US20070232533A1 - Use of Vinca Alkaloids and Salts Thereof - Google Patents

Use of Vinca Alkaloids and Salts Thereof Download PDF

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US20070232533A1
US20070232533A1 US10/556,309 US55630904A US2007232533A1 US 20070232533 A1 US20070232533 A1 US 20070232533A1 US 55630904 A US55630904 A US 55630904A US 2007232533 A1 US2007232533 A1 US 2007232533A1
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insulin
pancreas
conophylline
nicotinamide
cells
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Kazuo Umezawa
Hisako Ohgawara
Itaru Kojima
Takashi Koyano
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Keio University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to the use of vinca alkaloids and their salts. More specifically, the present invention relates to agents that enhance insulin-producing and/or -secreting abilities of non-neoplastic cells derived from the pancreas, therapeutic agents for diabetes, blood glucose level-lowering agents, methods for inducing differentiation of non-neoplastic cells derived from the pancreas, methods for enhancing insulin-producing and/or -secreting abilities of non-neoplastic cells derived from the pancreas, methods for producing pancreas-derived non-neoplastic cells whose insulin-producing ability has been enhanced, methods for culturing non-neoplastic cells derived from the pancreas, methods for producing insulin, pancreas-derived non-neoplastic cells that have been induced to differentiate, and pancreas-derived non-neoplastic cells whose insulin-producing and/or -secreting abilities have been enhanced.
  • Type 1 diabetes is caused by an autoimmune destruction of insulin-producing pancreatic ⁇ cells, resulting in an absolute lack of insulin.
  • type 2 diabetes is caused by the expression of insulin resistance in target tissues, such as muscle, fat, and liver, or by a decrease in blood insulin levels due to a decline in pancreatic ⁇ cell function.
  • target tissues such as muscle, fat, and liver
  • insulin injections are conventionally used to lower blood glucose levels in most cases. It is needless to say that, in such cases, four injections a day are laborious to patients.
  • the PPAR ⁇ inhibitor which reduces insulin resistance, is used in some cases. However, the inhibitor is not very effective and causes obesity as a side effect, as has been pointed out. In other cases, agents such as sulfonylurea, which promotes insulin release from ⁇ cells, are used, but they are also not very effective.
  • transplantation of cells or tissue has been considered promising as regenerative therapy. If large amounts of cells with insulin-releasing ability can be transplanted into type I diabetes patients, four insulin injections per day can be avoided for a long term. Meanwhile, transplantation of insulin-releasing cells is also effective for type 2 diabetes because, irrespective of insulin resistance in target tissues, normal insulin production supresses the increase of blood glucose level. It is expected that the efficacy of transplantation is far superior to that of treatment with agents currently used, e.g., sulfonylurea, that induce insulin release from ⁇ cells.
  • agents currently used e.g., sulfonylurea
  • Porcine pancreatic cells are expected to be utilized in regenerative therapy for diabetes because of their ease of availability, immunological properties, etc.
  • the technique for collecting large amounts of cells and inducing them to differentiate into insulin producing and releasing cells to a sufficient degree has never been known in any cell system.
  • pancreatic ⁇ cells are generated in the fetal period and then proliferate and differentiate very slowly (Herrera, P. L. et al., Development 127: 2317-2322 (2000)).
  • experiments have shown that when the pancreas is damaged ⁇ cells actively differentiate and proliferate. Differentiation and proliferation of ⁇ cells, together with growth of remnant ⁇ cells, occur from pancreatic ductal cells both in adult mice subjected to a 90% partial pancreatectomy and in mice that have developed impaired glucose tolerance due to ⁇ cell destruction induced by alloxan.
  • stem cells are also present in the adult pancreas and have regenerative capacity (Bonner-Wier S. et al., Diabetes 42: 1715-1720 (2000)).
  • pancreatic ⁇ cells While studies are under way on cells that can potentially serve as materials for pancreatic ⁇ cells, physiologically active substances that induce differentiation of pancreatic ⁇ cells are considered promising for clinical application in the field of regenerative medicine. Examples of substances that have thus far been known as inducers of differentiation into ⁇ cells include activin A, which belongs to the TGF- ⁇ superfamily (Demeterco, C. J. et al., Clin. Endo. 85: 3892-3897 (2000)); betacellulin (BTC), which belongs to the EGF family (Ishiyama, N. et al., Diabetologia 41: 623-628 (1998) and Yamamoto, K.
