CN1770975A - Methods of Improving Cell Viability - Google Patents

Abstract

Methods for increasing the viability of a transplant cell population by administering a hydrophilic bile acid, such as ursodeoxycholic acid (UDCA), salts thereof, and analogs thereof (e.g., glycoursodeoxycholic acid and tauroursodeoxycholic acid).

Description

Methods of Improving Cell Viability

Background technique

Cell transplantation (eg, of organs) is currently widely used in the treatment of a variety of human diseases. For example, heart transplants have been routinely performed on patients with heart defects. However, a major obstacle in transplantation is the lack of high-quality tissue for transplantation. Many cells (eg, organs) ultimately fail to engraft due to poor survival after harvest. Methods of increasing the viability of transplanted cells are therefore highly desirable.

An example of a disease for which transplantation is possible is Parkinson's disease. Parkinson's disease (PD) is a central nervous system disorder that affects 1 to 1.5 million Americans. PD can appear at any age, but the risk of developing PD increases with age. Furthermore, PD exists throughout the world and affects men slightly more commonly than women. Symptoms of PD can include stiffness, tremors, bradykinesia, difficulty walking and/or balance. People with PD often don't experience all of these symptoms, but more of some of them.

The exact cause of PD is unknown. PD is generally believed to be caused by a combination of genetic predisposition and as yet unidentified environmental triggers. When PD occurs, degenerative changes are found in the dopamine-producing area of the brain called the substantia nigra. Dopamine is a chemical that enables normal and free movement. PD is characterized by a severe deficiency of dopamine. It is generally believed that a deficiency of dopamine causes the symptoms of PD.

Transplantation of human embryonic dopamine neurons in PD patients is currently being evaluated in clinical trials (A. Björklund et al., Nat. Neurosci. 3:537-544, 2000.). Although it has been reported that a small number of PD patients show normalization of dopamine content in the brain and return to normal life after nerve transplantation (J.H.Kordower et al., J.Comp.Neurol.370:203-230,1996; P.Piccini etc., Nat.Neurosci.2: 1137-1140, 1999), most transplant patients show incomplete symptom recovery (C.R.Freed et al., N.Engl.J.Med.344:710-719, 2001; Lindvall, O., Cell Transpl. 4:393-400, 1995). Several factors may have contributed to this limitation. Among them, the loss of transplanted dopamine neurons is a major obstacle to achieving clinical improvement. Many animal experiments and clinical studies have shown that when dopamine neurons are transplanted into the brain using cell suspension technology (P.Brundin et al. Prog. Brain Res. 71:293-308, 1987; N.Nakao et al., Proc. Acad.Sci.USA91:12408-12412, 1994; H.Sauer et al., Restor.Neurol.Neurosci.2:123-135, 1991; H.Sauer et al., Exp.Brain Res.90:54-62, 1992 G.S.Schierle et al., Nat.Med.5:97-100,1999; A.Zuddas et al., Eur.J.Neurosci.3:72-85,1991) the survival rate of transplanted dopamine neurons is only about 10% .

In a recent review, it was estimated that a minimum of 80,000 dopamine neurons are estimated to be required for a significant clinical effect (A. Björklund et al., Nat. Neurosci. 3:537-544, 2000). However, some clinical studies have demonstrated that the number of surviving dopamine neurons in the brain is far less than in patient samples (C.R. Freed et al., N. Engl. J. Med. 344:710-719, 2001). Increased survival of dopamine neurons is therefore an appropriate topic (P. Brundin et al., Cell Transplant. 9: 179-195, 2000). Furthermore, the improvement in dopamine neuron survival may bypass the limited availability of human embryonic dopamine tissue, a barrier in the clinical application of neural transplantation.

There is strong evidence that apoptosis plays a role in the loss of transplanted dopamine neurons (M. Emgard et al., Exp. Neurol. 160, 279-288, 1999; T. J. 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). Furthermore, apoptosis usually occurs within the first few days after transplantation (M. Emgard et al., Exp. Neurol. 160, 279-288, 1999; G.S. Schierle et al., Nat. Med. 5: 97-100, 1999; W.M. Zawada et al., Brain Res. 786, 96-103, 1998). In an in vitro study, it was found that the apoptosis associated with the loss of dopamine neurons occurred mainly during the first 24 hours, and about 50% of the dopamine neurons were lost within the first 8 hours (R.L.Branton et al., Exp. Neurol. 160:88-98, 1999). These studies provide a possible explanation for the observed majority of cell death in substantia nigra grafts observed above (R.A.Barker et al., Exp.Neurol.141:79-93, 1996; W.-M.Duan et al., Exp. Brain Res. 104:227-242, 1995).

Since neural transplantation of human embryonic dopamine neurons in PD patients exhibits incomplete symptomatic recovery, there remains a need to reduce the loss of transplanted cells in patients. These requirements apply not only to neuronal transplantation in PD, but also to other transplantation applications.

Summary of the invention

The present invention provides methods of increasing the viability of a transplanted cell population transplanted into a recipient. Preferably the recipient is a human recipient and increasing the viability of the transplanted cell population comprises contacting the transplanted cell population with an effective amount of a compound selected from the group consisting of hydrophilic bile acids, salts thereof, analogs thereof, and combinations thereof.

