Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/37—Digestive system
  • A61K35/39—Pancreas; Islets of Langerhans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0676—Pancreatic cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/90—Polysaccharides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/998—Proteins not provided for elsewhere
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/999—Small molecules not provided for elsewhere

Definitions

• the subject matter disclosed generally relates to the use of anti-aging glycopeptides. More particularly, the subject matter relates to the use of anti-aging glycopeptides to enhance human pancreatic cells engraftment.
• Antifreeze biological compounds and particularly glycoproteins, exist in the natural environment. These compounds are present for example in some fishes, enabling them to survive in a low temperature environment (i.e. near zero or sub-zero temperatures).
• a low temperature environment i.e. near zero or sub-zero temperatures.
• scientists have been investigating how antifreeze compounds taken from the natural environment (fish, amphibians, plants, insects, etc.) have an influence on these phenomena.
• Research has focused on the synthesis of analogous compounds that are sufficiently stable and whose activity is at least equal to or even greater than the activity of the natural molecules, for commercial applications.
• AFP Anti-freeze proteins
• Anti-aging glycopeptides (AAGPTM) compounds are gem difluorinated C- glycopeptides which have been proposed to have applicability under harsh cellular stresses, such as nutrient deprivation, high temperature and cryopreservation, oxidative stress from hydrogen peroxide (H2O2), UV irradiation, and inflammation.
• harsh cellular stresses such as nutrient deprivation, high temperature and cryopreservation, oxidative stress from hydrogen peroxide (H2O2), UV irradiation, and inflammation.
• Beta cell transplantation is the transplantation of isolated beta islet cells from a donor pancreas and into another person. It is an experimental treatment for type 1 diabetes mellitus. Once transplanted, the islet beta cells begin to produce insulin, actively regulating the level of glucose in the blood. While significant progress has been made in the islet transplantation field, many obstacles remain that currently preclude its widespread application. Two of the most important limitations are the currently inadequate means for preventing islet rejection, and the limited supply of beta islet cells for transplantation. Current immunosuppressive regimens are capable of preventing beta islet cells failure for months to years, but the agents used in these treatments are expensive and may increase the risk for specific malignancies and opportunistic infections.
• an in vitro method for enhancing engraftment of isolated pancreatic cells, isolated pancreatic progenitor cells, or both comprising the step of:
• N is an integer between 1 and 5
• R 4 H, AAi, or AA AA 2 ,
• AAi and AA2 independently represent amino acids with a non-polar side chain
• R 1 , R 2 , R 3 are independent groups in which two of R 1 , R 2 and R 3 are selected from H, ChbPh, CH(CH 3 ) 2 , CH 2 CH(CH 3 )2 or CH(CH 3 )CH 2 CH 3 and the remaining R 1 , R 2 , R 3 is
• n is an integer between 3 and 4,
• Y, Y' are independent groups
• R H, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl or acetate group,
• R'" H, alkyl, or acetate group
• R 6 is H, CH 3 , CH2OH, CH 2 -glycoside group or CH 2 -OGP in which GP is a protector group selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group,
• R 7 OH, OGP', NH 2 , N 3 , NHGP' or NGP'GP" in which GP' and GP" are independently selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group,
• R 8 is a hydrogen atom H or a free or protected alcohol function
• n is an integer between 3 and 4,
• Y, Y' are independent groups
• R H, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group
• R'" H, alkyl, or acetate group
• R 6 is H, CH 3 , CH 2 OH, CH 2 -glycoside group, or CH 2 -OGP in which GP is a protector group selected from alkyl, benzyl, trimethylsilyl, tertbutyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group,
• R 7 OH, OGP', NH 2 , N 3 , NHGP', or NGP'GP" in which GP' and GP" are independently selected from alkyl, benzyl, trimethylsilyl, tertbutyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group,
• n is an integer between 3 and 4,
• Y, Y' are independent groups
• R H, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group
• R'" H, alkyl, or acetate group
• R 6 is H, CH 3 , CH 2 OH, CH 2 -glycoside group, or CH 2 -OGP in which GP is a protector group selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group,
• R 7 OH, OGP', NH 2 , N 3 , NHGP', or NGP'GP" in which GP' and GP" are independently selected from alkyl, benzyl, trimethylsilyl, tertbutyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group,
• R 8 is a hydrogen atom H or a free or protected alcohol function, if H, CH 3 , CH(CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , or CH(CH 3 )CH 2 CH 3
• n is an integer between 3 and 4
• Y, Y' are independent groups
• R H, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group
• R'" H, alkyl, or acetate group
• R 6 is H, CH 3 , CH 2 OH, CH 2 -glycoside group, or CH 2 -OGP in which GP is a protector group selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group,
• R 7 OH, OGP', NH 2 , N 3 , NHGP', or NGP'GP" in which GP' and GP" are independently selected from alkyl, benzyl, trimethylsilyl, tertbutyldimethylsilyl,
• R 8 is a hydrogen atom H or a free or protected alcohol function.
• an in vitro method for improving the insulin secretory function of isolated pancreatic beta cells prior to transplantation in a subject in need thereof comprising the step of:
• an in vitro method for protecting isolated pancreatic cells, isolated pancreatic progenitor cells, or both from immunosuppressant drug toxicity prior to a transplantation in a subject in need thereof comprising the step of:
• an in vitro method for decreasing an inflammatory response of isolated pancreatic cells, isolated pancreatic progenitor cells, or both prior to a transplantation in a subject in need thereof comprising the step of:
• a method of transplanting isolated pancreatic cells, isolated pancreatic progenitor cells, or both in a subject in need thereof comprising the steps of :
• step b) transplanting the treated isolated pancreatic cell of step a) in said subject in need thereof.
• the method may further comprise step a') before step a): a') isolating pancreatic cells, pancreatic progenitor cells, or both.
• the method may also further comprising step b') before step b) contacting the isolated pancreatic cell, the isolated pancreatic progenitor cells, or both of step a) with an immunosuppressant drug.
• the isolated pancreatic cell may be an isolated alpha cell, an isolated beta cell, an isolated delta cell, an isolated gamma cell, an epsilon cell, or a combination thereof, and preferably the isolated pancreatic cell may be an isolated beta cell.
• the immunosuppressant drug may be one of daclizumab, sirolimus, tacrolimus, cyclosporine, or combinations thereof.
• the subject may be a human subject.
• the isolated pancreatic cell may be isolated from a live donor, a cadaveric donor, or combinations thereof.