  • activin A which belongs to the TGF- ⁇ superfamily (Demeterco, C. J. et al., Clin. Endo. 85: 3892-3897 (2000))
  • betacellulin (BTC) which belongs to the EGF family (Ishiyama, N. et al., Diabetologia 41: 623-628 (
  • HGF hepatocyte growth factor
  • bFGF basic fibroblast growth factor
  • nicotinamide acts as a poly (ADP-ribose) synthetase inhibitor, promoting regeneration of pancreatic ⁇ cells (Watanabe, T. et al., Proc. Natl. Acad. Sci. USA 91: 3589-3592 (1994) and Sjoholm, A. et al., Endocrinology 135: 1559-1565 (1994)).
  • nicotinamide has also been reported to promote the expression of Reg protein (Watanabe, T. et al., Proc. Natl. Acad. Sci.
  • pancreatic ⁇ cells pancreatic ⁇ cells
  • ICCs pancreatic islet-like cell clusters
  • conophylline Umezawa, K. et al., Anticancer Res. 14: 2413-2418 (1994)
  • conophyllidine Kam, T. S. et al., J. Nat. Prod. 56: 1865-1871 (1993)
  • Conophylline is known to exhibit anti-tumor activity in animals (Umezawa, K. et al., Drugs Exptl. Clin. Res. 22: 35-40 (1996)).
  • the object of the present invention is to provide agents capable of inducing insulin production and/or secretion of non-neoplastic cells derived from the pancreas.
  • the present inventors have intensively studied to solve the above-mentioned problems and, as a result, found that vinca alkaloids markedly induce differentiation of normal pancreatic cells into insulin-producing and -releasing cells in vitro. Thus, the present invention has been accomplished.
  • vinca alkaloids refer to vinblastine and vincristine isolated from Vincarosea, an Apocynaceae family plant, and to alkaloids containing the backbone represented by the following structural formula: It should be noted that alkaloids refer to cyclic compounds produced by plants with a nitrogen atom (N) in the ring. Specific examples of vinca alkaloids include, but not limited to vinblastine, vincristine, conophylline, conophyllidine, conofoline, conophyllinine, taberhanine, pachysiphine etc., as shown in FIG. 1 .
  • Non-neoplastic cells derived from the pancreas refer to cells without tumorigenicity that are derived from the pancreas of individual organisms. Such cells include those harvested from the pancreas of organisms and those cultivated from the harvested cells (i.e., cultured cells). Cultured cells include both primary cultured cells and successively transferred cells.
  • “To increase insulin-producing ability and/or -secreting abilities of cells” is a concept including both causing cells that do not have insulin-producing and/or -secreting abilities to acquire insulin-producing and/or -secreting abilities and causing cells that have insulin-producing and/or -secreting abilities to enhance their insulin-producing and/or -secretion abilities.
  • To differentiate into ⁇ cells means that progenitor cells of ⁇ cells come to produce and secrete insulin.
  • conophylline a kind of vinca alkaloid, induces insulin production of pancreatic acinar carcinoma AR42 J-B13 cells, but the AR42 cells were found to be incapable of releasing insulin out of cells, though they do produce insulin. It was unpredictable that, under conditions in which only such weak effects are known, a vinca alkaloid induces insulin production, and further, can even release insulin out of cells when non-neoplastic cells derived from the pancreas were used instead.
  • Activin which is capable of inducing AR42J cells to produce insulin, does not exhibit the effect on porcine pancreatic cells.
  • LAF leukocyte activating factor
  • LAF leukocyte activating factor
  • the conditions under which cells are induced to differentiate are different depending on the individual cell type. For this reason, even those skilled in the art could not predict that a vinca alkaloid increases insulin production of non-neoplastic cells derived from pancreas and induces insulin to be secreted out of the cells.
  • AR42J cells were cancer cells, they could not be used for regenerative medicine from the viewpoint of safety even if their insulin-producing ability was increased.
  • the technique of increasing insulin-producing and -secreting abilities of non-neoplastic cells derived from the pancreas has been established by the present invention, which has made it possible to prepare large amounts of cells available for regenerative medicine.