The cells of the transplanted cell population can include, for example, differentiated cells and precursor cells. In addition, the cells of the transplanted cell population can include autologous cells, xenogeneic cells, or allogeneic cells. Additionally, the cells of the transplanted cell population may comprise autologous tissue, xenogenic tissue, or at least a portion of allogeneic tissue. For certain embodiments, the population of cells may be in the form of an organ, or part thereof, such as liver, heart, kidney, lung, and pancreas. Preferably the cell population is a human cell population.

Contacting can be performed in vitro, in vivo, and combinations thereof. As used herein, in vitro is different from in vivo. In vitro refers to an artificial environment in which a transplanted cell population is to be processed, such as cell culture in a tissue culture dish. In vivo refers to the natural environmental location of the population of cells being treated, such as in a mammalian body.

One aspect of the invention provides a method comprising contacting a population of transplanted cells with a compound prior to transplanting the population of transplanted cells into a recipient. This can be done before (in vivo) or after (in vitro) the transplanted cell population is removed from the donor. Alternatively, this can be done after the transplanted cell population has been transplanted into the recipient.

Additionally or alternatively, the invention provides a method comprising treating a subject to receive a transplanted cell population with a compound. For example, treating a subject with a compound can comprise administering the compound to the subject prior to transplanting the transplanted cell population into the subject. Additionally, as described above, treating a subject with a compound may comprise administering the compound to the subject after transplantation of the transplanted cell population into the subject. In the latter embodiment, the recipient may have been treated with the compound prior to transplantation of the transplanted cell population into the recipient. Preferably, treating the subject comprises treating the subject with the compound parenterally or orally.

The invention also provides a method of treating a donor and/or recipient of a population of transplanted cells with a compound selected from the group consisting of hydrophilic bile acids, their salts, their analogs, and combinations thereof. As described herein, hydrophilic bile acids useful in the present invention may include, but are not limited to, ursodeoxycholic acid and/or tauroursodeoxycholic acid.

One aspect of the invention provides a method comprising treating a donor of a population of transplanted cells with a compound. This can be done before (in vivo), during (in vivo) or after (in vitro) removal of the transplanted cell population from the donor. Additionally or additionally, the invention provides a method comprising treating a recipient of a transplanted cell population with a compound. This can be done before (in vivo/in vitro), during (in vivo) or after (in vivo) transplantation of the transplanted cell population into the recipient.

Another aspect of the invention is a method for treating a subject, preferably a human, suffering from a disease requiring cell replacement. For example the invention may provide a method for treating a subject, preferably a human, suffering from Parkinson's disease. The method may comprise contacting the transplanted cell population with an effective amount of a compound selected from the group consisting of ursodeoxycholic acid, salts thereof, analogs thereof, and combinations thereof to increase the viability of the transplanted cell population. The method then further comprises transplanting the population of transplanted cells into the recipient.

An example of an analog of ursodeoxycholic acid includes a conjugated derivative, wherein the conjugated derivative may be tauroursodeoxycholic acid. In a specific embodiment, the method of the present invention may comprise treating a human suffering from Parkinson's disease, wherein the method comprises ex vivo combining the transplanted cell population with an effective amount of taurursodeoxychol and a pharmaceutically acceptable carrier Acid exposure, wherein the transplanted cell population is differentiated cells. The method then further comprises transplanting the population of transplanted cells into a human.

As noted above, cell populations for transplantation in humans with Parkinson's disease can include differentiated cells and precursor cells. In addition, the cells of the transplanted cell population can include autologous cells, xenogeneic cells, or allogeneic cells. Additionally, the cells of the transplanted cell population may comprise autologous tissue, xenogenic tissue, or at least a portion of allogeneic tissue. The contacting step can be performed in vitro, in vivo, and combinations thereof. In one embodiment, the cell population is a human cell population.

Brief description of the drawings

Figure 1 is a bar graph summarizing the effect of TUDCA (50 [mu]M/ml) on the survival of TH-positive neurons (shaded bars) and total cells (blank bars) in ventral midbrain (VM) tissue cultures. Bars represent mean ± SEM of three independent experiments of four wells per culture condition. ^* p<0.01, significant difference from 7DIV+TUDCA culture (one-way ANOVA with post hoc Scheffé's F test).

Figure 2 is a bar graph showing the effect of TUDCA (50 [mu]M/ml) on apoptosis in VM tissue culture. Bars represent mean ± SEM of three independent experiments of four wells per culture condition. ^* p<0.01, significant difference from 2DIV and 7DIV+TUDCA cultures (one-way ANOVA with post hoc Scheffé's F test).

当与移植前的值比较时p＜0.01(成对Student t检验)。Figure 3 is a graph showing the net ipsilateral amphetamine-induced rotational asymmetry (full rotation per minute (min) minus ipsilateral lateral lesion side) for control and TUDC-treated groups over a 90-minute test period. Histogram of the rotation of the damaged side). Rats were tested before transplantation and 2 and 6 weeks after transplantation. Each bar represents group mean and error bars represent SEM. ^* p<0.01 when compared to pre-transplant values (paired Student t-test) and p<0.05 when compared to control group (one-way ANOVA with post-hoc Scheffé's F test). symbol

p<0.01 when compared to pre-transplant values (paired Student's t-test).

Figures 4A-4E are photomicrographs of coronal sections of transplanted striatum treated with TH-immunocytochemistry. Photographs show typical transplantation of sample mice in TUDCA-treated groups (A, C and E) and control groups (B and D) six weeks after transplantation. (A) and (B) show an overview of the transplanted brain at low magnification. The host striatal region adjacent to the graft was neurally grafted with TH-immunopositive fibers from the grafted neurons. Scale bars in B = 1 mm, in D = 100 μm and in E = 25 μm.

