EP1615497A2 - Methods of promoting cell viability - Google Patents

Methods of promoting cell viability

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Publication number
EP1615497A2
EP1615497A2 EP03723860A EP03723860A EP1615497A2 EP 1615497 A2 EP1615497 A2 EP 1615497A2 EP 03723860 A EP03723860 A EP 03723860A EP 03723860 A EP03723860 A EP 03723860A EP 1615497 A2 EP1615497 A2 EP 1615497A2
Authority
EP
European Patent Office
Prior art keywords
subject
cell population
treating
compound
transplant cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03723860A
Other languages
German (de)
English (en)
French (fr)
Inventor
Clifford J. Steer
Walter C. Low
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Minnesota Twin Cities
Original Assignee
University of Minnesota Twin Cities
University of Minnesota System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Minnesota Twin Cities, University of Minnesota System filed Critical University of Minnesota Twin Cities
Publication of EP1615497A2 publication Critical patent/EP1615497A2/en
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • 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
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/36—Lipids
    • 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

  • Parkinson's disease is a disorder of the central nervous system that affects between one and one-and-a-half million Americans. PD may appear at any age, but the risk for developing PD increases with age. In addition, PD occurs in all parts of the world, and affects men slightly more often than women.
  • the symptoms of PD may include rigidity, tremors, bradykinesia, difficulties in walking, and or difficulties with balance. People with PD usually do not experience all of these symptoms, but rather a subset of these symptoms.
  • PD results from a combination of genetic predisposition and an as yet unidentified environmental trigger.
  • degenerative changes are found in the area of the brain known as the substantia nigra, which produces dopamine.
  • Dopamine is a chemical substance that enables people to move normally and smoothly.
  • PD is characterized by a severe shortage of dopamine. It is believed that the deficiency of dopamine causes the symptoms of PD.
  • apoptosis plays a role in the loss of transplanted dopamine neurons (M. Emgard et al., Exp. Neurol. 160, 279-288, 1999; TJ. Mahalik et al., Exp. Neurol. 129:27-36, 1994; G.S. Schierle et al., Nat. Med. 5:97- 100, 1999; W.M. Zawada et al., Brain Res. 786, 96-103, 1998).
  • apoptosis normally takes place within the first several days after transplantation (M. Emgard et al., Exp. Neurol. 160, 279-288, 1999; G.S. Schierle et al., Nat.
  • the present invention provides a method for promoting the viability of a transplant cell population to be transplanted into a subject.
  • the subject is a human subject
  • promoting viability of a transplant cell population includes contacting the transplant cell population with an effective amount of a compound selected from the group consisting of a hydrophilic bile acid, a salt thereof, an analog thereof, and a combination thereof.
  • the cells of the transplant cell population can include, for example, differentiated cells and precursor cells.
  • the cells of the transplant cell population can include autologous cells, heterologous cells, or xenologous cells.
  • the cells of the transplant cell population can include at least a portion of autologous tissue, heterologous tissue, or xenologous tissue.
  • the cell population is in the form of an organ, or a part thereof, such as a liver, heart, kidney, lung, and pancreas, for example.
  • the cell population is a human cell population.
  • in vitro is to be distinguished from in vivo.
  • in vitro refers to an artificial environment location of the transplant cell population to be treated, such as a cell culture in a tissue culture dish.
  • in vivo refers to a natural environment location of the cell population to be treated, such as in a mammalian body.
  • One aspect of the present invention provides a method that includes contacting the transplant cell population with the compound prior to transplanting the transplant cell population in a subject. This can be done prior to (in vivo) or after (in vitro) removal of the transplant cell population from the donor. Alternatively, this can be done after the transplant cell population has been transplanted in the subject.
  • the present invention provides a method that includes treating the subject to receive the transplant cell population with the compound.
  • treating the subject with the compound can include administering the compound to the subject prior to transplanting the transplant cell population in the subject.
  • treating the subject with the compound can include administering the compound to the subject after transplanting the transplant cell population in the subject. In this latter example, the subject may have already been treated with the compound prior to transplanting the transplant cell population in the subject.
  • treating the subject includes treating the subject parenterally or orally with the compound.
  • the present invention also provides for treating a donor, and/or a subject, of a transplant cell population with a compound selected from the group consisting of a hydrophilic bile acid, a salt thereof, an analog thereof, and a combination thereof.
  • a hydrophilic bile acid useful for the present invention can include, but is not limited to, ursodeoxycholic acid and/or tauroursodeoxycholic acid.
  • One aspect of the present invention provides a method that includes treating the donor of the transplant cell population with the compound. This can be done prior to (in vivo), during (in vivo) or after (in vitro) removal of the transplant cell population from the donor. Alternatively or in addition to, the present invention provides a method that includes treating the subject of the transplant cell population with the compound. This can be done prior to (in vivo/in vitro), during (in vivo) or after (in vivo) transplanting the transplant cell population into the subject.
  • Another aspect of the present invention is a method for treating a subject, preferably a human, having a disease that requires cell replacement.
  • the present invention can provide a method for treating a subject, preferably a human, having Parkinson's disease.
  • the method can include contacting a transplant cell population with an effective amount of a compound selected from the group consisting of ursodeoxycholic acid, a salt thereof, an analog thereof, and a combination thereof to promote viability of the transplant cell population.