• the compound of formula I may be a compound of formula II:
• N is an integer between 1 and 5
• R 1 , R 2 , R 3 are independent groups in which two of R 1 , R 2 and R 3 are selected from H, CH 3 and the remaining R 1 , R 2 and R 3 is
• n is an integer between 3 and 4
• Y, Y' are independent groups
• R H, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group
• R'" H, alkyl, or acetate group
• R 6 is selected from H, CH 3 , CH 2 OH, CH 2 -OGP in which GP is a protector group selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group,
• R 7 OH, OGP', NH 2 , N 3 , NHGP', NGP'GP" in which GP' and GP" are independently selected from alkyl, benzyl, trimethylsilyl, tert- butyldimethylsilyl, tert-butyldiphenylsilyl or acetate group,
• R 8 is a hydrogen atom H or a free or protected alcohol function
• n is an integer between 3 and 4,
• Y, Y' are independent groups
• R H, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group
• R'" H, alkyl, or acetate group
• R 6 is selected from H, CH 3 , CH 2 OH, CH 2 -OGP in which GP is a protector group selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group,
• R 7 OH, OGP', NH 2 , N 3 , NHGP', NGP'GP" in which GP' and GP" are independently selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group,
• R 8 is a hydrogen atom H or a free or protected alcohol function
• n is an integer between 3 and 4,
• Y, Y' are independent groups
• R H, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group
• R'" H, alkyl, or acetate group
• R 6 is selected from H, CH 3 , CH 2 OH, or CH 2 -OGP in which GP is a protector group selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group,
• R 7 OH, OGP', NH 2 , N 3 , NHGP', or NGP'GP" in which GP' and GP" are independently selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group,
• R 8 is a hydrogen atom H or a free or protected alcohol function
• n is an integer between 3 and 4,
• Y, Y' are independent groups
• R H, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group
• R'" H, alkyl, or acetate group
• R 6 is selected from H, CH 3 , CH 2 OH, or CH 2 -OGP in which GP is a protector group selected from alkyl, benzyl, trimethylsilyl , tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group,
• R 7 OH, OGP', NH 2 , N 3 , NHGP', or NGP'GP" in which GP' and GP" are independently selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group,
• R 8 is a hydrogen atom or a free or protected alcohol function.
• the compound of formula I may be a compound of formula III:
• the contacting the isolated pancreatic cells may be with from about from about
• an isolated pancreatic cell prepared according to the method of the present invention.
• a method of transplanting isolated pancreatic cells, isolated pancreatic progenitor cells, or both in a subject in need thereof comprising the steps of :
• a method of treating diabetes comprising the step of :
• an isolated pancreatic cell, an isolated pancreatic progenitor cell, or both prepared according to the method of the present invention, for transplantation to a subject in need thereof.
• an isolated pancreatic cell an isolated pancreatic progenitor cell, or both prepared according to the method of the present inventiopn, for treatment of diabetes in a subject in need thereof.
• an isolated pancreatic cell prepared, an isolated pancreatic progenitor cell, or both according to the method of the present invention, for transplantation to a subject in need thereof
• an isolated pancreatic cell an isolated pancreatic progenitor cell, or both contacted with a gem-difluorinated C-glycopeptide compound of general formula I, or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above.
• the compound of general formula I as described above may be a compound of formula II as described above, and/or formula III as described above.
• the isolated pancreatic cell may be an isolated alpha cell, an isolated beta cell, an isolated delta cell, an isolated gamma cell, an epsilon cell, or a combination thereof, and preferably the isolated pancreatic cell may be an isolated beta cell.
• the isolated pancreatic cell, isolated pancreatic progenitor cell, or both may be contacted with from about from about 0.01 mg/ml to about 5 mg/ml of said compound of formula I, formula II or formula III, or from about from about 1 mg/ml to about 5 mg/ml of said compound of formula I, formula II or formula III.
• C-glycopeptide compound of general formula I as described above or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above, for enhancing engraftment of isolated pancreatic cells, isolated pancreatic progenitor cells, or both in a subject in need thereof.
• C-glycopeptide compound of general formula I as described above or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above, for improving the insulin secretory function of isolated pancreatic beta cells, isolated pancreatic progenitor cells, or both prior to transplantation in a subject in need thereof.
• the isolated pancreatic cell may be an isolated alpha cell, an isolated beta cell, an isolated delta cell, an isolated gamma cell, an epsilon cell, or a combination thereof, and preferably the isolated pancreatic cell may be an isolated beta cell.
• the immunosuppressant drug may be one of daclizumab, sirolimus, tacrolimus, cyclosporine, or combinations thereof.
• the subject may be a human subject.
• the isolated pancreatic cell, isolated pancreatic progenitor cell, or both may be isolated from a live donor, a cadaveric donor, or combinations thereof.
• the compound of general formula I as described above may be a compound of formula II as described above, and/or formula III as described above.
• the isolated pancreatic cell, isolated pancreatic progenitor cell, or both may be contacted with from about from about 0.01 mg/ml to about 5 mg/ml of said compound of formula I, formula II or formula III, or from about from about 1 mg/ml to about 5 mg/ml of said compound of formula I, formula II or formula III.
• a gem-difluorinated C- glycopeptide compound of general formula I as described above or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above, for use in enhancing engraftment of isolated pancreatic cells, isolated pancreatic progenitor cells, or both in a subject in need thereof.
• a gem-difluorinated C- glycopeptide compound of general formula I as described above or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above, for use in improving the insulin secretory function of isolated pancreatic beta cells, isolated pancreatic progenitor cells, or both prior to transplantation in a subject in need thereof.
• a gem-difluorinated C- glycopeptide compound of general formula I as described above or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above, for use in protecting isolated pancreatic cells, isolated pancreatic progenitor cells, or both from immunosuppressant drug toxicity prior to a transplantation in a subject in need thereof.
• a gem-difluorinated C- glycopeptide compound of general formula I as described above or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above, for use in decreasing an inflammatory response of isolated pancreatic cells, isolated pancreatic progenitor cells, or both prior to a transplantation in a subject in need thereof.
• the isolated pancreatic cell may be an isolated alpha cell, an isolated beta cell, an isolated delta cell, an isolated gamma cell, an epsilon cell, or a combination thereof, and preferably the isolated pancreatic cell may be an isolated beta cell.
• the immunosuppressant drug may be one of daclizumab, sirolimus, tacrolimus, cyclosporine, or combinations thereof.
• the subject may be a human subject.
• the isolated pancreatic cell, isolated pancreatic progenitor cell, or both may be isolated from a live donor, a cadaveric donor, or combinations thereof.
• the compound of general formula I as described above may be a compound of formula II as described above, and/or formula III as described above.
• the isolated pancreatic cell, isolated pancreatic progenitor cell, or both may be contacted with from about from about 0.01 mg/ml to about 5 mg/ml of said compound of formula I, formula II or formula III, or from about from about 1 mg/ml to about 5 mg/ml of said compound of formula I, formula II or formula III.
• composition » as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
• compositions or other compositions in general of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier.
• pharmaceutically acceptable or “acceptable” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
• pancreatic cell is intended to mean cells derived from the Islet of Langerhans from the pancreas from live or cadaveric donors isolated through known protocols and/or as described herein below, as well as cells of pancreatic origin grown in vivo, ex vivo and/or in vitro.