  • FIG. 1 shows the chemical structural formulae of several compounds belonging to the vinca alkaloid family.
  • FIG. 2 shows the results of immunostaining of fetal porcine pancreatic cells cultured in media to which the following were added for three weeks: vehicle (1) ; only nicotinamide (10 mM) (2); nicotinamide (10 mM) and HGF (10 ng/ml) (4);only conophylline (0.1 ⁇ g/ml) (5); conophylline (0.1 ⁇ g/ml) and HGF (10 ng/ml) (7); conophylline (0.1 ⁇ g/ml), nicotinamide (10 mM), and HGF (10 ng/ml) (8).
  • N and CNP denote nicotinamide and conophylline, respectively.
  • FIG. 3 shows the results of ELISA measurement of the amount of insulin produced by fetal porcine pancreatic cells cultured in media to which the following were added: vehicle (white circle); nicotinamide and HGF(white triangle); only conophylline (black circle); conophylline, nicotinamide, and HGF (black triangle).
  • N and CNP denote nicotinamide and conophylline, respectively.
  • FIG. 4 shows the effect of conophylline on blood glucose levels of streptozotocin-administered rats.
  • Embodiments of the present invention accomplished based on the above-described findings are hereinafter described in detail by giving Examples. Unless otherwise explained, methods described in standard sets of protocols such as J. Sambrook and E. F. Fritsch & T. Maniatis (Ed.),“Molecular Cloning, a Laboratory Manual (3rd edition), Cold Spring Harbor Press and Cold Spring Harbor, N.Y. (2001); and F. M. Ausubel, R. Brent, R. E. Scientific, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (Ed.), “Current Protocols in Molecular Biology,” John Wiley & Sons Ltd., or alternatively, modified/changed methods from these are used. When using commercial reagent kits and measuring apparatus, unless otherwise explained, attached protocols to them are used.
  • FIG. 1 The chemical structural formulae of some compounds belonging to the vinca alkaloid family are shown in FIG. 1 .
  • Vinblastine and vincristine can be isolated and purified from Vinca rosea Linn by the method described in Neuss N, Gorman M, Hargrove W, et al., & Manning R E (1964) J. Am. Chem. Soc. 86: 1440-1442,
  • Conophylline can be isolated and purified from leaves of Ervatamia microphylla, an Apocynaceae family plant, in the manner as will be described later in Production example 1 (a method modified from Umezawa, K. et al. Anticancer Res. 14: 2413-2418 (1994)). About 4 kg of Ervatamia microphylla leaves yields about 500 mg of conophylline crystals.
  • Conophyllidine can be prepared from leaves of Ervatamia microphillae in the same manner as conophylline (Kam, T. S. et al. J. Nat. Prod. 56: 1865-1871 (1993)).
  • Conofoline and pachysiphine can be prepared by the methods described in Kam T S, Anuradha S. Alkaloids from Tabernaemontana divaricata, and Phytochemistry (1995) 40: 313-6.
  • Conophyllinine and taberhanine can be prepared by the methods described in Kam T S, Pang H S, Lim T M, Biologically active indole and bisindole alkaloids from Tabernaemontana divaricata. Org. Biomol. Chem. (2003) 21; 1(8): 1292-7.
  • salts of vinca alkaloids include hydrochlorides and sulphates, which are pharmacologically acceptable. These salts can be produce by known methods.
  • Non-neoplastic cells derived from the pancreas are prepared.
  • Non-neoplastic cells derived from the pancreas may be derived from mammals. Such mammals include primates, such as humans and monkeys, as well as non-primates, such as pigs, cattle, dogs, and rats.
  • Cells may be derived from healthy animals or from patients who need treatment. Patients are not limited to humans; they may be unhealthy non-human animals.
  • preferred animals are fetuses and neonates (for example, in the case of pigs, neonatal pigs within 3 days of birth)
  • Non-neoplastic cells may be exocrine cells or endocrine cells, but endocrine cells are preferred. Further, among endocrine cells, ⁇ cells and their progenitor cells are more preferred.