  • the method then further includes transplanting the transplant cell population into the subject.
  • an analog of ursodeoxycholic acid includes a conjugated derivative, where the conjugated derivative can be tauroursodeoxycholic acid.
  • the method of the present invention can include treating a human having Parkinson's disease, where the method includes contacting a transplant cell population in vitro with an effective amount of the tauroursodeoxycholic acid in combination with a pharmaceutically acceptable carrier, where the transplant cell population are differentiated cells. The method further includes transplanting the transplant cell population into the human.
  • the transplant cell population for the human having Parkinson's disease can include differentiated cells and precursor cells.
  • the cells of the transplant cell population can include autologous cells, heterologous cells, or xenologous cells.
  • the cells of the transplant cell population can include at least a portion of autologous tissue, heterologous tissue, or xenologous tissue.
  • the contacting step can occur in vitro, in vivo, and a combination thereof.
  • the cell population is a human cell population.
  • Fig. 1 is a bar diagram summarizing the effects of TUDCA (50 ⁇ M/ml) on the survival of TH-positive neurons (shadow bars) and total cells (open bars) in ventral mesencephalic (NM) tissue cultures. Bars represent the mean ⁇ S.E.M. of three independent experiments with quadruplicate wells for each culturing condition, f * p ⁇ 0.01, significant difference from the 7 DIN+TUDCA cultures (one-factor A ⁇ ONA with post-hoc Scheffe's F-test).
  • Fig. 2 is a bar diagram illustrating the effects of TUDCA (50 ⁇ M/ml) on apoptosis in VM tissue cultures. Bars represent the mean ⁇ SEM of three independent experiments with quadruplicate wells for each culturing condition. * p ⁇ 0.01, significant difference from the 2 DIN and 7 DIV+TUDCA cultures (one-factor A ⁇ ONA with post-hoc Scheffe's F-test).
  • Fig. 3 is a bar diagram illustrating net ipsilateral amphetamine-induced rotation asymmetry (full turns per minute (min) contralateral to the lesion subtracted from turns ipsilateral to the lesion) over the 90-min test session for the control and the TUDCA- treated groups.
  • the rats were tested before grafting and 2 and 6 weeks after grafting.
  • Each bar represents the group mean and the enor bars denote S.E.M. * p ⁇ 0.01 (paired Student t-test) when compared to pregrafting value and p ⁇ 0.05 (one-factor A ⁇ ONA with post-hoc Scheffe's F-test) when compared to the control group.
  • the symbols f J p ⁇ 0.01 when compared to pregrafting values.
  • Figs. 4A-4E are photomicrographs of coronal sections through the grafted striatum processed for TH-immunocytochemistry.
  • the photographs illustrate typical grafts from a representative rat in the TUDCA-treated (A, C, and E) and control (B and D) groups six weeks after transplantation.
  • (A) and (B) demonstrate a overview of grafted brain at a low magnification.
  • the host striatal areas adjacent to the grafts are reinnervated by TH-immunopositive fibres from transplanted neurons.
  • 5 is a bar diagram of the mean number of TH-immunopositive cells and the graft volume in the control and TUDCA-treated groups six weeks after transplantation.
  • the bars represent the group mean value ⁇ S.E.M. * p ⁇ 0.01 (one-factor ANONA with post-hoc Scheffe's F-test) when compared to the TUDCA-treated group.
  • Fig. 6 is a bar diagram illustrating the mean number of apoptotic cells in the graft areas for the control and TUDCA-treated groups 4 days post-transplantation.
  • the number of apoptotic cells in the graft areas was significantly smaller in the TUDCA- treated group than in the control group.
  • An asterisk indicates ) ⁇ 0.01 (one-factor A ⁇ ONA with post-hoc Scheffe's F-test) compared to the control group.
  • Enor bars represent S.E.M.
  • the present invention provides such a method for promoting the viability of a transplant cell population.
  • the method can include contacting the transplant cell population with an effective amount of a compound selected from the group consisting of a hydrophilic bile acid, a salt thereof, an analog thereof, and/or a combination thereof prior to, during, or after transplanting the transplant cell population into a subject.
  • transplant cell population viability includes maintaining, prolonging and/or improving the survival and/or proliferation of the transplant cell population to be transplanted, or those that have been transplanted, into the subject.
  • viability refers to maintaining the normal function of cells in a transplant cell population, typically in vivo, however the term is meant to include in vitro as well.
  • prolonging means that transplant cell population for transplantation are preserved by treatment using the method of the invention as compared to a similar transplant cell population that has not been so treated. While not wanting to be bound by a particular theory, it is believed that contacting the transplant cell population for transplantation with the compound of the present invention inhibits programmed cell death, thereby prolonging the viability of the transplant cell population.
  • transplant cell population includes, but is not limited to, a population of individual cells and cells present in tissue and/or an organ that has been or can be transplanted into a subject.
  • the cells of the transplant cell population could include matrix structures (e.g., proteins, polysaccharides, peptides, and other molecules) found in tissues and/or organs.