• the cells of the Islet of Langerhans include alpha cells, producing the hormone glucagon and representing about 15-20% of the islet cells, the beta cells producing the hormones insulin and amylin, and representing about 65-80% of the islet cells, the delta cells producing the hormone somatostatin and representing about 3-10% of the islet cells, the PP cells (also known as gamma cells) producing the hormone pancreatic polypeptide (3-5% of the islet cells, and the epsilon cells producing the hormone ghrelin, representing ⁇ 1 % of the islet cells.
• pancreatic beta cell and "isolated pancreatic beta cell” is intended to mean cells derived from pancreas from live or cadaveric donors isolated through known protocols and/or as described herein below, as well as beta cells of pancreatic origin grown in vivo, ex vivo and/or in vitro.
• pancreatic progenitor and “isolated pancreatic progenitor” is intended to mean cells derived from any suitable sources, such as embryonic stem cells (of human or other origin), that have differentiated or are differentiate naturally or through known protocols into pancreatic progenitor cells in vivo, ex vivo or in vitro.
• the isolated pancreatic progenitor cells have the potential to become pancreatic beta cells through further differentiation naturally or through treatment with known protocols, in vivo, ex vivo or in vitro.
• the isolated pancreatic progenitor cells may be obtained in vitro through a differentiation protocol, and further differentiated into pancreatic beta cells in vitro through a differentiation protocol.
• the isolated pancreatic progenitor cells may be obtained in vitro through a differentiation protocol, and further differentiated into pancreatic beta cells in vivo after implantation/transplantation into a patient in need thereof.
• alkyl as well as other groups having the prefix “alk”, such as alkoxy and alkanoyl, means carbon chains which may be linear or branched, and combinations thereof, unless the carbon chain is defined otherwise.
• alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and the like.
• the term alkyl also includes cycloalkyl groups, and combinations of linear or branched alkyl chains combined with cycloalkyl structures. When no number of carbon atoms is specified, C1-6 is intended.
• Cycloalkyl is a subset of alkyl and means a saturated carbocyclic ring having a specified number of carbon atoms.
• Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.
• a cycloalkyi group generally is monocyclic unless stated otherwise. Cycloalkyi groups are saturated unless otherwise defined.
• alkoxy refers to straight or branched chain alkoxides of the number of carbon atoms specified (e.g., C1-6 alkoxy), or any number within this range [i.e., methoxy (MeO-), ethoxy, isopropoxy, etc.].
• alkylthio refers to straight or branched chain alkylsulfides of the number of carbon atoms specified (e.g., C1-6 alkylthio), or any number within this range [i.e., methylthio (MeS-), ethylthio, isopropylthio, etc.].
• alkylamino refers to straight or branched alkylamines of the number of carbon atoms specified (e.g., C1-6 alkylamino), or any number within this range [i.e., methylamino, ethylamino, isopropylamino, t-butylamino, etc.].
• alkylsulfonyl refers to straight or branched chain alkylsulfones of the number of carbon atoms specified (e.g., C1-6 alkylsulfonyl), or any number within this range [i.e., methylsulfonyl (MeSC ), ethylsulfonyl, isopropylsulfonyl, etc.].
• alkylsulfinyl refers to straight or branched chain alkylsulfoxides of the number of carbon atoms specified (e.g., C1-6 alkylsulfinyl), or any number within this range [i.e., methylsulfinyl (MeSO-), ethylsulfinyl, isopropylsulfinyl, etc.].
• alkyloxycarbonyl refers to straight or branched chain esters of a carboxylic acid derivative of the present invention of the number of carbon atoms specified (e.g., Ci-6 alkyloxycarbonyl), or any number within this range [i.e., methyloxycarbonyl (MeOCO), ethyloxycarbonyl, or butyloxycarbonyl].
• Aryl means a mono- or polycyclic aromatic ring system containing carbon ring atoms.
• the preferred aryls are monocyclic or bicyclic 6-10 membered aromatic ring systems. Phenyl and naphthyl are preferred aryls. The most preferred aryl is phenyl.
• Heterocyclyl refer to saturated or unsaturated non-aromatic rings or ring systems containing at least one heteroatom selected from O, S and N, further including the oxidized forms of sulfur, namely SO and SO2.
• heterocycles include tetrahydrofuran (THF), dihydrofuran, 1 ,4-dioxane, morpholine, 1 ,4-dithiane, piperazine, piperidine, 1 ,3- dioxolane, imidazolidine, imidazoline, pyrroline, pyrrolidine, tetrahydropyran, dihydropyran, oxathiolane, dithiolane, 1 ,3-dioxane, 1 ,3-dithiane, oxathiane, thiomorpholine, 2-oxopiperidin-1- yl, 2-oxopyrrolidin-1-yl, 2-oxoazetidin-1-yl, 1
• Heteroaryl means an aromatic or partially aromatic heterocycle that contains at least one ring heteroatom selected from O, S and N. Heteroaryls thus include heteroaryls fused to other kinds of rings, such as aryls, cycloalkyls and heterocycles that are not aromatic.
• heteroaryl groups include: pyrrolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridyl, oxazolyl, oxadiazolyl (in particular, 1 ,3,4-oxadiazol-2-yl and 1 ,2,4-oxadiazol-3-yl), thiadiazolyl, thiazolyl, imidazolyl, triazolyl, tetrazolyl, furyl, triazinyl, thienyl, pyrimidyl, benzisoxazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, dihydrobenzofuranyl, indolinyl, pyridazinyl, indazolyl, isoindolyl, dihydrobenzothienyl, indolizinyl, cinnolinyl, phthalazinyl, quinazolinyl, naphth
• Halogen refers to fluorine, chlorine, bromine and iodine. Chlorine and fluorine are generally preferred. Fluorine is most preferred when the halogens are substituted on an alkyl or alkoxy group (e.g. CF3O and CF3CH2O).
• Figure 1 illustrates the use of AAGPTM to enhance human beta cell Health.
• Beta islet cells recovery after 24h with or without AAGPTM (n 6 per group);
• B) Beta islet cells viability (as measured with Syto® green/EB) after 24h with or without AAGPTM (n 6 per group); and
• C-D) Glucose-stimulated insulin release with or without AAGPTM (n 3 per group).
• Figure 2 illustrates the use of AAGPTM to protect human beta islet cell health Islet against tacrolimus (Tac) toxicity.
• FIG. 3 illustrates the use of AAGPTM to improve transplant outcome.
• Figure 4 illustrates the in vitro assessment of human islets in culture with or without AAGPTM supplementation.
• B Functional assessment with perifusion curves comparing glucose-stimulated insulin secretion (GSIS).
• Figure 5 illustrates in vitro assessment of human islets in culture with or without
• ROS reactive oxygen species
• FIG. 7 illustrates that AAGPTM effect is not the result of direct drug inhibition with tacrolimus.
• Figure 8 illustrates that acute exposure to tacrolimus does not affect intracellular content of calcium but decreases exocytosis. Intra-islet calcium concentration remains unchanged despite the experimental interventions.
• A Comparative intracellular calcium concentration and
• FIG. 9 illustrates the proinflammatory cytokines and chemokines acutely expressed (24h) after transplantation.