  • Isolation of non-neoplastic cells from the pancreas of healthy mammalian animals may be performed, for example, as follows: The pancreas is removed from a mammal and connective tissue is detached. The pancreas is dissected, buffer is added, and the mixture is stirred. After stirring, the supernatant is discarded, an enzyme Liberase is added, and the mixture is stirred again. Subsequently, a cycle of centrifugation, discarding of the supernatant, and addition of PBS is repeated. Cells are suspended with PBS and this cell suspension is overlaid on Histopaque, followed by centrifugation.
  • pancreatic endocrine cells forms a belt-like white layer at the interface between cell suspension and Histopaque, this layer is harvested.
  • the sample is centrifuged and the supernatant is discarded.
  • the cells is suspended in medium and transferred to the culture chamber to be incubated. Subsequently, spheroids (loose cell aggregates) are formed by stirring for an appropriate time, followed by incubation. Cells detached from the chamber and floating in the medium are used for the subsequent operations.
  • the cells floating in the culture chamber are transferred to the centrifuge tube, which is let to stand still until spheroids have sunk to the bottom. Then the supernatant is discarded and medium was added, followed by light shaking. The tube is let to stand still and the spheroids are allowed to settle down to the bottom. After this operation is repeated 2 or 3 times, the cell lysate is centrifuged and the supernatant is discarded.
  • the residual cells are cultured under the condition of 5% CO 2 and 37° C. in medium supplemented with a vinca alkaloid or its pharmacologically acceptable salt.
  • the culture may be stationary culture. Examples of suitable media include RPMI medium etc.
  • nicotinamide and hepatocyte growth factor (HGF) in addition to a vinca alkaloid or its pharmacologically acceptable salt.
  • the medium may be changed every 4 to 7 days and the culture is incubated for 7 to 35 days.
  • the morphology of the cells may be examined at suitable time intervals during the incubation period. Further, when the medium is changed, the culture solution may be recovered and subjected to the measurement of the insulin amount in the medium.
  • the methods for separating and culturing cells described above can also be applied when non-neoplastic cells derived from patients' pancreas are prepared. It should be noted that, to obtain non-neoplastic cells derived from patients' pancreas, a method is suggested in which tissue fragments are harvested from patients' pancreas so that non-neoplastic cells are isolated from these tissue fragments by the above-described methods.
  • non-neoplastic cells derived from the pancreas in the presence of a vinca alkaloid or its salt, differentiation of non-neoplastic cells derived from the pancreas can be induced.
  • non-neoplastic cells derived from the pancreas can be induced to differentiate into insulin-producing and -releasing cells (e.g., ⁇ cells).
  • insulin-producing and/or -secreting abilities of normal pancreatic cells can be increased.
  • insulin can be isolated and purified from cultures (cultured cells or a medium) by known methods.
  • pancreas-derived non-neoplastic cells whose insulin-producing and/or -secreting abilities have been increased by culturing in the presence of a vinca alkaloid or its salt are capable of producing 10 ng or more, preferably 25 ng or more, and more preferably 55 ng or more in 1 ml of medium when they were cultured at the concentration of 2.5 ⁇ 10 5 cells per 1 ml of medium for 7 to 35 days.
  • porcine pancreatic cells are expected to be used in regenerative therapy for diabetes because of their ease of availability, immunological properties, etc.
  • the method for collecting large amounts of cells to induce them to differentiate into insulin-producing and -releasing cells to a sufficient degree has never been known in any cell system.
  • the technique according to the present invention makes it possible to induce large amounts of cells to differentiate into insulin-producing and -releasing cells to a sufficient degree. Accordingly, insulin-producing and -releasing cells obtained by the methods according to the present invention can be used in regenerative medicine for diabetes.
  • Insulin-producing and -releasing cells obtained by the methods according to the present invention may be suspended in a solution and/or embedded in a support matrix and then administered to subjects.
  • the solution in which insulin-producing and -releasing cells are suspended may be a pharmacologically acceptable carrier and diluent, such as saline, a buffer solution, etc.
  • a preservative e.g., p-hydroxybenzoic acid ester, chlorobutanol, thiromesal, etc.
  • a stabilizer e.g., L-arcorbic acid, etc.
  • a support matrix in which insulin-producing and -releasing cells are embedded may be a matrix that is recipient-compatible and that degrades into a product not harmful to the recipient.