Figure 5 is a bar graph of the mean number of TH-immunopositive cells and graft volume in the control group and the TUDCA-treated group six weeks after transplantation. Each bar represents group mean ± SEM. ^* p<0.01 when compared to TUDCA-treated group (one-way ANOVA with post-hoc Scheffé's F test).

Fig. 6 is a bar graph showing the mean number of apoptotic cells in the transplanted area of the control group and the TUDCA-treated group 4 days after transplantation. The number of apoptotic cells in the transplanted area in the TUDCA-treated group was significantly less than that in the control group. Asterisks indicate p<0.01 compared to control group (one-way ANOVA with post hoc Scheffé's F test). Error bars represent S.E.M.

Detailed Description of Illustrative Embodiments

Currently, effective treatments are needed to increase the viability of transplanted cell populations for the treatment of various medical conditions. The present invention provides such methods for increasing the viability of a transplanted cell population. The method may comprise contacting the transplanted cell population with an effective amount of a compound selected from the group consisting of hydrophilic bile acids, salts thereof, analogs thereof, and/or combinations thereof before, during or after transplantation of the transplanted cell population into the recipient.

As used herein, "increasing" the viability of a transplanted cell population includes maintaining, prolonging and/or improving the survival and/or proliferation of a transplanted cell population that is about to be transplanted, or has been transplanted into a recipient. Furthermore, the term "viability" refers to the maintenance of normal function of cells in a transplanted cell population generally in vivo, however the term also includes in vitro.

The term "prolongation" refers to the preservation of a transplanted cell population for transplantation by treatment with the methods of the invention, compared to a similar transplanted cell population that has not undergone such treatment. While not wishing to be bound by a particular theory, it is believed that contacting a transplanted cell population for transplantation with a compound of the invention inhibits apoptosis, thereby prolonging the viability of the transplanted cell population.

As used herein, a "transplanted cell population" includes, but is not limited to, individual cell populations that have been or can be transplanted into a recipient as well as cell populations present in tissues and/or organs. Likewise, the cells of the transplanted cell population may include matrix structures (eg, proteins, polysaccharides, polypeptides, and other molecules) found in tissues and/or organs. As used herein, "a," "at least one," and "one or more" are used interchangeably.

Patients with medical conditions that can be improved by increased viability of transplanted cell populations include those with neurodegenerative diseases such as Parkinson's and Alzheimer's diseases; Huntington's disease; multiple sclerosis; amyotrophic lateral Sclerosis; cerebellar ataxia; degeneration of lysosomal storage; cancer; birth defects; those requiring organ and/or tissue transplantation; spinal cord injury; ischemic injury such as stroke, ischemic kidney disease, and heart disease; burns; autoimmunity disease; diabetes; patients with inflammatory diseases such as osteoarthritis and rheumatoid arthritis. This list is not exhaustive however, and other medical conditions known to be ameliorated by increased viability of transplanted cell populations for transplantation are also considered to be within the scope of the present invention.

For determining the increase in viability of a transplanted cell population, as defined herein, it can be assessed by measuring the ATP level and protein synthesis capacity of the cells in the transplanted cell population. ATP levels affect energy production during and after transplantation. Protein synthesis capacity is a routine indicator of cell viability because protein synthesis requires the integration of several complex biochemical pathways.

Preferred transplant cell populations may include transplant cell populations obtained from donors for transplantation into recipients. Cell populations for transplantation can be derived from pediatric, adult or fetal tissue. Preferred cell populations for transplantation may include, but are not limited to, those derived from blood and all 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 ganglion, posterior pituitary gland, adrenal medulla, and pineal gland; connective tissue, including skin, skin stem cells, ligaments, tendons, and fibroblasts; muscle tissue, including bone, skeletal muscle stem cells , smooth muscle, and cardiac tissue or cells thereof; endocrine tissue, including anterior pituitary gland, thyroid, parathyroid, adrenal cortex, pancreas and its subunits (eg, islet cells), testis, ovary, placenta, and each of these tissues Portions of endocrine cells; blood vessels, including arteries, veins, capillaries, and cells derived from these vessels; lung tissue; heart tissue; brain tissue; heart valves; liver; kidney; intestine; bone; immune tissue, including blood cells, bone marrow, and spleen Adipose tissue, including adult progenitor cells (eg, adult stem cells) derived from adipose tissue; eye and parts thereof; cell populations of reproductive tract tissue and urinary tissue. Intact organs are a common form of transplanted cell populations.

The term "organ" as used herein is meant to include whole or a portion of an intact multi-cellular organ such as a kidney, liver, brain or heart; a suspension of cells derived from a multi-cellular organ; and a suspension of blood cells or hematopoietic progenitor cells . "Tissue" as used herein refers to an aggregate comprising a population of transplanted cells and their associated intracellular matrix (eg, collagen, proteins, polysaccharides, etc.). Transplanted cell populations may also include single cells that have been sorted and/or isolated from the tissue and/or organ from which they were derived.

For those transplant cell populations that include sorted and/or isolated cells, the transplant cell population for transplantation into a recipient can include differentiated cells and/or precursor cells (eg, undifferentiated cells). For example, differentiated cells can include, but are not limited to, cells described herein, including, but not limited to, cardiomyocytes, muscle cells, smooth muscle cells, epidermal cells, neurons including dopamine neurons, pancreatic cells, bone marrow cells , hepatic and non-hepatic cells. For example, precursor cells can include, but are not limited to, stem cells, pluripotent stem cells, embryonic stem cells, and adult stem cells.