  • matrix structures e.g., proteins, polysaccharides, peptides, and other molecules
  • Subjects having a medical condition that can benefit by improved viability of a transplant cell population includes those with neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease; Huntington's disease; multiple sclerosis; amyotrophic lateral sclerosis; cerebellar ataxia; lysosomal storage disorders; cancer; birth defects; those in need of organ and/or tissue transplants; spinal cord injury; ischemic injury such as stroke, ischemic kidney disease, and heart disease; burns; autoimmune diseases; diabetes; inflammatory diseases such as osteoarthritis and rheumatoid arthritis, to name only a few.
  • This list is not exhaustive and other medical conditions known to benefit from improved viability of the transplant cell population to be transplanted are also considered to be within the scope of the present invention.
  • the transplant cell population can include a transplant cell population obtained from a donor for transplantation into a subject.
  • the transplant cell population may be derived from children, adults, or fetal tissue.
  • the transplant cell population can include, but is not limited to, a population of cells derived from blood and all of its components, including erythrocytes, leukocytes, platelets, serum, hematopoietic stem cells; central nervous tissue, including brain and spinal cord tissue, neurons, glia, and neural stem cells; peripheral nervous tissue, including ganglia, posterior pituitary gland, adrenal medulla, and pineal; connective tissue, including skin, skin stem cells, ligaments, tendons, and fibroblasts; muscle tissue, including skeletal, skeletal muscle stem cells, smooth, and cardiac tissues or the cells therefrom; endocrine tissue, including anterior pituitary gland, thyroid gland, parathyroid gland, adrenal cortex, pancreas and its subparts (e.g., islet cells), testes, ovaries, placenta, and the endocrine cells that are a part of each of these tissues; blood vessels, including arteries, veins, capillaries, and the cells from these vessels; lung tissue;
  • organ is meant to include all or part of intact multi- cellular organs such as kidney, liver, brain, or heart; cell suspensions derived from multi-cellular organs; as well as suspensions of blood cells or hematopoietic precursor cells.
  • tissue is meant to include aggregations of a transplant cell population with their associated intracellular matrix (e.g., collagen, proteins, polysaccharides, etc.). The transplant cell population can also include individual cells that have been separated and/or isolated from the tissue and/or organ from which they were derived.
  • the transplant cell population for transplantation into a subject can include differentiated cells and/or precursor cells (e.g., undifferentiated cells).
  • differentiated cells can include, but are not limited to, the cells discussed herein, including but not limited to, myocardial cells, muscle cells, smooth muscle cells, epidermal cells, neurons including dopamine neurons, pancreatic cells, bone marrow cells, hepatic and nonhepatic cells.
  • precursor cells can include, but are not limited to, stem cells, pluripotent stem cells, embryonic stem cells, and adult stem cells.
  • cells of the transplant cell population useful with the present invention include, but are not limited to, autologous cells, heterologous cells, xenologous cells, or combinations thereof.
  • cells can also include those cells that form a portion of tissues and/or organs. So, for the present invention, the cells can include at least a portion of autologous tissue, heterologous tissue, xenologous tissue, and/or combinations thereof, wherein the tissue can be transplanted into the subject.
  • subjects can include mammals.
  • a subject as used herein is a human.
  • donor can include mammals used as a source of biological material, such as a part of, or all of, a cell population (including organs) for transplanting into a subject.
  • the donor can include, but is not limited to, a human, a porcine, a non-human primate, a bovine, or a combination thereof.
  • the donor can be living during the donation of the cell population.
  • a transplant cell population as defined herein for transplantation can be derived from any species.
  • the present invention is useful for preserving a transplant cell population for use in same species transplant in a subject such as human and other human donors (allografts and autologous or heterologous grafts) or to a human subject from another species such as sheep, pig, cow, or non- human primate (xenografts), for example.
  • a transplant cell population for transplant includes, but is not limited to, heart, liver, kidney, lung, pancreas, pancreatic islets, brain, cornea, bone, intestine, skin, blood, and cells from such organs and tissues.
  • contacting the transplant cell population with an effective amount of one or more compounds of the present invention can be accomplished in vitro and/or in vivo.
  • the transplant cell population can be contacted with a compound according to the present invention in vivo prior to transplanting the transplant cell population in the subject.
  • One preferred way of accomplishing this includes treating the donor of a transplant cell population with one or more compounds of the present invention.
  • the donor of the transplant cell population can be treated with the compound of the present invention prior to (in vivo), during (in vivo) or after (in vitro) removal of the transplant cell population from the donor. So, the transplant cell population could be contacted with a compound of the present invention while still in the donor (in vivo).
  • the transplant cell population could be contacted with a compound of the present invention during removal, or once the transplant cell population is removed from the donor.
  • benefit to the donor may be realized by administering the compounds of the present invention before and/or after removal of a part of the organ.
  • the transplant cell population is perfused and/or immersed or otherwise contacted with a compound of the present invention during harvesting, storing, growing and/or transplanting of the transplant cell population.
  • a compound of the present invention can be used in vivo to treat the subject receiving the transplant cell population. This can be done prior to (in vivo/in vitro), during (in vivo) or after (in vivo) transplanting the transplant cell population into the subject. So, it is possible that a subject receiving a transplant cell population also receives treatment with a compound of the present invention.