• Concentration of I L-1 ⁇ (A), IL-6 (B), TNF (C) and Keratinocyte chemokine (KC) (D) were measured locally to the graft by homogenization.
• Concentrations of I L- ⁇ , IL-6 and Keratinocyte chemokine (KC) were significantly lower in the engrafted islets previously treated with AAGPTM.
• Figure 10 illustrates the apoptosis acutely measured 24h post-transplant.
• Figure 11 illustrates the post-transplant graft function in immunodefficient mice receiving minimal human islet mass (-1000 IEQ). Islets were previously treated with AAGPTM and tracrolimus accordingly.
• A Pooled blood glucose profiles demonstrating long-term graft function (60 days). The dotted line indicates graft recovery nephrectomy on day 60.
• B Intraperitoneal glucose tolerance test (IPGTT) to evaluate metabolic response after receiving a glucose bolus.
• IPGTT Intraperitoneal glucose tolerance test
• Beta cell transplantation is the transplantation of isolated beta islet cells from a donor pancreas into another person. It is an experimental treatment for type 1 diabetes mellitus. Once transplanted, the islet beta cells begin to produce insulin, actively regulating the level of glucose in the blood.
• Islets are usually infused into the patient's liver. If the cells are not from a genetically identical donor the patient's body will recognize them as foreign and the immune system will begin to attack them as with any transplant rejection. To prevent this, immunosuppressant drugs are used. While significant progress has been made in the islet transplantation field, many obstacles remain that currently preclude its widespread application. Two of the most important limitations are the currently inadequate means for preventing islet rejection, and the limited supply of beta islet cells for transplantation. Current immunosuppressive regimens are capable of preventing beta islet cells failure for months to years, but the agents used in these treatments are expensive and may increase the risk for specific malignancies and opportunistic infections.
• the most commonly used agents include daclizumab (ZenapaxTM), sirolimus (RapamuneTM), cyclosporine and tacrolimus (PrografTM)] are also known to impair normal beta islet cells function and/or insulin action.
• an in vitro method for enhancing engraftment of isolated pancreatic cells, isolated pancreatic progenitor cells, or both comprising the step of:
• N is an integer between 1 and 5
• AAi and AA2 independently represent amino acids with a non-polar side chain
• R 1 , R 2 , R 3 are independent groups in which two of R 1 , R 2 and R 3 are selected from H, ChbPh, CH(CH 3 ) 2 , CH 2 CH(CH 3 )2 or CH(CH 3 )CH 2 CH 3 and the remaining R 1 , R 2 , R 3 is
• n is an integer between 3 and 4,
• Y, Y' are independent groups
• R H, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl or acetate group,
• R'" H, alkyl, or acetate group
• R 6 is H, CH 3 , CH2OH, CH 2 -glycoside group or CH 2 -OGP in which GP is a protector group selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group
• R 7 OH, OGP ⁇ NH 2 , N 3 , NHGP' or NGP'GP" in which GP' and GP" are independently selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group,
• R 8 is a hydrogen atom H or a free or protected alcohol function
• n is an integer between 3 and 4,
• Y, Y' are independent groups
• R H, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group
• R'" H, alkyl, or acetate group
• R 6 is H, CH 3 , CH 2 OH, CH 2 -glycoside group, or CH 2 -OGP in which GP is a protector group selected from alkyl, benzyl, trimethylsilyl, tertbutyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group,
• R 7 OH, OGP', NH 2 , N 3 , NHGP', or NGP'GP" in which GP' and GP" are independently selected from alkyl, benzyl, trimethylsilyl, tertbutyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group
• n is an integer between 3 and 4,
• Y, Y' are independent groups
• R H, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group
• R'" H, alkyl, or acetate group
• R 6 is H, CH 3 , CH2OH, CH 2 -glycoside group, or CH 2 -OGP in which GP is a protector group selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group,
• R 7 OH, OGP', NH 2 , N 3 , NHGP', or NGP'GP" in which GP' and GP" are independently selected from alkyl, benzyl, trimethylsilyl, tertbutyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group,
• R 8 is a hydrogen atom H or a free or protected alcohol function, if H, CH 3 , CH(CH 3 ) 2 , CH 2 CH(CH 3 )2, or CH(CH 3 )CH 2 CH 3
• n is an integer between 3 and 4
• Y, Y' are independent groups
• R H, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group
• R'" H, alkyl, or acetate group
• R 6 is H, CH 3 , CH 2 OH, CH 2 -glycoside group, or CH 2 -OGP in which GP is a protector group selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group,
• R 7 OH, OGP', NH 2 , N 3 , NHGP', or NGP'GP" in which GP' and GP" are independently selected from alkyl, benzyl, trimethylsilyl, tertbutyldimethylsilyl,
• R 8 is a hydrogen atom H or a free or protected alcohol function.
• an in vitro method for improving the insulin secretory function of isolated pancreatic beta cells prior to transplantation in a subject in need thereof comprising the step of: a) contacting an isolated pancreatic beta cell with a gem-difluorinated C- glycopeptide compound of general formula I, or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above.
• an in vitro method for protecting isolated pancreatic cells, isolated pancreatic progenitor cells, or both from immunosuppressant drug toxicity prior to a transplantation in a subject in need thereof comprising the step of:
• an in vitro method for decreasing an inflammatory response of isolated pancreatic cells, isolated pancreatic progenitor cells, or both prior to a transplantation in a subject in need thereof comprising the step of:
• a method of transplanting isolated pancreatic cells, isolated pancreatic progenitor cells, or both in a subject in need thereof comprising the steps of :
• step b) transplanting the treated isolated pancreatic cell of step a) in said subject in need thereof.
• the method of transplanting of the present invention may further comprise step a') before step a): a') isolating pancreatic cells, pancreatic progenitor cells, or both.
• the method of transplanting of the present invention may also further comprise step b') before step b) : contacting the isolated pancreatic cell, the isolated pancreatic progenitor cells, or both of step a) with an immunosuppressant drug.
• the transplantation procedure may be performed according to any know and suitable transplantation procedure, as well as future transplantation procedures that be suitably adapted to the present cell types.
• the transplantation procedure may be "the Edmonton protocol".
• the islet cell transplant procedure known as “the Edmonton protocol” involves the transplantation of cadaveric islet cell into the patient's hepatic portal vein. Due to the allogeneic nature of the transplantation, immune suppression through the use of calcineurin inhibitors such as Tacrolimus is applied. There is a period post-transplant during the engraftment process where the transplanted cells are avascular and under severe stress, it is during this period where failure to graft is likely to occur. Other sites for transplantation have that been used including sub renal capsule and sub dermal. According to embodiments, in the case of islet or beta progenitor cell transplants the primary transplant site is still the portal vein due to the large number of cells required to achieve the desired effect.
• the isolated pancreatic cell may be an isolated alpha cell, an isolated beta cell, an isolated delta cell, an isolated gamma cell, an epsilon cell, or a combination thereof, and preferably an isolated pancreatic cell is an isolated beta cell.