Preferred cells for use in the transplant cell populations of the present invention include, but are not limited to, autologous cells, allogeneic cells, allogeneic cells, or combinations thereof. As noted above, cells may also include those cells that form part of a tissue and/or organ. Thus, for the present invention, cells may comprise at least a portion of autologous tissue, allogeneic tissue, allogeneic tissue, and/or combinations thereof, wherein the tissue may be transplanted into the recipient.

As used herein, "recipient" may include mammals. Preferably the receptor used herein is human. A "donor" as used herein may include a mammal used as a source of biological material, such as a portion or all of a cell population (including an organ) for transplantation into a recipient. Donors can include, but are not limited to, humans, pigs, non-human primates, cattle, or combinations thereof. Additionally, the donor can be alive during the donation of the cell population.

Furthermore, it is understood that the population of transplanted cells for transplantation as defined herein may be derived from any species. The invention can be used for preservation in allografts for recipients such as humans as well as other donors (allografts and autologous or allografts) or for example from another species such as sheep, pigs, cows or non-human Primate (xenograft) grafted cell populations into human recipients. The transplanted cell populations for transplantation include, but are not limited to, heart, liver, kidney, lung, pancreas, islets, brain, cornea, bone, intestine, skin, blood and cells from the above organs and tissues.

Preferably, contacting the transplanted cell population with an effective amount of one or more compounds of the invention can be accomplished in vitro and/or in vivo. For example, a population of transplanted cells can be contacted with a compound of the invention in vivo prior to transplanting the population of transplanted cells into a recipient. A preferred method of accomplishing this involves treating a donor of a transplanted cell population with one or more compounds of the invention.

In one aspect of the invention, a donor of a transplant cell population may be treated with a compound of the invention before (in vivo), during (in vivo) or after (in vitro) removal of the transplant cell population from the donor. Thus, the transplanted cell population can be contacted with the compounds of the invention while still in the donor (in vivo). Alternatively, the transplanted cell population can be contacted with a compound of the invention during removal or when the transplanted cell population is removed from the donor. For partial organ donors, for example, benefiting the donor can be achieved by administering the compounds of the invention before and/or after partial removal of the organ. Preferably the transplanted cell population is perfused and/or infiltrated or contacted with a compound of the invention during harvest, storage, culture and/or transplantation of the transplanted cell population.

In addition, the compounds of the present invention are useful in the in vivo treatment of recipients of transplanted cell populations. This can be done before (in vivo/in vitro), during (in vivo) or after (in vivo) transplantation of the transplanted cell population into the recipient. Thus, it is possible that recipients receiving transplanted cell populations also receive treatment with the compounds of the invention. For example, the recipient can be administered the compound before (i.e., pre-cell population transplantation), during (i.e., while transplanting the transplantation cell population) and/or after (i.e., post-cell population transplantation) the transplanted cell population. The compounds of the invention are administered to a recipient for systemic or local treatment.

In another embodiment, a population of transplanted cells can be contacted with a compound of the invention in vivo following transplantation of the population of transplanted cells into a recipient. Thus, once a transplanted cell population has been implanted in a recipient, the transplanted cell population can be contacted with a compound of the invention. Furthermore, the compounds of the invention may be used in different combinations of the in vivo/in vitro treatment modalities described herein.

In a specific embodiment, starting at a predetermined time prior to transplantation, the recipient may be administered immunosuppressive drugs to enhance transplant acceptance. Recipients may be administered one or more compounds of the invention immediately prior to transplantation. The donor's allografted cell population can then be flushed with a solution comprising at least one compound of the invention, eg, tauroursodeoxycholic acid (TUDCA). Following standard surgical transplantation, recipients are usually maintained with conventional immunosuppressive drugs and, optionally, with compounds of the present invention. Immunosuppressant doses may be reduced on the basis of clinical signs and symptoms related to the immune response.

As used herein, the term "effective amount" includes dosage levels of the compounds of the present invention effective to increase the viability of a transplanted cell population. Although the inventors do not wish to be bound by any theory, it is generally believed that an "effective amount" is an amount effective to prevent, reduce, inhibit or arrest apoptosis of cells to be transplanted and/or cells that have been transplanted. Effective dosages of the desired compounds described herein can be determined by comparing their in vitro and in vivo activity in animal models. Methods for extrapolating effective doses from mice and other animals to humans are known in the art.

It will be appreciated, however, that a particular "effective amount" for any particular recipient (or donor) and/or transplanted cell population will depend on a variety of factors, including the activity of the particular compound used; the transplanted cell population; the transplanted cell population Conditions of harvest, isolation, storage, and/or incubation of transplanted cell populations while maintained in vitro; and age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion when used in vivo , the drug combination and the severity of the recipient (or donor) medical condition to be treated.

Preferably, the transplanted cell population is contacted with a compound of the invention during storage. Preservation of a transplanted cell population can include conditions under which the transplanted cell population is maintained. Alternatively, preservation of the transplanted cell population can also include conditions of the transplanted cell population that promote growth and proliferation of the transplanted cell population. In either case, the transplanted cell population can be contacted with an effective amount of a compound of the invention.

To increase the survival of transplanted cell populations prior to transplantation, the compounds of the present invention may be used with cell culture media for the cultivation of cells transplanted into the recipient. The concentration of the compound depends on several factors, including solubility and activity.