  • the subject could be treated systemically or locally with the compound by administering the compound of the present invention to the subject prior to (i.e., pre-cell population transplant), during (i.e., concurrent with the time of transplanting the transplant cell population), and/or after (i.e., post-cell population transplant) the transplantation of the transplant cell population.
  • the transplant cell population can be contacted with a compound according to the present invention in vivo after transplanting the transplant cell population in the subject. So, the transplant cell population could be contacted with a compound of the present invention once the transplanted cell population has been implanted into the subject.
  • the subject may be dosed with immunosuppressive pharmaceuticals to enhance graft acceptance.
  • the subject may be dosed with one or more compounds of the present invention.
  • the donor heterologous transplant cell population can then be flushed with a solution containing at least one compound of the present invention, e.g., tauroursodeoxycholic acid (TUDCA).
  • a solution containing at least one compound of the present invention e.g., tauroursodeoxycholic acid (TUDCA).
  • TDCA tauroursodeoxycholic acid
  • the subject is typically maintained on routine immunosuppression pharmaceuticals and optionally, a compound of the present invention. Based on clinical signs and symptoms related to immune responsiveness, the immunosuppressants can be reduced in dosage.
  • an "effective amount” as used herein includes useful dosage levels of the compound of the present invention that will be effective to promote the viability of a transplant cell population. Although the inventors do not wish to be bound by theory, it is believed that an "effective amount” is one effective to prevent, reduce, inhibit, or suppress apoptosis of cells to be transplanted and/or cells that have been transplanted. Useful dosages of the desired compound described herein can be determined by comparing its in vitro activity and its in vivo activity in animal models. Methods for extrapolation of effective dosages in mice, and other animals, to humans are known in the art.
  • the specific "effective amount" for any particular subject (or donor) and/or transplant cell population will depend upon a variety of factors including the activity of the specific compound employed; the transplant cell population; the conditions under which the transplant cell population are being harvested, isolated, stored, and/or incubated when the transplant cell population is maintained in vitro; and when used in vivo, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the medical condition for the subject (or donor) being treated.
  • the transplant cell population can be contacted with a compound of the present invention during storage.
  • Storage of a transplant cell population can include conditions under which the transplant cell population is maintained.
  • storage of the transplant cell population can also include conditions under which the transplant cell population is placed in order to encourage the growth and proliferation of the transplant cell population.
  • the transplant cell population can be contacted with an effective amount of a compound of the present invention.
  • a compound of the present invention may be used with cell culture media used in the culture of the cells to be transplanted into the subject. Concentration of a compound will depend on a variety of factors, including solubility and activity.
  • hydrophilic bile acids are those more hydrophilic than deoxycholic acid (DCA). This can be determined by evaluating the partition coefficient between water and octanol, with the more hydrophilic bile acids being more favorable toward water. Alternatively, the more hydrophilic bile acids have earlier retention times on a reverse-phase column using high performance liquid chromatography. A particularly prefened hydrophilic bile acid includes ursodeoxycholic acid. Examples of analogs of hydrophilic bile acids include conjugated derivatives of bile acids.
  • hydrophilic bile acids may not be useful in all methods of the present invention, they can be evaluated readily by testing their ability to inhibit apoptosis in cell cultures using agents known to induce apoptosis.
  • Two particularly prefened conjugated derivatives include glyco- and tauroursodeoxycholic acid.
  • Ursodeoxycholic acid is an endogenous bile acid that has been in clinical use over the last few decades for the treatment of a variety of liver diseases.
  • Conjugated derivatives of UDCA include ursodeoxycholic acid 3-sulfate, ursodeoxycholic acid 7-sulfate, ursodeoxycholic acid 3,7-disulfate, tauroursodeoxycholic acid (TUDCA), and glycoursodeoxycholic acid.
  • the compound described herein can be formulated in pharmaceutical compositions.
  • a transplant cell population can be then contacted with the pharmaceutical composition containing a compound of the present invention.
  • the pharmaceutical composition containing a compound of the present invention can be administered to a subject, typically a mammal such as a human subject, in a variety of forms adapted to the chosen route of administration.
  • the formulations include those suitable for in vitro cell culture as well as, oral, rectal, vaginal, topical, nasal, ophthalmic, parenteral (including subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal, intraventricular, direct injection into brain tissue, etc.) administration.
  • the formulations may be conveniently presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Typically, such methods include the step of bringing the active compound into association with a carrier, which can include one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid canier, a finely divided solid canier, or both, and then, if necessary, shaping the product into a desired formulation.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as tablets, troches, capsules, lozenges, wafers, or cachets, each containing a predetermined amount of the apoptosis limiting compound as a powder, in granular form, incorporated within liposomes, or as a solution or suspension in an aqueous liquid or non-aqueous liquid such as a syrup, an elixir, an emulsion, or a draught.
  • the tablets, troches, pills, capsules, and the like may also contain one or more of the following: a binder such as gum tragacanth, acacia, corn starch, or gelatin; an excipient such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid, and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, fructose, lactose, or aspartame; and a natural or artificial flavoring agent.
  • a binder such as gum tragacanth, acacia, corn starch, or gelatin
  • an excipient such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid, and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, fructose, lactose, or aspartame
  • Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form.
  • tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar, and the like.