• the immunosuppressant drug may be one of daclizumab, sirolimus, tacrolimus, cyclosporine, or combinations thereof.
• subject may be a human subject
• the isolated pancreatic cell may be isolated from a live donor, a cadaveric donor, or combinations thereof.
• the gem-difluorinated C-glycopeptide compound of general formula I may be a compound of general formula II: in which: N is an integer between 1 and 5,
• R 1 , R 2 , R 3 are independent groups in which two of R 1 , R 2 and R 3 are selected from H, CH 3 and the remaining R 1 , R 2 and R 3 is
• n is an integer between 3 and 4,
• Y, Y' are independent groups
• R H, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group
• R'" H, alkyl, or acetate group
• R 6 is selected from H, CH 3 , CH 2 OH, CH 2 -OGP in which GP is a protector group selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group
• R 7 OH, OGP ⁇ NH 2 , N 3 , NHGP', NGP'GP" in which GP' and GP" are independently selected from alkyl, benzyl, trimethylsilyl, tert- butyldimethylsilyl, tert-butyldiphenylsilyl or acetate group,
• R 8 is a hydrogen atom H or a free or protected alcohol function
• n is an integer between 3 and 4,
• Y, Y' are independent groups
• R H, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group
• R'" H, alkyl, or acetate group
• R 6 is selected from H, CH 3 , CH 2 OH, CH 2 -OGP in which GP is a protector group selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group,
• R 7 OH, OGP', NH 2 , N 3 , NHGP', NGP'GP" in which GP' and GP" are independently selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group, R 8 is a hydrogen atom H or a free or protected alcohol function,
• n is an integer between 3 and 4,
• Y, Y' are independent groups
• R H, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group
• R'" H, alkyl, or acetate group
• R 6 is selected from H, CH 3 , CH 2 OH, or CH 2 -OGP in which GP is a protector group selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group,
• R 7 OH, OGP', NH 2 , N 3 , NHGP', or NGP'GP" in which GP' and GP" are independently selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group,
• R 8 is a hydrogen atom H or a free or protected alcohol function
• n is an integer between 3 and 4,
• Y, Y' are independent groups
• R H, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group
• R'" H, alkyl, or acetate group
• R 6 is selected from H, CH 3 , CH 2 OH, or CH 2 -OGP in which GP is a protector group selected from alkyl, benzyl, trimethylsilyl , tert-butyldimethylsilyl, tert- butyldiphenylsilyl, or acetate group,
• R 7 OH, OGP', NH 2 , N 3 , NHGP', or NGP'GP" in which GP' and GP" are independently selected from alkyl, benzyl, trimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or acetate group,
• R 8 is a hydrogen atom or a free or protected alcohol function.
• a preferred gem-difluorinated C-glycopeptide compound of general formula I is a compound of formula III, also named AAGPTM :
• an isolated pancreatic cell an isolated pancreatic progenitor cell, or both prepared according to the methods of the present invention.
• a method of transplanting isolated pancreatic cells, isolated pancreatic progenitor cells, or both in a subject in need thereof comprising the steps of :
• a method of treating diabetes comprising the step of : a) transplanting an isolated pancreatic cell, an isolated pancreatic progenitor cell, or both prepared according to the method of the present invention, in said subject in need thereof.
• the isolated pancreatic cell may be an isolated alpha cell, an isolated beta cell, an isolated delta cell, an isolated gamma cell, an epsilon cell, or a combination thereof, and preferably an isolated pancreatic cell is an isolated beta cell.
• an isolated pancreatic cell an isolated pancreatic progenitor cell, or both prepared according to the method of the present invention, for transplantation to a subject in need thereof.
• an isolated pancreatic cell an isolated pancreatic progenitor cell, or both prepared according to the method of the present invention, for treatment of diabetes in a subject in need thereof.
• an isolated pancreatic cell prepared, an isolated pancreatic progenitor cell, or both according to the method of the present invention, for transplantation to a subject in need thereof.
• the isolated pancreatic cell may be an isolated alpha cell, an isolated beta cell, an isolated delta cell, an isolated gamma cell, an epsilon cell, or a combination thereof, and preferably an isolated pancreatic cell is an isolated beta cell.
• an isolated pancreatic cell an isolated pancreatic progenitor cell, or both contacted with a gem-difluorinated C-glycopeptide compound of general formula I, or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above.
• a gem-difluorinated C-glycopeptide compound of general formula I as described above or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above, for enhancing engraftment of isolated pancreatic cells, isolated pancreatic progenitor cells, or both in a subject in need thereof.
• a gem-difluorinated C-glycopeptide compound of general formula I as described above or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above, for improving the insulin secretory function of isolated pancreatic beta cells, isolated pancreatic progenitor cells, or both prior to transplantation in a subject in need thereof.
• a gem-difluorinated C-glycopeptide compound of general formula I as described above or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above, for protecting isolated pancreatic cells, isolated pancreatic progenitor cells, or both from immunosuppressant drug toxicity prior to a transplantation in a subject in need thereof.
• a gem-difluorinated C-glycopeptide compound of general formula I as described above or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above, for decreasing an inflammatory response of isolated pancreatic cells, isolated pancreatic progenitor cells, or both prior to a transplantation in a subject in need thereof.
• a gem-difluorinated C- glycopeptide compound of general formula I as described above or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above, for use in enhancing engraftment of isolated pancreatic cells, isolated pancreatic progenitor cells, or both in a subject in need thereof:
• a gem-difluorinated C- glycopeptide compound of general formula I as described above or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above, for use in improving the insulin secretory function of isolated pancreatic beta cells, isolated pancreatic progenitor cells, or both prior to transplantation in a subject in need thereof:
• a gem-difluorinated C- glycopeptide compound of general formula I as described above or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above, for use in protecting isolated pancreatic cells, isolated pancreatic progenitor cells, or both from immunosuppressant drug toxicity prior to a transplantation in a subject in need thereof:
• a gem-difluorinated C-glycopeptide compound of general formula I as described above or a pharmaceutically acceptable base, addition salt with an acid, hydrate or solvate of the compound of general formula I as described above, for use in decreasing an inflammatory response of isolated pancreatic cells, isolated pancreatic progenitor cells, or both prior to a transplantation in a subject in need thereof:
• the compound of formula I may be a compound of formula II and/or a compound of formula III:
• the isolated pancreatic cell, isolated pancreatic progenitor cell, or both may be contacted with from about from about 0.01 mg/ml to about 5 mg/ml of said compound of formula I, formula II or formula III, or from about 1 mg/ml to about 5 mg/ml of said compound of formula I, formula II or formula III.
• the immunosuppressant drug may be one of daclizumab, sirolimus, tacrolimus, cyclosporine, or combinations thereof.
• the subject may be a human subject, and the isolated pancreatic cell may be isolated from a live donor, a cadaveric donor, or combinations thereof.
• the isolated pancreatic cell may be an isolated alpha cell, an isolated beta cell, an isolated delta cell, an isolated gamma cell, an epsilon cell, or a combination thereof, and preferably an isolated pancreatic cell is an isolated beta cell.