Preferably the methods of the invention involve the use of hydrophilic bile acids, their salts, their analogs or combinations thereof. As used herein, hydrophilic bile acids are more hydrophilic than deoxycholic acid (DCA). This can be determined by evaluating the partition coefficient between water and octanol with more hydrophilic bile acids that are more water-oriented. Alternatively, more hydrophilic bile acids have earlier retention times on reverse-phase columns for HPLC. A particularly preferred hydrophilic bile acid includes ursodeoxycholic acid. Examples of hydrophilic bile acid analogs include conjugated derivatives of bile acids. While not all hydrophilic bile acids are useful in all methods of the invention, they can be readily assessed by testing their ability to inhibit apoptosis in cell culture using agents known to induce apoptosis. Two particularly preferred conjugated derivatives include glycine- and tauro-ursodeoxycholic acid.

Ursodeoxycholic acid (UDCA) is an endogenous bile acid that has been clinically used in the past decades to treat a variety of liver diseases. Conjugated derivatives of UDCA include ursodeoxycholic acid 3-sulfate, ursodeoxycholic acid 7-sulfate, ursodeoxycholic acid 3,7-pyrosulfate, tauroursodeoxycholic acid (TUDCA) and glycosylated ursodeoxycholic acid.

In general for the preferred embodiments, the compounds described herein can be formulated as pharmaceutical compositions. The transplanted cell population can then be contacted with a pharmaceutical composition comprising a compound of the invention, according to the methods of the invention. Furthermore, pharmaceutical compositions comprising compounds of the present invention may be administered to a recipient, typically a mammalian such as a human recipient, in a variety of forms suitable for the chosen route of administration. Formulations include those suitable for in vitro cell culture and for oral, rectal, vaginal, topical, nasal, ophthalmic, parenteral (including subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal, intraventricular, direct injection into brain tissue, etc. etc.) granted.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. In general the methods include the step of bringing into association the active substance with a carrier, which may include one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately incorporating the active material into liquid carriers, finely divided solid carriers or both, and then, if necessary, shaping the product into the desired dosage form.

Dosage forms suitable for oral administration according to the present invention may be discrete units such as tablets, troches, capsules, lozenges, flakes or cachets, each containing a predetermined amount of an apoptosis-limiting compound, which is incorporated into a lipid in particulate form. In vivo powders, or solutions or suspensions in aqueous or non-aqueous liquids such as syrups, elixirs, emulsions, or tractions.

Tablets, lozenges, pills, capsules, etc. may also contain one or more of the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, alginic acid, etc.; lubricants such as magnesium stearate; sweeteners such as sucrose, fructose, lactose or aspartame; and natural or synthetic flavorings. When the unit dosage form is a capsule, it may further contain a liquid carrier such as vegetable oil or polyethylene glycol. Various other materials may be present to coat or otherwise modify the physical form of the solid unit dosage form. For example, tablets, pills or capsules may be coated with gel, wax, shellac, sugar, and the like. Syrups or elixirs may contain one or more sweeteners, preservatives such as methyl- or propylparaben, agents that delay the crystallization of sugars, agents that increase the solubility of any other ingredient such as polyols, such as glycerin or sorbitol Sugar alcohols, dyes and flavorings. Materials used in preparing any unit dosage form are substantially nontoxic in the amounts employed. The compounds may, if desired, be incorporated into sustained-release formulations and devices.

Compounds suitable for use in the methods of the invention may also be added directly to the food of the subject's diet as additives, supplements, and the like. Thus, the present invention further provides a food. Although processed foods have been used as a source of added or enhanced nutrition, any food is suitable for this purpose, such as bread, cereals, milk, etc. are suitable for this purpose.

Dosage forms suitable for convenient parenteral administration include sterile aqueous formulations of the desired compound or sterile powder suspensions comprising the compound, which are preferably isotonic with the blood of the recipient. Isotonic agents that can be included in liquid dosage forms 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. Suspensions of the desired compound can be prepared in water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, glycerides, and mixtures thereof. The final dosage form is sterile, liquid and stable under the conditions of manufacture and storage. The necessary fluidity can be achieved, for example, by using liposomes, by using a suitable particle size in suspension or by using surfactants. Sterilization of liquid dosage forms can be accomplished by any convenient method that retains the biological activity of the desired compound, preferably by filter sterilization. Preferred methods for the preparation of powders include vacuum drying and freeze-drying of sterile injectable solutions. Subsequent microbial contamination can be utilized with various antimicrobial agents, for example, antibacterial agents including parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like, antivirals and antifungals agent to prevent. Absorption of the desired compound over an extended period of time can be brought about by the inclusion of delaying agents, for example, aluminum monostearate and gelatin.

Nasal spray forms may contain purified aqueous solutions of the desired compound and preservatives and isotonic agents. The above dosage forms are preferably adjusted to a pH and isotonic state compatible with the nasal mucosa. Ophthalmic dosage forms are prepared by a method similar to the nasal spray formulation, except that the pH and isotonic conditions are preferably adjusted to match those of the eye. Formulations for rectal or vaginal administration may be presented as suppositories with suitable carriers such as cocoa butter or hydrogenated fats or hydrogenated fatty carboxylic acids.