  • a syrup or elixir may contain one or more of a sweetening agent, a preservative such as methyl- or propylparaben, an agent to retard crystallization of the sugar, an agent to increase the solubility of any other ingredient, such as a polyhydric alcohol, for example glycerol or sorbitol, a dye, and flavoring agent.
  • the material used in preparing any unit dosage form is substantially nontoxic in the amounts employed.
  • the compound may be incorporated into sustained-release preparations and devices if desired.
  • a compound suitable for use in the methods of the invention can also be incorporated directly into the food of a subject's diet, as an additive, supplement, or the like.
  • the invention further provides a food product. Any food can be suitable for this purpose, although processed foods already in use as sources of nutritional supplementation or fortification, such as breads, cereals, milk, and the like, are convenient to use for this purpose.
  • Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the desired compound, or dispersions of sterile powders comprising the desired compound, which are preferably isotonic with the blood of the subject.
  • Isotonic agents that can be included in the liquid preparation include sugars, buffers, and salts such as sodium chloride.
  • Solutions of the desired compound can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions of the desired compound can be prepared in water, ethanol, a polyol (such as glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, glycerol esters, and mixtures thereof.
  • the ultimate dosage form is sterile, fluid, and stable under the conditions of manufacture and storage.
  • the necessary fluidity can be achieved, for example, by using liposomes, by employing the appropriate particle size in the case of dispersions, or by using surfactants.
  • Sterilization of a liquid preparation can be achieved by any convenient method that preserves the bioactivity of the desired compound, preferably by filter sterilization.
  • Prefened methods for preparing powders include vacuum drying and freeze drying of the sterile injectible solutions.
  • Subsequent microbial contamination can be prevented using various antimicrobial agents, for example, antibacterial, antiviral and antifungal agents including parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • Absorption of the desired compounds over a prolonged period can be achieved by including agents for delaying, for example, aluminum monostearate and gelatin.
  • Nasal spray formulations can include purified aqueous solutions of the desired compound with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucous membranes. Ophthalmic formulations are prepared by a similar method to the nasal spray, except that the pH and isotonic factors are preferably adjusted to match that of the eye. Formulations for rectal or vaginal administration may be presented as a suppository with a suitable carrier such as cocoa butter, or hydrogenated fats or hydrogenated fatty carboxylic acids.
  • a compound of the present invention can be modified by appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system, brain), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of exertion.
  • a compound may be altered to pro-drug form such that the desired compound is created in the body of the subject as the result of the action of metabolic or other biochemical processes on the pro-drug.
  • pro-drug forms include ketal, acetal, oxime, and hydrazone forms of a compound that contains ketone or aldehyde groups.
  • the dosage level of the compound can be dependent on the route of delivering the compound.
  • the dosage level of the compound of the present invention is preferably on the order of about 10 milligrams or greater, 15 milligrams or greater, or 50 milligrams or greater per kilogram of body weight per day.
  • the dosage level of compound of the present invention for oral delivery is preferably on the order of about 100 milligrams or less, 50 milligrams or less, or 15 milligrams or less per kilogram of body weight per day.
  • the dosage level of compound of the present invention for oral delivery has a range on the order of about 10 milligrams to about 100 milligrams per kilogram of body weight per day.
  • the effective amount by oral administration is on the order of about 500 milligrams to about 1000 milligrams per subject per day.
  • the dosage level of the compound of the present invention is preferably even higher than those delivered orally.
  • the dosage level of the compound of the present invention when delivered intravenously can be on the order of about 10 milligrams or greater, 15 milligrams or greater, 50 milligrams or greater, or 100 milligrams or greater per kilogram of body weight per day.
  • the dosage level of compound of the present invention for intravenous delivery is preferably on the order of about 200 milligrams or less, 100 milligrams or less, 50 milligrams or less, or 15 milligrams or less per kilogram of body weight per day.
  • the dosage level of compound of the present invention for intravenous delivery has a range on the order of about 10 milligrams to about 200 milligrams per kilogram of body weight per day. Dosage levels greater than or less than those recited herein are also possible.
  • the compound When a compound of the present invention is delivered to a subject, the compound can be delivered in one or multiple dosages for injection, infusion, and/or ingestion.
  • PD Parkinson's disease
  • PD degenerative changes are found in the substantia nigra.
  • the substantia nigra is an area of the brain that produces dopamine, a chemical substance that enables people to move normally and smoothly.
  • PD is characterized by a severe shortage of dopamine. It is believed that the deficiency of dopamine causes the symptoms of PD.
  • the compounds described herein are believed to play a role in modulating the apoptotic threshold in both hepatic and nonhepatic cells, it was unexpected that they could be used for the treatment of PD because of the unknown origin of the degenerative changes of the substantia nigra. Similarly, it was unexpected that they could be used in promoting viability of a transplant cell population because of concerns of apoptosis of the transplant cell population during the time necessary for engraphment of the transplanted cell population. In other words, it was surprising that the compounds, and the amount of compounds used, of the present invention were able to inhibit apoptosis of the transplanted cell population long enough for engraphment of the transplant cell population to take place in the subject. In addition, it was unexpected that they could be used in promoting viability of the transplant cell population because of the unknown causes that are involved with the loss of transplanted dopamine neurons.
  • Substantial evidence indicates that apoptosis plays a role in the loss of dopamine neurons in vitro and in vivo.