• the invention includes the compounds as shown, and also includes (where possible) individual diastereomers, enantiomers, and epimers of the compounds, and mixtures of diastereomers and/or enantiomers thereof including racemic mixtures. Although the specific stereochemistries disclosed herein are preferred, other stereoisomers, including diastereomers, enantiomers, epimers, and mixtures of these may also be useful. Inactive or less active diastereoisomers and enantiomers are useful for scientific studies relating to the targets and/or the mechanism of activation.
• the compounds disclosed herein may be used in pharmaceutical compositions comprising (a) the compound(s) or pharmaceutically acceptable salts thereof, and (b) a pharmaceutically acceptable carrier.
• the compounds may be used in pharmaceutical compositions that include one or more other active pharmaceutical ingredients.
• the compounds may also be used in pharmaceutical compositions in which the compound of Formula I, II or III, or a pharmaceutically acceptable salt thereof is the only active ingredient.
• Compounds of structural Formula I, structural Formula II and/or structural Formula III may contain one or more asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers.
• the present invention is meant to comprehend all such isomeric forms of the compounds of structural Formula I, structural Formula II and/or structural Formula III.
• Compounds of structural Formula I, structural Formula II and/or structural Formula III may be separated into their individual diastereoisomers by, for example, fractional crystallization from a suitable solvent, for example methanol or ethyl acetate or a mixture thereof, or via chiral chromatography using an optically active stationary phase.
• Absolute stereochemistry may be determined by X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing an asymmetric center of known absolute configuration.
• any stereoisomer of a compound of the general structural Formula I, structural Formula II and/or structural Formula III may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known absolute configuration.
• racemic mixtures of the compounds may be separated so that the individual enantiomers are isolated.
• the separation can be carried out by methods well known in the art, such as the coupling of a racemic mixture of compounds to an enantiomerically pure compound to form a diastereomeric mixture, followed by separation of the individual diastereomers by standard methods, such as fractional crystallization or chromatography.
• the coupling reaction is often the formation of salts using an enantiomerically pure acid or base.
• the diasteromeric derivatives may then be converted to the pure enantiomers by cleavage of the added chiral residue.
• the racemic mixture of the compounds can also be separated directly by chromatographic methods utilizing chiral stationary phases, which methods are well known in the art.
• Some of the compounds described herein may exist as tautomers, which have different points of attachment of hydrogen accompanied by one or more double bond shifts.
• a ketone and its enol form are keto-enol tautomers.
• the individual tautomers as well as mixtures thereof are encompassed with compounds of the present invention.
• the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature.
• the present invention is meant to include all suitable isotopic variations of the compounds of generic Formula I, Formula II and/or Formula III.
• different isotopic forms of hydrogen (H) include protium ( 1 H) and deuterium ( 2 H). Protium is the predominant hydrogen isotope found in nature.
• Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples.
• Isotopically-enriched compounds within generic Formula I, Formula II and/or Formula III can be prepared without undue experimentation by conventional techniques well known to those skilled in the art.
• references to the compounds of structural Formula I, Formula II and/or Formula III are meant to also include the pharmaceutically acceptable salts, and also salts that are not pharmaceutically acceptable when they are used as precursors to the free compounds or their pharmaceutically acceptable salts or in other synthetic manipulations.
• pharmaceutically acceptable salt refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts of basic compounds encompassed within the term “pharmaceutically acceptable salt” refer to non-toxic salts of the compounds of this invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid.
• Representative salts of basic compounds of the present invention include, but are not limited to, the following: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, camsylate, carbonate, chloride, clavulanate, citrate, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate),
• suitable pharmaceutically acceptable salts thereof include, but are not limited to, salts derived from inorganic bases including aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, mangamous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts.
• Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, cyclic amines, and basic ion-exchange resins, such as arginine, betaine, caffeine, choline, ⁇ , ⁇ -dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2- dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.
• basic ion-exchange resins such as arginine, betaine, caffeine, choline, ⁇ , ⁇
• esters of carboxylic acid derivatives such as methyl, ethyl, or pivaloyloxymethyl
• acyl derivatives of alcohols such as acetyl, pivaloyl, benzoyl, and aminoacyl
• esters and acyl groups known in the art for modifying the solubility or hydrolysis characteristics for use as sustained-release or prodrug formulations.
• Solvates, in particular hydrates, of the compounds of structural Formula I, Formula II and/or Formula III are included in the present invention as well.
• the compounds of structural Formula I, Formula II and/or Formula III may be included in various formulations for use as medicaments.
• Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions.
• excipients are suspending agents, for example sodium carboxymethyl-cellulose, methylcellulose, hydroxypropylmethy-cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.
• dispersing or wetting agents may be a naturally-occurring phosphatide, for example lec
• the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.
• preservatives for example ethyl, or n-propyl, p-hydroxybenzoate
• coloring agents for example ethyl, or n-propyl, p-hydroxybenzoate
• coloring agents for example ethyl, or n-propyl, p-hydroxybenzoate
• flavoring agents such as sucrose, saccharin or aspartame.
• sweetening agents such as sucrose, saccharin or aspartame.
• Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin.
• the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
• Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
• a dispersing or wetting agent exemplified by those already mentioned above.
• Additional excipients for example sweetening, flavoring and coloring agents, may also be present.
• the pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsion.
• the oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these.
• Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
• the emulsions may also contain sweetening and flavouring agents.
• the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension.
• This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
• the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1 ,3-butane diol.
• the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil may be employed including synthetic mono- or diglycerides.
• fatty acids such as oleic acid find use in the preparation of injectables.
• the cells are isolated using methods known in the art for their preparation.
• the cells may be isolated from donors (live or cadaveric donors) using mixtures of enzymes such as Collagenase I and Collagenase II, Thermolysin, non-clostridial neutral protease, or other enzymes being used for such purpose.
• the isolated cells may then be cultured under normal tissue culture conditions in standard tissue culture flasks.
• the cells, pancreatic progenitor cells, or both may be treated with a gem-difluorinated C-glycopeptide compound of general formula I - preferably, the compound of Formula II, and most preferably the compound of formula III in concentrations varying from about 0.01 mg/ml to about 5 mg/ml; or from about 0.1 mg/ml to about 5 mg/ml; or from about 0.5 mg/ml to about 5 mg/ml; or from about 1 mg/ml to about 5 mg/ml; or from about 3 mg/ml to about 5 mg/ml; or from about 0.01 mg/ml to about 3 mg/ml, or from about 0.1 mg/ml to about 3 mg/ml, or from about 0.5 mg/ml to about 3 mg/ml, or from about 1 mg/ml to about 3 mg/ml, or from about 0.01 mg/ml to about 1 mg/ml; or from about 0.1 mg/ml to about 1 mg/m/ml;
• the amounts above are considered to be therapeutically effective amounts for the purpose of the present inventions.
• the cells are contacted with the gem- difluorinated C-glycopeptide compound for a time sufficient to effect improvements on cell viability and survival rate.