In addition, the compounds of the present invention may be modified with appropriate functionalities to enhance their selective biological characteristics. Such modifications are known in the art and include those that increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system, brain), increase oral availability, increase solubility to allow injection by injection Modifications for drug delivery, altering metabolism, and altering utilization. In addition, a compound can be altered to a prodrug form such that the desired compound is produced in the recipient as a result of metabolism of the prodrug or other biochemical process. Some examples of prodrug forms include ketal, acetal, oxime and hydrazone forms of compounds containing ketone or aldehyde groups.

Where a compound of the invention can be delivered in vivo, the dosage level of the compound will depend on the route of delivery of the compound. For example, when delivered orally, dosage levels of the compounds of the invention are preferably on the order of about 10 milligrams or more, 15 milligrams or more, or 50 milligrams or more per kilogram of body weight per day. Furthermore, in a preferred embodiment, dosage levels of the compounds of the invention for oral delivery are preferably on the order of about 100 milligrams or less, 50 milligrams or less, or 15 milligrams or less per kilogram body weight per day. Dosage levels of the compounds of the invention for oral delivery are preferably in the range of about 10 milligrams to about 100 milligrams per kilogram body weight per day. In one embodiment, the effective amount administered orally is on the order of about 500 mg to about 1000 mg per subject per day.

In another embodiment, when the compound of the invention is delivered intravenously, the dosage level of the compound of the invention is preferably higher than when it is delivered orally. For example, when delivered intravenously, dosage levels of the compounds of the invention may be on the order of about 10 mg or more, 15 mg or more, or 100 mg or more per kilogram of body weight per day. Furthermore, in a preferred embodiment, the dosage level of the compounds of the invention for intravenous delivery is preferably about 200 mg or less, 100 mg or less, 50 mg or less, or 15 mg or more per kilogram of body weight per day. Less or so. Dosage levels of the compounds of the invention for intravenous delivery are preferably in the range of about 10 milligrams to about 200 milligrams per kilogram of body weight per day.

Dosage levels higher or lower than those described herein are also feasible. When the compounds of the present invention are delivered to a recipient, the compounds may be delivered in single or multiple doses for injection, infusion and/or oral administration.

One example where it would be useful to increase the viability of transplanted cell populations for transplantation into recipients is in the treatment of patients with Parkinson's disease (PD). In PD, degenerative lesions are found in the substantia nigra. The substantia nigra is the area of the brain that produces dopamine, a chemical that enables normal and free movement. PD is characterized by a severe deficiency of dopamine. It is generally believed that a deficiency of dopamine causes the symptoms of PD. Although the compounds described here are thought to act on modulating apoptotic thresholds in hepatic and non-hepatic cells, it was unexpected that they could be useful in the treatment of PD due to the unknown cause of substantia nigra degeneration. Also, it is surprising that it can be used to increase the viability of a transplanted cell population due to its involvement in the apoptosis of the transplanted cell population during the time required for engraftment of the transplanted cell population. In other words, it is surprising that the compounds of the invention and the amount of compounds used can inhibit apoptosis of the transplanted cell population long enough for engraftment of the transplanted cell population to occur in the recipient. Furthermore, it was unexpected that for unknown reasons related to the loss of transplanted dopamine neurons, it could be used to increase the viability of transplanted cell populations.

There is convincing evidence that apoptosis plays a role in the loss of dopamine neurons both in vitro and in vivo. Thus, one example where the present invention can be used to maintain the viability of cells to be transplanted into a recipient is the administration of an effective amount of a hydrophilic bile acid, a salt thereof, an analog thereof, or a combination thereof to block the Enhanced survival of dopamine neurons and improved function of substantia nigra grafts through apoptotic pathway. Anti-apoptotic agents can be used in the preparation of transplanted cell populations prior to transplantation or during the first few days after transplantation to prevent apoptosis and improve graft survival.

Preferably ursodeoxycholic acid (UDCA), its salts, its analogs (e.g., the conjugated derivative tauroursodeoxycholic acid (TUDCA)) and combinations thereof are useful for increasing the yield of transplanted cell populations for the treatment of PD patients. viability. For example, human embryonic dopamine neuron suspension obtained by standard methods in the art can be processed by the method of the present invention and used for neural transplantation in recipients. For example as an in vitro therapy, human dopamine neurons (autologous reconstitution) or dopamine neurons derived from other receptors alone (heterologous reconstitution) can be expanded for the treatment or prevention of PD. As disclosed herein, TUDCA exhibits anti-apoptotic properties, wherein the addition of TUDCA to the cell suspension prior to transplantation resulted in increased survival of substantia nigra grafts. This demonstrates that pretreatment of cell suspensions with TUDCA reduces apoptosis and increases survival of transplanted cells, leading to improved behavioral rehabilitation.

The advantages of the invention are illustrated by the following examples. However, the specific materials and amounts, as well as other conditions and details, set forth in the examples are to be construed as those broadly applied in the art and should not be construed to unduly limit the present invention.

Example

In the following examples, the effect of hydrophilic bile acids on apoptosis in neural networks is further characterized and the nature and mechanism of tauroursodeoxycholic acid (TUDCA)-induced neuronal protection are described. Specifically, TUDCA treatment caused a significant reduction in dopamine neuronal cell death and degeneration. Furthermore, a deoxynucleotidyl transferase (TdT)-mediated 2′-deoxyuridine 5′-triphosphate (dUTP)-biotin nick end labeling (TUNEL) assay demonstrated that apoptosis in TUDCA-treated cultures The number of dead cells was significantly less than in control cultures.