  • one example where the present invention can be useful in maintaining the viability of a cell to be transplanted into a subject would be in administering an effective amount of hydrophilic bile acid, a salt thereof, an analog thereof, or a combination thereof, to block apoptotic pathways in the cultures and in the grafts, leading to the enhancement of dopamine neuron survival and the improvement of nigral graft function.
  • Anti-apoptotic agents can be applied to the preparation of transplant cell population prior to transplantation or during the first few days after transplantation to prevent apoptosis and improve graft survival.
  • ursodeoxycholic acid UDCA
  • salts thereof analogs thereof (e.g., conjugate derivative tauroursodeoxycholic acid (TUDCA)), and combinations thereof are useful in promoting viability of a transplant cell population used in treating subjects with PD.
  • a suspension of human embryonic dopamine neurons obtained by standard methods in the art can be treated by the method of the invention and can be used for neural transplantation in a subject.
  • human dopamine neurons autologous reconstitution
  • derived from an individual other than the subject heterologous reconstitution
  • TUDCA displays anti-apoptotic properties, where supplementation of TUDCA to cell suspensions prior to transplantation can lead to enhanced survival of nigral grafts. This demonstrates that pre-treatment of the cell suspension with TUDCA can reduce apoptosis and increase the survival of grafted cells, resulting in an improvement of behavioral recovery.
  • TUDCA tauroursodeoxycholic acid
  • cell suspension with TUDCA exhibited a significant reduction in amphetamine-induced rotation scores when compared to pre-transplantation value.
  • the number of apoptotic cells was much smaller in the graft areas in the TUDCA-treated groups than in the control group 4 days after transplantation.
  • ventral mesencephalic (VM) tissue was dissected from Sprague-Dawley (SD) (Charles River Labs, Wilmington, MA) rat embryos at embryonic day 14 under sterile conditions as described (A. Bjorklund et al., Acta. Physiol. Scand. Suppl. 522:9-18; 1983; P. Brundin et al., In: Conn, P. M. ed. Methods in Neurosciences, Lesions and transplantation. Academic Press, San Diego, Vol 7, 1991:305-326; E. M. Grasbon-Frodl et al., Brain Res. Bull.
  • TUDCA was added to a final concentration of 50 ⁇ M to the culture medium when the culture medium was switched to serum-free conditions after two days in vitro. Neural survival and apoptosis were determined by counting TH-positive cells and TUNEL-positive cells in the cultures two or seven days in vitro. A total of three separate series of cell culture experiments were performed.
  • 6-OHDA 6-hydroxy dopamine
  • DA mesostriatal dopamine
  • 12 rats were normal.
  • the rats were housed 2 per cage under a 12 hour day-night cycle with ad libitum access to food and water. They were maintained and treated in accordance with published National Institutes of Health guidelines.
  • TUDCA-treated rats a 50 ⁇ M concentration of TUDCA was added to the media when nigral tissue was trypsinized and dissociated. Two microliters of cell suspension containing TUDCA were then stereotaxically injected into the striatum of SD rats. For the control animals, the same amount of vehicle solution was added to the media.
  • TdT terminal deoxynucleotidyl transferase
  • TUNEL dUTP-biotin nick end labeling
  • an amphetamine-induced rotation test was used to assess the completeness of the 6-OHDA lesions prior to grafting, and repeated two and six weeks after transplantation to monitor functional effects of the neural grafts.
  • the rats were sacrificed after the last session of the rotational behavioral test and the brain tissue was processed for tyrosine hydroxylase (TH)-immunocytochemistry. Graft survival was assessed by counting the number of TH-positive neurons in the grafts.
  • TH tyrosine hydroxylase
  • VM tissue from 24-32 embryos was incubated in 0.1 % trypsin (Sigma, St. Louis, MO)/0.05 % DNase (Sigma, St. Louis MO) at 37 degrees Celsius (°C) for 20 minutes (min), and mechanically dissociated using a 1 milliliter (ml) Gilson pipette. Following dissociation, the cells were centrifuged at 600 rotation per minute (rpm) for 5 min and the pellet was resuspended in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Carlsbad, CA). The cell number and viabilities of dissociated cells were assessed with a hemocytometer using trypan blue dye exclusion.
  • trypsin Sigma, St. Louis, MO
  • DNase Sigma, St. Louis MO
  • the number of 100,000 cells/cm (178,000 cells per well) were plated on to four- well chamber slides (Nunc, Rochester, NY) precoated with 10 milligram per milliliter (mg/ml) poly-d-lysine (Sigma, St. Louis MO). The viabilities of dissociated cells were over 95%.
  • Cell cultures were incubated for 2 days in DMEM supplemented with 10% fetal calf serum at 37°C in a 95% air/5% CO humidified atmosphere. After 2 days in vitro, the culture medium was switched to serum-free N2 medium, consisting of DMEM/Ham's F 12(1:1) mixture (Gibco, Carlsbad, CA). At this time point, some cultures were supplemented with TUDCA.
  • 6-OHDA hydrochloride salt, Sigma, St. Louis MO
  • a first injection of 2.5 microliter ( ⁇ l) of 6-OHDA (3 microgram/microliter ( ⁇ g/ ⁇ l), free base, in 0.2 mg/ml ascorbate-saline) was performed at the following coordinates: 4.4 millimeter (mm) caudal to bregma; 1.2 mm lateral to midline; 7.8 mm ventral to the dural surface; with the tooth-bar set at 2.4 mm below the interaural line.