  • Materials for the matrix may include natural polymers, synthetic polymers, etc. Examples of natural polymers include collagen, gelatin, etc. Examples of synthetic polymers include polyglycolic acid, polylactic acid, etc.
  • the matrix can be in the form of, but not limited to, film, sheet, particle, paste, etc.
  • Vinca alkaloids and their pharmacologically acceptable salts can increase insulin-producing and/or -secreting abilities of non-neoplastic cells derived from the pancreas. Further, vinca alkaloids and their pharmacologically acceptable salts can also decrease blood glucose levels. Therefore, these compounds may be administered to human and other animals as drugs (e.g., therapeutic agents for diabetes) or may be used as reagents in experiments. These compounds may be used alone or in combination with other agents (e.g., other therapeutic agents for diabetes).
  • nicotinamide and/or hepatocyte growth factor may be simultaneously administered.
  • a vinca alkaloid or its pharmacologically acceptable salt When administered to humans, a vinca alkaloid or its pharmacologically acceptable salt may be administered orally.
  • the dosage is, for example, 0.1 to 10 mg/kg bw daily in a single dose or divided doses. However, the amount of a dose and number of administration can be suitably changed depending on the symptoms, age, dosage regimen, etc.
  • Vinca alkaloids and their pharmacologically acceptable salts may be administered orally in preparations, such as tablets, capsules, granule, powder, syrups, etc. Alternatively, they may be administered parenterally by intraperitoneal or intravenous injection in preparations such as injectable formulations, suppositories, etc.
  • the content of a vinca alkaloid or its pharmacologically acceptable salt (active ingredient) in a preparation can vary between 1 to 90% by weight.
  • the preparation is in the form of a tablet, a capsule, a granule, a powder, etc.
  • the content of an active ingredient is preferably 5 to 80% by weight.
  • the content of an active ingredient is preferably 1 to 30% by weight.
  • the content of an active ingredient is preferably 1 to 10% by weight.
  • Vinca alkaloids and their pharmacologically acceptable salts are formulated by known methods using the following formulation additives: excipients (sugars such as lactose, sucrose, glucose, and mannitol; starches such as potato, wheat, and corn; inorganic substances such as calcium carbonate, calcium sulfate, and sodium hydrocarbonate; cellulose crystal; etc.), binders (starch-paste liquid, gum arabic, gelatin, sodium arginate, methylcellulose, ethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropylcellulose, carmellose, etc.), lubricants (magnesium stearate, talc, hydrogenerated vegetable oil, macrogol, and silicone oil), disintegrators (starch, agar, gelatin powder, cellulose crystal, sodium carboxymethylcellulose, calcium carboxymethylcellulose, calcium carbonate, sodium hydrocarbonate, sodium arginate, etc.), correctives (lactose, sucrose, glucose, manni
  • Vinca alkaloids and their pharmacologically acceptable salts can be used to prevent and/or treat diseases (e.g., diabetes and arteriosclerosis) associated with lack of insulin. Vinca alkaloids and their pharmacologically acceptable salts can also be used in studies of insulin production and/or secretion of pancreatic cells. Vinca alkaloids and their pharmacologically acceptable salt can further be used for blood glucose level-lowering agents as well as for therapeutic agents for complications resulting from prolonged high blood glucose levels, such as retinopathy of the eyes, nephropathy, neuropathy, gangrene, arteriosclerosis, etc.
  • diseases e.g., diabetes and arteriosclerosis
  • Vinca alkaloids and their pharmacologically acceptable salts can also be used in studies of insulin production and/or secretion of pancreatic cells. Vinca alkaloids and their pharmacologically acceptable salt can further be used for blood glucose level-lowering agents as well as for therapeutic agents for complications resulting from prolonged high blood glucose levels, such as retinopathy
  • Conophylline was isolated and purified from leaves of Ervatamia microphylla, an Apocynaceae family plant, (harvested in Khon Khen, Thailand) in the following manner.
  • Active substance was extracted from about 4 kg of Ervatamia micorophylla leaves with 100 L of chloroform to afford about 130 g of oily substance.