• the time sufficient may be from about 12 hours to 120 hours, or from about 12 hours to about 96 hours, or from about 12 hours to about 72 hours, or from about 12 hours to about 48 hours, or from about 12 hours to about 24 hours, or about 120 hours, or about 96 hours, or about 72 hours, or about 48 hours, or about 24 hours, or about 12 hours.
• a cell preparation prepared according to the method of the present invention in a pharmaceutically acceptable carrier.
• the cell preparation may be used for the preparation of a medicament for a cell transplantation.
• the cell preparation may be used for a cell transplantation.
• a method of transplantation comprising transplanting a cell preparation of the present invention to a subject in need thereof.
• the subject may be a mammal, and preferably a human.
• Islets are cultured in humidified atmosphere of 95% air and 5% CO2 at 37°C, at a density of 200 lE/mL using standard tissue culture flask.
• the AAGPTM concentration is 3mg/mL.
• Several tests are conducted on day 1 post-islet isolation, including: Islet recovery, membrane integrity viability stain (Syto® EB) as well as Glucose-Stimulated Insulin Release. The results are shown in Figs. 1A-D.
• Islets are cultured in humidified atmosphere of 95% air and 5% CO2 at 37°C, at a density of 200 lE/mL using standard tissue culture flask. AAGPTM concentration is 3mg/mL.
• AAGPTM concentration is 3mg/mL.
• AAGPTM formulation shows no toxicity on human islet cells.
• AAGPTM seems to enhance islet survival in culture.
• AAGPTM protects islet exposed to tacrolimus in vitro.
• Islets are used in all experiments. Islets are isolated from deceased donors in the clinical Good Manufacturing Practice (GMP) facility within the Clinical Islet Transplant Program, University of Alberta. Briefly, pancreata are procured from multiorgan deceased donors and preserved in histidine-tryptophan-ketoglutarate (HTK, Custodiol. Metapharm, Brandford, ON, Canada) solution or Static Preservation Solution (SPS-1 , Itasca, IL, USA).
• GMP clinical Good Manufacturing Practice
• HTK histidine-tryptophan-ketoglutarate
• SPS-1 Static Preservation Solution
• Islets are counted with dithizone staining (3mg/mL final concentration, Sigma- Aldrich, ON, Canada) using an optical graticule at the beginning and end of culture.
• the total number of islets is converted to Islet Equivalents (IEQ) (standardized to a diameter of 150 ⁇ ) (Islet isolation for clinical transplantation.
• IEQ Islet Equivalents
• CMRL-1066 Mediatech, Manassas, VA, USA
• AAGPTM 3mg/mL (ProtoKinetix), a synthetic analog of AFP.
• AAGPTM is highly soluble in all media and has proven to be more stable while retaining high bioactive properties comparable to natural AFP. The other group served as control.
• islets are assessed for recovery, viability and function. Recovery rate is calculated as the percentage of surviving islets after 24hr culture in comparison to the initial count for each condition. Viability is assessed using a fluorescent membrane integrity assay with counter-stains syto green/ethidium bromide (Cedarlane Laboratories, Burlington, ON, and Sigma-Aldrich, ON) (Islet isolation for clinical transplantation. In: Shahidul. M, (ed). The islets of Langerhans. New York: Springer, 2010; Ranuncoli A, et al. Cell transplantation 2000;9(3):409-414;Ricordi C et al. Acta diabetologica latina 1990;27(3): 185-195; and Barnett MJ et al. Cell transplantation 2004; 13(5):481-488).
• Islet secretory function is assessed by monitoring in vitro insulin secretory profile during continuous glucose stimulation, as described by Cabrera et al Cell transplantation 2008; 16(10): 1039-1048.
• This in vitro perifusion assay is a modality of continuous Glucose- Stimulated Insulin Secretion (GSIS) test to stress islets in alternating normoglycemic and hyperglycemic environment to model physiological islet response.
• GSIS Glucose- Stimulated Insulin Secretion
• Supernatants from the perifusion assay are collected and insulin levels are determined using a commercially available ELISA kit (Insulin ELISA kit. Mercodia Inc. Pittsburgh, PA, USA). Results are expressed as fold- change of insulin secretion compared to the low (2.8mMol) glucose stimulation baseline, normalized for 100 IEQ.
• AAGPTM The cytoprotective capabilities of AAGPTM are evaluated against tacrolimus- induced toxicity (Johnson JD et al, Cell transplantation 2009; 18(8): 833-845). Islets from six different research-grade human preparations are separated into three groups: one AAGPTM- supplemented group and two non-supplemented controls. Islets are kept in similar culture conditions for 24hr followed by the addition of tacrolimus (Prograf, Astellas Pharma Canada Inc., Markham, ON, Canada) in the AAGPTM group and in one of the control groups (positive control).
• tacrolimus Prograf, Astellas Pharma Canada Inc., Markham, ON, Canada
• Tacrolimus is added to the culture media at a concentration of 10ng/mL, a clinically relevant dose, and all groups are cultured for an additional 24hr, followed by perifusion and determination of pro-inflammatory cytokines, oxidative stress and apoptosis. The results are presented in Figs. ??.
• Frozen samples from all groups are utilized to determine reactive oxygen species (ROS) in the culture media, using the Acridan LumigenTM PS-3 assay (Amershan ECL Plus kit, Fisher Scientific Inc. Ottawa, ON, Canada) (18).
• Acridan LumigenTM PS-3 is excited by reactive oxygen and nitrogen species (RNS) in the presence of hydrogen peroxide, producing chemiluminescense at 430nm.
• RNS reactive oxygen and nitrogen species
• Media samples are flash-frozen with liquid nitrogen and stored until the assay is performed.
• CMRL culture media alone is used as a control, and results are expressed as fold-change increase compared to the control media.
• Apoptosis is measured in all groups using a cleaved caspase-3 spectrophotometric assay and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. All determinations are made on normalized groups of 100 IEQ and their corresponding culture media.
• Frozen media samples are used to determine cleaved caspase-3 increase using a spectrophotometric assay (EMD MilliporeTM. Billerica, MA, USA). Again, CMRL culture media alone with additives and no cells is used as a control, and results are expressed as fold-change increase compared to the control media.
• EMD MilliporeTM Billerica, MA, USA
• TUNEL staining (DeadEndTM Apoptosis Detection System, Promega, Madison, Wl) islets are fixed in formalin, processed and embedded in paraffin. Co-staining is performed to identify the graft (insulin), apoptotic cells and all nuclei present in the field. Apoptosis is quantified by percentage of positive TUNEL stained area using ImageJ software.
• mice Eight to 12 week immunodeficient mice (B6.129S7-Rag1 tm1Mom ) are obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and housed under specific pathogen-free conditions with access to food and water ad libitum. Animals are cared for according to the guidelines of the Canadian Council on Animal Care, and ethical approval is obtained from the animal welfare committee at the University of Alberta.