  • a second injection of 2 ⁇ l of 6-OHDA was performed at the following coordinates; 4.0 millimeter (mm) caudal to bregma; 0.8 mm lateral to midline; 8.0 mm ventral to the dural surface; with the tooth-bar set at 3.4 mm above the interaural line.
  • the 6-OHDA was infused at a rate of 1 ⁇ l/min, and the cannula was left in place for an additional 4 min before it was withdrawn.
  • Nigral Tissue Preparation and Transplantation 5 A cell suspension technique was used to perform the neural transplants as previously described (A. Bjorklund et al., Acta. Physiol. Scand. Suppl. 522:9-18; 1983; P. Brundin et al., In: P.M. Conn, ed., Methods in Neurosciences, Lesions and Transplantation, Academic Press, San Diego, Vol 7, 1991 :305-326). Briefly, VM tissue was obtained from embryos with a crown-to-rump length of 13-14 mm, 0 conesponding to a gestational age of embryonic day 14.
  • HBSS Hank's balanced salt solution
  • Uterine horns were removed by hysterectomy from the animals under deep chloral hydrate anesthesia (250 mg/kg, i.p.) and placed in plastic tubes containing HBSS.
  • the embryos were removed from the uterus and 5 embryonic brains were individually transfened to a petri-dish with a dark background.
  • the VM was dissected out from each brain under a dissection microscope using iridectomy scissors and fine watchmaker's forceps. The dissected pieces were pooled and incubated in 0.1% trypsin (Sigma, St.
  • the avidin-biotin complex immunoperoxidase technique was used to visualize immunocytochemical staining as described previously (W.-M. Duan et al., Neuroscience 100:521-530; 2000).
  • cultures were rinsed once with phosphate buffered saline (PBS, 0.2 Molar, pH 7.4), followed by fixation with 4% formaldehyde for 20 min at room temperature. Then cultures were processed for immunocytochemistry.
  • rats were deeply anesthetized with chloral hydrate at a lethal dose (500 mg/kg, body weight, i.p.) and transcardially perfused with 0.1 Molar PBS followed by cold 4% formaldehyde.
  • the brains were then removed and post-fixed for 4 hours in the same fixative, and placed in 20% sucrose at 4 degree Celsius until they sank. Sections were coronally cut at 30 micrometer thickness on a freezing sliding microtome. Throughout the region of the graft, four adjacent series of sections were collected in four glass vials. The following primary antibodies were used against TH (1 :500 Pel-Freez, Rogers AR). Biotinylated goat anti-rabbit (rat-absorbed) immunoglobulins (1:200) (Vector Laboratories, Inc., Burlingame, CA) were used as the secondary antibody.
  • Sections were incubated in ABC solution (Nectastain ABC Elite kit, Vector Laboratories Inc.) followed by development with 3,3'-diaminobenzidine solution (Vectastain DAB kit, Vector Laboratories Inc.) to visualize the immunoreactive products. After staining, sections were mounted on superfrost microscope slides (Fisher Scientific, Pittsburgh, PA), dehydrated through ascending graded concentrations of alcohol, cleared in xylene, and cover-slipped using DPX mounting medium (Fluka, Switzerland).
  • ABC solution Nectastain ABC Elite kit, Vector Laboratories Inc.
  • 3,3'-diaminobenzidine solution Vectastain DAB kit, Vector Laboratories Inc.
  • TUNEL assay was performed by using an in situ Apoptosis Detection Kit (sold under the trade designation ApopTag) according to the manufacturer's protocol (Intergen, Purchase, NY) as described previously (W.-M. Duan et al., Neuroscience 100:521-530; 2000). Briefly, 3-4 sections containing graft tissue were selected from each grafted animal 4 days post-grafting. They were mounted on superfrost microscope slides (Fisher Scientific, Pittsburgh, PA) for the following staining protocol. The sections were first quenched in 3% hydrogen peroxide in PBS to remove endogenous peroxidase, and then subsequently incubated in working strength TdT enzyme and peroxidase-conjugated antibody against digoxigenin solution.
  • ApopTag in situ Apoptosis Detection Kit
  • 3,3'- diaminobenzidine (Sigma, St. Louis MO) was used as a chromogen to visualize the reactive products. After counterstaining with methyl green, the sections were dehydrated in alcohol and cleared in xylene and cover-slipped using Permount mounting medium (Fisher Scientific, Fair Lown, NJ).
  • the number of TH-positive cells and the total cells were assessed at x20 and x40 magnification, respectively, with the aid of a
  • TH- positive neurons in the grafts were counted on every fourth section using a lOx objective lens in a Nikon light microscope (Nikon, Japan). Only cells were counted when they exhibit at least one neurite or have a visible nucleus.
  • TUNEL-positive cells were assessed at x400 magnification with the aid of a 400- ⁇ m square, reticule grid. Six fields were selected from each culture well in a systematic fashion. For the transplantation experiments, TUNEL-positive cells in the graft areas were counted in 3-4 sections per animal. Average number of TUNEL-positive cells was calculated and represented a value for single animals.