  • This oily substance was chromatographed on a silica gel column (purification was performed by a total of 5 rounds of column chromatography using about 500 g of silica gel), eluting sequentially with chloroform:methanol (40:1 and 20:1). Subsequently, using morphological changes of K-ras-NRK cells as an activity marker, the fractions exhibiting this biological activity were recovered.
  • the crude purified product obtained (about 40 g) was chromatographed on a silica gel column with n-hexane:ethyl acetate (1:2 and 0:1) (using about 500 g of silica gel; purchased from Merck Co.) to afford 1.5 g of active fractions.
  • the active fractions were then chromatographed on a silica gel column (using 150 g of silica gel) with n-hexane:ethyl acetate:chloroform (9:3:1 and 6:3:1), active fractions were recovered, and concentrated to afford about 500 mg of crystals.
  • Neonatal pigs within 72 hours of birth were obtained from the (Takayama) pig farm.
  • the whole pancreas (ventral and dorsal pancreas) was removed under general anesthesia immediately after animals were brought to the operating room.
  • the pancreas was transferred to a 10 ml beaker and dissected into small pieces with ophthalmic scissors.
  • the dissected pieces were transferred to a 100 ml conical flask, to which 50 to 60 ml of phosphate buffered saline (PBS) was added.
  • PBS phosphate buffered saline
  • the cells collected in the centrifugal sedimentum was suspended in 25 to 50 ml of PBS, 25 ml of cell suspension was overlaid gently on 10 ml of Histopaque 1077 (Sigma), followed by centrifugation at 1800 rpm for 10 min.
  • Pancreatic (endocrine) cells form a band-like white layer at the interface between cell suspension and Histopaque (cell separation and purification methods)
  • Cells present at the interface are recovered with a Pasteur pipette, suspended in RPMI 1640 supplemented with 10 mM Nicotinamide and 10% heat-inactivated FBS, collected by centrifugation at 1200 rpm for 5 min. These cells were resuspended in the same medium and then subjected to stationary culture in a 75 ml culture flask (5% CO 2 incubator, 37° C.) for a whole day and night.
  • Cells floating in the flask were removed, and cells adhering to the bottom were detached with EDTA-Trypsin and collected.
  • Cells were suspended in each of media (groups 1 to 8) and stationary culture was performed by plating cells (1.25 ⁇ 10 3 cells/ml) in culture chambers (2 ml each; 5% CO 2 -incubator, 37° C.).
  • the culture medium was exchanged every four days and the observation was made for three weeks, during which time the morphology of cells was observed every week and the appearance of pancreatic ⁇ -cells was confirmed by immunofluorescence using peroxidase (Experiment example 1). Coloration was performed using 3-amino-9-ethyl carbazole (AEC; 0.75 mg/ml) as a substrate. In addition, the medium was recovered every time it was exchanged (every four days) for measurement of the amount of insulin secreted into the medium by ELISA (Experiment example II).
  • FIG. 2 shows the results of Example 1 It has been reported that nicotinamide (Akiyama, T. et al., Proc. Natl. Acad. Sci. USA 98: 48-53 (2001)) and HGF (Ocana, A. G. et al., J. Biol. Chem. 275: 1226-1232 (2000)) promote differentiation of insulin-producing cells in the limited experimental systems, but their effects are weak. As shown in FIG.
  • the mixture of nicotinamide and conophylline (6) produced a smaller number of insulin-producing cells than the aforementioned triple mixture the mixture (6) did; however, nicotinamide-conophylline mixture had a marked increase in that number, compared with the others (not shown in the figure).
  • FIG. 3 shows the results of Example II. As indicated in FIG. 3 , nicotinamide combined with either HGF or conophylline cultured for 8 to 20 days was able to produce some amount of insulin in the medium. Furthermore, nicotinamide combined with HGF and conophylline had a marked increased in the amount of insulin release, compared with other combinations or alone.
  • conophyllidine like conophylline, has been confirmed to induce morphological changes involved in insulin production of pancreatic acinar carcinoma AR42J cells, suggesting that conophyllidine, like conophylline, has the effect of increasing insulin-producing and/or -secreting abilities of non-neoplastic cells derived from the pancreas.
