finally, they were added to a 1.6% sodium citrate solution and stirred for 2 minutes. This resulted in encapsulated islets (preparation) in which the islets were covered with a triple membrane consisting of an alginate membrane, a polyornithine membrane, and another alginate membrane. The resulting encapsulated islets were cultured at 37°C in a CO 2 incubator for 25 days.

[0044] 2. Preparation of Five-layer Membrane Preparation 1 Pancreatic islets isolated from 14-day-old pigs were suspended in 1.7 w / v% sodium alginate solution (Pronova) at a concentration of 16,000 IEQ / mL. The suspension was passed through a needle and dropped into 109 mM calcium chloride solution, while the airflow was used to cut the suspension. The solidified alginate beads were collected from the calcium chloride solution and added to a 0.075 w / v% poly-L-ornithine (PLO) (molecular weight 5,000-15,000) solution and stirred for 10 minutes. The beads were then collected, added to a 0.038 w / v% PLO solution, stirred for 6 minutes, collected, and then added to a 0.17 w / v% sodium alginate solution and stirred for 6 minutes. The beads were then collected, added again to a 0.038 w / v% PLO solution, stirred for 6 minutes, collected, and added to a 0.17 w / v% sodium alginate solution and stirred for 6 minutes. Finally, the mixture was added to a 1.6% sodium citrate solution and stirred for 2 minutes to obtain encapsulated islets covered with a five-layer membrane consisting of an alginate membrane, a polyornithine membrane, an alginate membrane, a polyornithine membrane, and another alginate membrane (quintuple membrane preparation 1). The resulting encapsulated islets were cultured at 37°C in a CO2 incubator for 25 days.

[0045] 3. Preparation of Five-layer Membrane Preparation 2 Pancreatic islets from juvenile pigs isolated from 14-day-old pigs were suspended in 1.7 w / v% sodium alginate solution (Pronova) at a concentration of 16,000 IEQ / mL. The suspension was passed through a needle and dropped into 109 mM calcium chloride solution while being cut by airflow. The solidified alginate beads were collected from the calcium chloride solution and added to a 0.10 w / v% poly-L-ornithine (PLO) (molecular weight 5,000-15,000) solution and stirred for 10 minutes. The beads were then collected, added to a 0.05 w / v% PLO solution, stirred for 6 minutes, collected, and then added to a 0.17 w / v% sodium alginate solution and stirred for 6 minutes. The beads were then collected, added again to a 0.038 w / v% PLO solution, stirred for 6 minutes, collected, and added to a 0.17 w / v% sodium alginate solution and stirred for 6 minutes. Finally, the mixture was added to a 1.6% sodium citrate solution and stirred for 2 minutes to obtain encapsulated islets covered with a five-layer membrane consisting of an alginate membrane, a polyornithine membrane, an alginate membrane, a polyornithine membrane, and another alginate membrane (quintuple membrane preparation 2). The resulting encapsulated islets were cultured at 37°C in a CO2 incubator for 25 days.

[0046] 4. Preparation of Five-layer Membrane Preparation 3 Pancreatic islets isolated from 14-day-old pigs were suspended in 1.7 w / v% sodium alginate solution (Pronova) at a concentration of 16,000 IEQ / mL. The suspension was passed through a needle and dropped into 109 mM calcium chloride solution, using airflow to cut the suspension. The solidified alginate beads were collected from the calcium chloride solution and added to a 0.15 w / v% poly-L-ornithine (PLO) (molecular weight 5,000-15,000) solution and stirred for 10 minutes. The beads were then collected, added to a 0.075 w / v% PLO solution, stirred for 6 minutes, collected, and then added to a 0.17 w / v% sodium alginate solution and stirred for 6 minutes. The beads were then collected, added again to a 0.038 w / v% PLO solution, stirred for 6 minutes, collected, and added to a 0.17 w / v% sodium alginate solution and stirred for 6 minutes. Finally, the mixture was added to a 1.6% sodium citrate solution and stirred for 2 minutes to obtain encapsulated islets covered with a five-layer membrane consisting of an alginate membrane, a polyornithine membrane, an alginate membrane, a polyornithine membrane, and another alginate membrane (quintuple membrane preparation 4). The resulting encapsulated islets were cultured at 37°C in a CO2 incubator for 25 days.