  • the volume of intrastriatal neural grafts was analyzed using a computer assisted image analysis system as described previously (W.-M. Duan et al., Eur. J. Neurosci. 10:2595-2606; 1998). Briefly, all the TH-immunostained sections with grafts were digitized using a lx objective lens under a Nikon light microscope (Nikon, Japan) that is connected with a high resolution digital camera (COOLPIX 950, Nikon, Japan). The images were first collected and stored in a CompactFlash card.
  • the CompactFlash card was read by using a CompactFlash card reader connected with a Pentium III PC (Dell, Dimension XPS T700r, USA) and the images were analyzed using a software package (Scion Image, Version Beta 4.0.2, Scion Corporation, Frederick, MD). In each section, the graft was manually outlined on the screen and the surface area measured. The number of pixels was subsequently converted into square millimeters. The graft volume was calculated based on graft area, section thickness and frequency.
  • TH-positive cells possessed short processes, indicating that they were in an early developmental stage. Some processes contacted processes from other cells in their vicinity (data not shown).
  • dopamine neurons exhibited more mature morphology. They sent out several long processes with clear varicosities (data not shown). At this time-point, some of the cell bodies and processes appeared granular under phase-contrast, possibly indicating that they were undergoing degeneration. There was a significant cell loss for TH- positive neurons and total cells in 7 DIV control cultures (p ⁇ 0.01, one-factor ANOVA with post-hoc Scheffe's F-test).
  • Fig. 5 summarizes the mean number of TH-immunoreactive neurons in the grafts in the control and the TUDCA-treated groups 6 weeks after grafting.
  • the mean number of TH-immunoreactive neurons in the grafts was significantly greater in the TUDCA-treated group than in the control group.
  • TH- immunopositive neurons were located at the periphery of the grafts, leaving the center of the grafts relatively devoid of TH-immunoreactivity (Fig. 4D).
  • Fig. 4D the majority of TH- immunopositive neurons were located at the periphery of the grafts, leaving the center of the grafts relatively devoid of TH-immunoreactivity.
  • Most TUDCA-treated animals showed an even distribution of TH-immunoreactive neurons in the graft area (Fig. 4C).
  • the TH-immunoreactive neurons possessed multipolar cell bodies with several clearly stained neurites (Fig. 4E).
  • the areas of the host striatum that were reinnervated by the grafts were found to be larger in the TUDCA-treated group than in the control group (Fig. 4A and B).
  • the mean number of TUNEL-positive cells in the grafted areas is summarized in Fig. 6 for the control and TUDCA-treated groups 4 days post-transplantation.
  • F (1; 10) 20.06
  • a large number of apoptotic cells with D ⁇ A fragmentation in nuclei were clustered and located within the grafts in the control group (data not shown). Only a few apoptotic cells were observed in several patches within the grafts in the TUDCA-treated group (data not shown).
  • the culture system where neuronal death was induced by serum deprivation in mesencephalic cultures is well known as an in vitro model to induce apoptosis.
  • This in vitro model has been used extensively to examine the effects of anti-apoptotic agents and neurotrophic factors on apoptosis that occurs in the NM tissue cultures and the survival of dopamine neurons (R.L. Branton et al., Exp. Neurol. 160:88-98; 1999; E.D. Clarkson et al., Neuroreport 7:145-149; 1995; E.D. Clarkson et al., Cell Tissue Res. 289:207-210; 1997; G.S. Schierle et al., Nat. Med.
  • Nigral grafts that are transplanted into the striatum are heterotopic grafts.
  • the new environment in the striatum may not favor nigral graft survival and the neural grafts may lack sufficient neurotrophic support.
  • TUDCA supplemented medium used during the preparation of cell suspension increased the survival of neural grafts by approximately three-fold and the graft function was also improved.
  • TUDCA has been shown to prevent mitochondrial swelling and disruption of the outer mitochondrial membrane. Membrane stability can inhibit pro-apoptotic molecules, cytochrome c release and lead to changes in cytochrome c-mediated downstream events, such as caspase activity. It is believed that TUDCA significantly reduces 3-nitropropionic acid (3-NP)-mediated neuronal cell death in striatal tissue cultures.
  • TUDCA can reduce striatal degeneration mainly through anti-apoptotic mechanisms and ameliorate neurological deficits in a 3-NP-lesioned rat model of Huntington's disease (CD. Keene et al., Exp. Neurol. 171; 351-360, 2001).
  • CD. Keene et al., Exp. Neurol. 171; 351-360, 2001 As the majority of transplanted dopamine neurons die immediately following transplantation, an effort has been focused on the enhancement of graft survival to obtain optimum graft benefit. Indeed, numerous experimental studies have shown that the extent of behavioral recovery strongly correlates with the graft survival (P. Brundin et al., In: S.B. Dunnett and A.
  • TUDCA was also effective in reducing apoptosis of pancreatic islet cells after 2 days in vitro.
  • the number of TUNEL-positive cells was approximately 29 in non-treated controls, but only 12 in cells exposed to TUDCA.
  • the more than 50% reduction by apoptosis by TUDCA was achieved after exposure of cells to TUDCA during the isolation procedure, followed by incubation with the bile acid during culture in vitro.

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