  • AR42J-B13 cells (endowed by Dr. Itaru Kojima, Institute for Cellular and Molecular Regulation, Gunma University) a highly sensitive subclone of AR42J, were cultured at 37° C. in a 5% CO 2 incubator with 20 ml of culture medium DMEM supplemented with 100 mg/l kanamycin, 0.6 g/l glutamine, 100 unit/ml penicillin G, 5 mM NaHCO 3 , and 10% FBS.
  • the cells were transferred every 2 to 3 days to maintain 2.5% to 5% of confluency.
  • the cell transfer was performed as follows: After the medium was removed, the cells were washed twice in PBS ⁇ (Ca 2+ , Mg 2+ -free PBS; 8 g/l, NaCl, 0.2 g/l KCl, 0.916 g/Na 2 HPO 4 , 0.2 g/l KH 2 PO 4 ). Subsequently, the cells were detached using 2 ml of trypsin-EDTA solution, and then trypsin was inactivated by addition of 8 ml of the medium.
  • DMSO dimethyl sulfoxide
  • the morphology of the viable cells was observed and photographed at 150 ⁇ magnification with a camera linked to the microscope. Subsequently, cells were detached with trypsin and all the solution was transferred to the aforementioned Eppendorf tube for trypan blue exclusion test. Further, the photograph was enlarged 2.5 times and a morphological change was defined as having occurred when the full length of a cell in the diameter became 1.5 times; thus, the rates of morphological changes were determined.
  • Cells that had been prepared at a density of 2 ⁇ 10 5 cells/ml were plated at 500 ⁇ l per well on 8-well plates, followed by addition of either 0.1 ⁇ g/ml conophylline or 0.1 ⁇ g/ml conophylline plus 100 pM HGF. Subsequently, the cells were incubated at 37° C. in a 5% CO 2 incubator for 72 hours to differentiate. After the medium was removed, the cells were washed once with PBS ⁇ for fluorescent antibodies (8 g/l NaCl, 50.45 g/l NaH 2 PO 4 .2H 2 O, 1.28 g/l Na 2 HPO 4 ) and fixed with 3% formaldehyde at 4° C.
  • fluorescent antibodies 8 g/l NaCl, 50.45 g/l NaH 2 PO 4 .2H 2 O, 1.28 g/l Na 2 HPO 4
  • the cells were washed twice with PBS ⁇ for fluorescent antibodies and blocked by adding 1 ml of 1% BSA solution (a solution of PBS ⁇ for fluorescent antibodies) per well, followed by incubation at room temperature for 1 hour. To quench with 50 mM glycine (a solution of PBS ⁇ for fluorescent antibodies), cells were incubated for 5 min, followed by washing 3 times. Next, the solution in the plastic wells was removed, 50 ⁇ l per well of alpha-insulin antibody diluted 100-fold with a 10-fold diluted blocking buffer was placed taking care not to allow cells to dry, and the plates were allowed to stand still at room temperature for 1 hour.
  • BSA solution a solution of PBS ⁇ for fluorescent antibodies
  • a well of cells induced to differentiate with 2 nM activin A and 100 pM HGF was prepared and preserved without primary antibody for the purpose of comparing the influence of the nonspecific binding of the secondary antibody.
  • slides were transferred to the chamber and washed three times with PBS ⁇ for fluorescent antibodies for 5 min while shaking on a shaker.
  • Hoechst 33258 was added to Cy3-conjugated anti-guinea pig antibody diluted 100-fold with a 10-fold diluted blocking solution at a 1:100 dilution, and the antibody was overlaid as was the primary antibody.
  • the slides were shielded from light with aluminum foil, allowed to stand still at room temperature for 1 hour and then placed in the shielded chamber, followed by washing three times for 10 min with TNT buffer (0.1 M Tris-HCl, 0.15 MNaCl, 0.05% Tween 20; pH 7.5) under light shielding on the shaker.
  • TNT buffer 0.1 M Tris-HCl, 0.15 MNaCl, 0.05% Tween 20; pH 7.5
  • the slides were overlaid with 50% glycerol in PBS ⁇ , covered with coverslips, and photographed under the microscope.