• Diabetes is chemically induced by intraperitoneal injection of streptozotocin (STZ, Sigma-Aldrich, ON, Canada) at a dose of 180mg/kg. Animals are considered diabetic after two consecutive blood glucose measurements are documented ⁇ 18mmol/L.
• STZ streptozotocin
• mice per group underwent nephrectomy and are euthanized 24hr post- transplant to perform acute determination of proinflammatory cytokines, cleaved capase-3 and TUNEL in the graft area.
• islet graft is excised from the kidney, weighed, flash frozen in liquid nitrogen and stored at -80°C.
• Tissue samples are subsequently lysed using 1 ml of lysis buffer (0.15M NaCI, 1 mM Tris-HCL, 0.1 % SDS, 0.1 % Triton X-100, 20mM Sodium deoxycholate, 5mM EDTA) per 200mg of tissue.
• Lysates are then homogenized (PowerGen, Fisher Scientific, ON, Canada) on ice for 30sec x 2 replications, and sonicated (VirSonic, VirTis, NY, USA) with 10 quick pulses while on ice.
• Cellular debris is pelleted by centrifugation at 14,000 rpm for 10min at 4°C and the resulting supernatant is collected and placed in a microcentrifuge tube containing 10 ⁇ of a protease inhibitor cocktail (Sigma-Aldrich Canada Co., Oakville, ON, Canada) per 1 ml of lysate (1 : 100).
• Non-fasting blood glucose is monitored in the remaining 7 mice per group three times a week using a portable glucometer (OneTouch Ultra 2, LifeScan, Canada) over 60 days.
• Normoglycemia is defined when two consecutive readings are below 11.3mmol/L.
• IPGTT Intraperitoneal Glucose-Tolerance Tests
• Preserved grafts are then processed as previously described and residual insulin content is determined using human insulin ELISA as a measure of surviving islets.
• a toxicity islet assay is performed and cytoprotective capabilities of AAGPTM are evaluated.
• isolated human islets from 6 different preparations are cultured for 24h in media supplemented with or without AAGPTM, followed by 24hr exposure to tacrolimus at a clinically relevant dose of 10ng/mL.
• islets are cultured in the presence of culture media only (Tac-), tacrolimus only (Tac+) or AAGPTM with tacrolimus (Tac+AAGPTM). After the defined culture period all groups are characterized for in vitro survival, viability, function and oxidative stress.
• Possible impact of AAGPTM on oxidative stress occurring to human islets during culture and exposure to tacrolimus is measured by fold-increase in extracellular ROS.
• Isolated human islets from 6 different preparations were cultured in media supplemented with or without AAGPTM for 24 hours, were supplemented with tacrolimus, for another 24 hours. After a total 48 hours culture period all groups were characterized for in vitro survival, viability, function and oxidative stress.
• Tac+ group showed a significantly impaired insulin secretion on day 3, not observed in the Tac+AAGPTM series (stimulation index 1.4 vs. 9.7, p ⁇ 0.01 , Figure 6B).
• this decrease in insulin secretion was transient and differences among groups disappeared by the 7th day when tacrolimus was no longer present, adjusting for an expected overall diminished islet potency at the end of the culture period.
• the intracellular insulin content remained stable and comparable throughout the groups, indicating no changes to biosynthesis of insulin in beta cells ( Figures 6C and 6D).
• KC secretion involved in neutrophil recruitment, is associated with major secretion changes.
• I L-1 ⁇ and IL-6 KC is significantly overexpressed in the Tac+ group and again, significantly reduced in the presence of AAGPTM (9.79 vs. 3.58pg/mL/g-tissue, p ⁇ 0.001) ( Figure 9D). No significant differences between groups are observed when comparing in vitro cytokine and chemokine expression (data not shown).
• Acutely removed grafts (24h after transplant) are also analyzed for intra-graft apoptosis Fold-change increase in levels of cleaved casapse-3 are determined relative to basal levels in kidneys of naive mice. There is a significant increase in levels of caspase-3 in the Tac+ group with respect to Tac+AAGPTM, which essentially showed very similar levels to those of islets never exposed to tacrolimus (3.14 vs. 1.46, p ⁇ 0.001) (Figure 10B).
• Tac- and Tac+AAGPTM Sixty days post-transplant study groups underwent IPGTT to evaluate islet transplant function.
• insulin content is determined as an indicator of the residual islet mass after 60 days of transplant.
• Figure 11 C shows marked differences between groups with substantial insulin content reduction in grafts exposed to tacrolimus. Again, presence of AAGPTM is beneficial in islet protection despite exposure to tacrolimus (Tac+ vs. Tac+AAGPTM, 30.86 vs. 100.8ng/mL, p ⁇ 0.01).
• AAGPTM islet culture media affords considerable protection of human islet survival and in vitro function. Furthermore, AAGPTM protected human islets from tacrolimus-related injury, with benefit in vivo including engraftment, and markers of acute inflammation, apoptosis and long-term graft function.
• Tacrolimus is a widely used immunosuppressant in the field of islet transplantation and a key element used routinely to reduce risk of acute, chronic rejection and recurrent autoimmunity (Shapiro AM et al. The New England journal of medicine 2000;343(4):230-238.).
• CNIs chronic rejection and recurrent autoimmunity
• the prolonged use of this and other CNIs have been associated with side effects, mainly nephrotoxicity and post-transplant diabetes (Chand DH et al. Pediatric transplantation 2001 ;5(1):32-36).
• tacrolimus and cyclosporine-related injury have been reported in islets, characterized by several mechanisms including calcineurin/nuclear factor of activated T-cells (NFAT) signaling inhibition (Oetjen E, et al. Molecular pharmacology 2003;63(6): 1289-1295), insulin gene suppression (Hernandez-Fisac I et al. American journal of transplantation 2007;7(1 1):2455-2462), mitochondrial arrest (Rostambeigi N et al. Transplantation 2011 ;91 (6):615-623) and decreased post-transplant vascularization (Nishimura R et al. PloS one 2013;8(4):e56799).
• NFAT calcineurin/nuclear factor of activated T-cells
• Islets are highly susceptible to hypoxia, occurring from donation through culture and intraportal transplantation. This phenomenon is mainly attributed to their high oxygen demand and size, responsible for creating detrimental pC>2 gradients depending on the seeding density during culture (Papas KK et al. Transplantation proceedings 2005;37(8):3412-3414). Islets are prone to oxidative stress due to decreased antioxidant capacity (Sklavos MM et al. Diabetes 2010;59(7): 1731-1738). These elements contribute to islet loss during culture and post-transplant. The findings show increased in oxidative stress in Tac+ group, as demonstrated by increased extracellular ROS in the culture media..
• Tacrolimus has also been associated with increased cell-death in islets (Johnson JD et al. Cell transplantation 2009; 18(8): 833-845).
• the present experiments measured the fold change of cleaved caspase-3 in the graft as an indication of apoptosis, followed by TUN EL staining of the same grafts. Results consistently indicated reduced apoptosis in the AAGPTM- supplemented group at similar levels to naive human islets never exposed to tacrolimus.

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