In addition, cell suspensions containing TUDCA showed a significant decrease in amphetamine-induced rotation scores when compared to pre-transplantation values. There was a significant increase (approximately 3-fold) in the number of tyrosine hydroxylase (TH)-positive cells in the nerve grafts for the TUDCA-treated group at 6 weeks post-transplantation when compared to controls. Four days after transplantation, the number of apoptotic cells in the transplanted area in the TUDCA-treated group was significantly less than that in the control group. These data demonstrate that pretreatment of cell suspensions with TUDCA reduces apoptosis and increases survival of transplanted cells, leading to improved behavioral rehabilitation.

Materials and methods

experimental design

For the present invention, cell culture and transplantation experiments were performed. In vitro and in vivo experiments, embryos were 14 days old under sterile conditions as described (A. Björklund et al., Acta. Physiol. Scand. Suppl. 522: 9-18; 1983; P. Conn, P.M.ed. Methods in Neurosciences, Lesions and transplantation. Academic Press, San Diego, Vol7, 1991: 305-326; E.M. Grasbon-Frodl et al., Brain Res. Bull. 39: 341-347; 1996) Anatomy of Sprague- Ventral midbrain (VM) tissues of Dawley (SD) (Charles River Labs, Wilmington, MA) rat embryos. For each experiment, obtained tissue sections were equally divided into two experimental groups: control and TUDCA-treated groups. The study was approved by the Animal Care Committee of the University of Minnesota and conducted under the auspices of Research Animal Resources, an American Association-approved institution for accreditation of laboratory animal care.

Cell Culture Experiment

In order to examine the effect of TUDCA on the apoptosis and survival rate of dopamine neurons in VM tissue culture under serum-free conditions and to determine the appropriate dose of TUDCA, primary neuron cultures were prepared from VM tissue of SD rat embryos and cultured in the presence of Incubation was performed with and without TUDCA (Calbiochem, La Jolla, CA, free base, dissolved in 0.15M (where "M" is molar concentration) NaHCO3 buffer) was added. In experiments, the dose-response effect of TUDCA was studied and a concentration of 50 μΜ (micro-molar) was found to be suitable. Therefore, when the medium was changed to serum-free conditions after two days in vitro, TUDCA was added to the medium at a final concentration of 50 μM. Neural survival and apoptosis were determined by counting TH-positive cells and TUNEL-positive cells in cultures of two or seven days in vitro. A total of three different series of cell culture experiments were performed.

transplant experiment

A total of 24 adult female Sprague-Dawley (SD) rats weighing approximately 250 g at the start of the experiment were used as recipients of nerve grafts. Of these, 12 rats suffered massive unilateral 6-hydroxydopamine (6-OHDA) impairment of the mesostriatal dopamine (DA) system while the other 12 were normal.

6-OHDA-lesioned rats and normal rats were randomly assigned to TUDCA-treated group (n=6 in each group) or control vehicle group (n=6 in each group), respectively. Two rats per cage were arranged under a 12-h diurnal cycle and unlimited access to food and water. It was grown and handled according to published National Institutes of Health guidelines.

For TUDCA-treated rats, TUDCA was added at a concentration of 50 [mu]M after the substantia nigra tissue was trypsinized and isolated. Two microliters of cell suspension containing TUDCA were then injected stereotaxically into the striatum of SD rats. For control animals, the same amount of vehicle solution was added to the medium. In the first series of transplantation experiments, normal rats receiving nerve grafts were sacrificed 4 days after transplantation and prepared for deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) Brain slices were assayed to measure apoptosis in the transplanted area. In the second series of transplantation experiments, the completeness of the 6-OHDA lesion was assessed before transplantation using the amphetamine-induced rotation test and was repeated at two and six weeks post-transplantation to monitor the functional effect of the nerve graft . Rats were sacrificed after the final rotational behavioral testing session and brain tissue processed for tyrosine hydroxylase (TH)-immunocytochemistry. Graft survival was assessed by counting TH-positive neurons in the graft.

Tissue culture of ventral midbrain

For each series of experiments, VM tissues collected from 24-32 embryos were incubated at 37 degrees Celsius (° C.) in 0.1% trypsin (Sigma, St. Louis, MO)/0.05% DNase (Sigma, St. Louis, MO) 20 minutes and mechanically dissociate with a 1 milliliter (ml) Gilson pipette. After isolation, cells were centrifuged at 600 revolutions per minute (rpm) for 5 minutes and the pellet was resuspended in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Carlsbad, CA). Cell number and viability of isolated cells were assessed with a hemocytometer using trypan blue dye exclusion. Spread at 100,000 cells/ ^cm (178,000 cells per well) into four-well chambers precoated with 10 milligrams per milliliter (mg/ml) poly-D-lysine (Sigma, St. Louis MO) On glass slides (Nunc, Rochester, NY). The viability of the isolated cells was over 95%. Cell cultures were incubated in DMEM supplemented with 10% fetal calf serum for 2 days at 37 °C in a humidified atmosphere of 95% air/5% _CO2 . After 2 days in vitro, the medium was changed to serum-free N2 medium consisting of a DMEM/Ham's F12 (1:1) mixture (Gibco, Carlsbad, CA).