  • the results of the immunofluorescence revealed the following: After 72 hours, when 100 pM HGF was used alone, no red coloration indicating insulin was observed in cells. When 0.1 ⁇ g/ml conophylline and 100 pM HGF were used in combination, however, red coloration of insulin was observed in the cytoplasm excluding the nucleus. Further, the coloration was seen scattered as if insulin was confined in granules in the cytoplasm. No insulin coloration was observed in the vicinity of the cell membrane involved in insulin release.
  • conophylline is capable of inducing morphological changes of ARJ42 cells, thereby inducing ARJ cells to produce insulin, but that conophylline is incapable of inducing insulin secretion out of cells.
  • Example 2 revealed that conophylline has the effect of inducing pancreatic cells to produce and release insulin in vitro. To examine whether the administration of conophylline induces insulin production in vivo as well, changes in blood glucose levels caused by the administration of conophylline in vivo were measured.
  • mice Ten 1-day old Wistar rats (purchased from Japan SLC, Shizuoka, Japan) were intraperitoneally injected with streptozotocin (purchased from Wako Pure Chemical Industries, Ltd., dissolved in 0.05 mM citrate buffer (pH 4.5)) at 85 ⁇ g/g BW per rat. Streptozotocin decreases insulin production by destroying pancreatic ⁇ cells, thereby inducing diabetes.
  • the day of streptozotocin injection was defined as day 0. Starting from the next day (day 1), five rats were injected subcutaneously with a solution (ethanol) of conophylline at 5 ⁇ g/g BW for seven consecutive days and their random blood glucose levels were measured daily. Five rats in the control group received the same volume of solvent (Control).
  • conophylline alone exerted an effect, as compared with the results obtained in vitro. This suggests that when conophylline has been administered into the body, endogenous nicotinamide and/or hepatocyte growth factor (HGF) have been utilized.
  • HGF hepatocyte growth factor
  • the agents according to the present invention can induce differentiation of non-neoplastic cells derived from the pancreas. Further, the agents according to the present invention can increase insulin-producing and/or -secreting abilities of non-neoplastic cells derived from the pancreas.

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100196468A1 (en) * 2007-07-11 2010-08-05 Pierre Fabre Medicament Stable pharmaceutical composition comprising a hydrosoluble vinflunine salt
US20100286184A1 (en) * 2006-03-07 2010-11-11 Kazuo Umezawa Aqueous solution of conophylline and/or conophyllidine
US20160122762A1 (en) * 2014-10-27 2016-05-05 University Of Iowa Research Foundation Methods of treating atherosclerosis
CN115073481A (zh) * 2021-03-13 2022-09-20 中国科学院昆明植物研究所 一种呋喃白坚木碱二聚体或其药学上可接受的盐及其制备方法和应用、药物组合物

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JPH11322602A (ja) 1998-05-13 1999-11-24 Kazuo Umezawa 転写因子ＮＦκＢ活性化阻害剤
US6159443A (en) 1999-04-29 2000-12-12 Vanderbilt University X-ray guided drug delivery
JP4086492B2 (ja) * 2001-10-02 2008-05-14 協和醗酵工業株式会社 糖尿病治療用医薬
JP2004121165A (ja) * 2002-10-07 2004-04-22 Asahi Kasei Corp 膵幹様細胞の分化誘導方法

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100286184A1 (en) * 2006-03-07 2010-11-11 Kazuo Umezawa Aqueous solution of conophylline and/or conophyllidine
US8178135B2 (en) 2006-03-07 2012-05-15 Keio University Aqueous solution of conophylline and/or conophyllidine
US20100196468A1 (en) * 2007-07-11 2010-08-05 Pierre Fabre Medicament Stable pharmaceutical composition comprising a hydrosoluble vinflunine salt
US9173842B2 (en) * 2007-07-11 2015-11-03 Pierre Fabre Medicament Stable pharmaceutical composition comprising a hydrosoluble vinflunine salt
US20160122762A1 (en) * 2014-10-27 2016-05-05 University Of Iowa Research Foundation Methods of treating atherosclerosis
CN115073481A (zh) * 2021-03-13 2022-09-20 中国科学院昆明植物研究所 一种呋喃白坚木碱二聚体或其药学上可接受的盐及其制备方法和应用、药物组合物

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US20090239301A1 (en) 2009-09-24
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EP1623986A1 (fr) 2006-02-08
US8394629B2 (en) 2013-03-12

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