[0047] 5. Capsule Size Measurement Encapsulated islets were placed in a 6-well plate, and capsules were photographed at 2x magnification using a stereomicroscope (M165FC, Leica). The diameters of 30 capsules were measured using the circle analysis tool in Leica Application Suite V3, and the average value was calculated.

[0048] 6. Capsule Strength Measurement Twenty to fifty encapsulated islets were placed in a 35 mm dish, and the capsules were crushed from above using a viscosity measuring device. The force at which the capsules were broken was measured. The applied force was divided by the number of capsules to determine the strength per capsule.

[0049] 7. Measurement of Islet Encapsulation Volume Pancreatic islets were placed in a 6-well plate and the number of islets 50 μm or larger was counted in 50 μm increments. The IEQ was calculated by multiplying each islet count by a factor determined for each size. The sum of these was taken as the total IEQ, and sodium alginate was added to adjust the total IEQ to 16,000 IEQ.

[0050] 8. Efficacy evaluation using diabetic mice C57BL / 6J mice with STZ-induced diabetes underwent a small abdominal incision along the midline. Encapsulated islets suspended in saline were transplanted into the incision at a dose of 10,000 IEQ / body using a sterile dropper. After transplantation, the mice were housed for 12 weeks, and blood glucose levels were measured over time by collecting blood samples from the tail vein. As shown in FIG. 1, it was confirmed that administration of the pentalayer membrane preparation was able to suppress the rise in blood glucose levels in diabetic mice for a longer period of time than administration of the triple membrane preparation.

[0051] 9. Measurement of fibrosis rate A small incision was made along the midline in the abdomen of healthy C57BL / 6J mice. Encapsulated islets were transplanted into the incision at a dose of 1,000 capsules / body using a sterile dropper. One week after transplantation, the encapsulated islets were collected from the abdominal cavity into a 125 mL container containing Hanks Balanced Salt Solution (HBSS). The supernatant was removed and washed with HBSS. The suspended encapsulated islets were placed in a 35 mm dish, and the number of capsules covered with fibroblasts was counted. The number of fibrotic capsules was divided by the total number of capsules in the dish, and the average value for the two dishes was expressed as the fibrosis rate. As shown in FIG. 2, it was confirmed that fibrosis of encapsulated islets was significantly suppressed when the diameter of the islets exceeded approximately 400 μm.

Claims

1. The core is covered with a five-layer membrane. the core comprises a pancreatic islet; The first, third and fifth membranes from the inside of the five-layer membrane contain alginic acid, and the second and fourth membranes from the inside contain polyornithine. formulation.

2. 10. The formulation of claim 1, wherein the average diameter of the formulation is 400 μm or greater.

3. 3. The formulation according to claim 1 or 2, wherein the average diameter of the formulation is 400 μm or more and 500 μm or less.

4. The preparation according to any one of claims 1 to 3, which is for treating diabetes.

5. The preparation according to any one of claims 1 to 4, wherein the pancreatic islets are obtained from a young pig aged 1 to 3 weeks.

6. (a) preparing a sodium alginate solution A containing pancreatic islets; (b) adding the sodium alginate solution A dropwise to a divalent cation solution and recovering gelled particles; (c) adding the particles recovered in step (b) to poly-L-ornithine solution A, stirring for a predetermined time, and then recovering the particles; (d) adding the particles recovered in step (c) to sodium alginate solution B, stirring for a predetermined time, and then recovering the particles; (e) adding the particles recovered in step (d) to poly-L-ornithine solution B, stirring for a predetermined time, and then recovering the particles; (f) adding the particles recovered in step (e) to sodium alginate solution C, stirring for a predetermined time, and then recovering the particles; and (g) adding the particles collected in step (f) to a sodium citrate solution, stirring for a predetermined period of time, and then collecting the particles. A method for producing the formulation according to any one of claims 1 to 5, comprising:

7. The method according to claim 6, wherein the poly-L-ornithine concentration of the poly-L-ornithine solution A is 0.05 w / v% or more and less than 1 w / v%.

8. The method according to claim 6 or 7, wherein in step (d), the particles recovered in step (c) are added to poly-L-ornithine solution A', stirred for a predetermined time, recovered, and then added to sodium alginate solution B.

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