At this time point, some cultures were supplemented with TUDCA.

unilateral 6-hydroxydopamine injury

As previously described (W.-M.Duan et al., Neuroscience 57:261-274; 1993), under magnesium chloride mixture anesthesia (0.3ml/100g body weight i.p.) 6-OHDA (hydrochloride, Sigma, St. Louis MO) twice into the right ascending mesostriatal DA pathway. Briefly, the first injection of 2.5 microliters (μl) of 6-OHDA (3 micrograms/microliter (μg/μl) of free base in 0.2 mg/ml ascorbate-saline was performed at the following sites: posterior to bregma 4.4 millimeters (mm); 1.2 mm lateral to the midline; 7.8 mm ventral to the dura; the tooth-bar was set 2.4 mm below the interauricular line. A second injection of 2 μl 6-OHDA was performed at the following site: anterior 4.0 millimeters (mm) posterior to the bregma; 0.8 mm lateral to the midline; 8.0 mm ventral to the dura mater; the alveolar setting is 3.4 mm above the interauricular line. 6-OHDA is advanced at a speed of 1 μl/min and remains at the injection site before pulling out Catheter 4 minutes.

Amphetamine-induced spin test

Two to three weeks after injury surgery, rats were given 5 milligrams per kilogram (mg/kg) of dextroamphetamine sulfate (Sigma, St. Louis MO) in saline, i.p. The cylinders were monitored for 90 minutes of their rotational behavior (U. Ungerstedt et al., Brain Res. 24:485-493; 1970). Twelve rats exhibiting a net rotational asymmetry of 7 complete ipsilateral lesion rotations per minute were selected and scored according to the net rotational asymmetry (number of rotations to the lateral lesion minus the ipsilateral lesion The balance of the number of rotations of the side) is divided into two groups. Rotation tests were subsequently repeated at two different time points mentioned in the experimental design. Rats showing a reduction in rotational asymmetry to less than 50% of the pre-transplant value were considered to have functional grafts.

Substantia nigra tissue preparation and transplantation

As previously described (A. Björklund 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, Vol7, 1991: 305-326) utilize cell suspension technology to carry out neural transplantation. Briefly, VM tissue was obtained from embryos with a brain-to-rump length of 13-14 mm, corresponding to a gestational age of 14 days. Dissection and preparation of the donor tissue was performed under sterile conditions in Hank's Balanced Salt Solution (HBSS) Gibco, Carlsbad, CA). Uterine horns were removed by hysterectomy of animals under deep chloral hydrate anesthesia (250 mg/kg, i.p.) and placed in plastic tubes containing HBSS. Embryos were removed from the uterus and embryonic brains were transferred individually into petri-dishes with a black background. Dissect the VM from each brain using iridectomy scissors and fine watchmaker forceps under a dissecting microscope. The dissected sections were collected and incubated in 0.1% trypsin (Sigma, St. Louis MO)/0.05% DNase (Sigma, St. Louis MO)/HBSS at 37°C for 20 minutes. After 4-5 rinses with 0.05% DNase/HBSS, the tissue was gradually dissociated into a mixture of single cells and small cell aggregates using a baked-polished Pasteur pipette with an inner diameter of 0.5-1.0 mm. The final solution of the cell suspension was adjusted so that five microliters of HBSS was added to each VM tissue dissection. Before and after transplantation, the viability of the cell suspension was assessed by the trypan blue dye exclusion method. Viability of all cell suspensions in this study was greater than 95%.

Two microliters of the cell suspension (containing about 100,000 cells, corresponding to one-third of a VM) were stereotaxically transplanted into recipients anesthetized with magnesium chloride (3 ml/kg, i.p.) immobilized in a Kopf stereotaxic apparatus. in the right striatum of the mouse. Using a 10-microliter Hamilton microsyringe (Hamilton Co., Reno, NV) equipped with a steel jacket (inner diameter = 0.25 mm, outer diameter = 0.47 mm), injections were performed at the following sites: 1.0 mm rostral to bregma; midline 3.0 mm lateral; 4.5 mm ventral to the dura surface, alveolar set to zero. Give injections longer than 2 minutes and leave the needle for 2-4 minutes before withdrawal.

Immunocytochemistry

The avidin-biotin complexed immunoperoxidase technique was used to visualize immunocytochemical staining as previously described (W.-M. Duan et al., Neuroscience 100:521-530; 2000). For cell culture experiments, cultures were rinsed once with phosphate-buffered saline (PBS, 0.2 M, pH 7.4) and then fixed with 4% formaldehyde for 20 minutes at room temperature. Cultures were then processed for immunocytochemistry. For transplantation experiments, rats were deeply anesthetized with chloral hydrate at a lethal dose (500 mg/kg body weight, i.p.) and intragastrically perfused with 0.1 M PBS followed by cold 4% formaldehyde. Brains were then removed, fixed in the same fixative for 4 hours, and placed in 20% sucrose at 4°C until sedimentation. Sections were coronally sectioned on a cryo-slide microtome to a thickness of 30 microns. Over the entire area of the graft, four adjacent serial sections were collected in four small glass tubes. The following primary antibodies were used against TH (1:500 Pel-Freez, Rogers AR). Biotinylated goat anti-rabbit (rat-absorbed) immunoglobulin (1:200) (Vector Laboratories, Inc., Burlingame, CA) was used as the secondary antibody. Sections were incubated in ABC solution (Vectastain ABC Elite kit, Vector Laboratories Inc.), and then developed with 3,3′-diaminobenzidine solution (Vectastain DAB kit, Vector Laboratories Inc.) until immunoreactive products were visible. After staining, sections were mounted on supercooled microscope slides (Fisher Scientific, Pittsburgh, PA), dehydrated through an increasing gradient of alcohol-xylene concentrations, and mounted with DPX mounting medium (Fluka, Switzerland).

Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay

As previously described (W.-M.Duan et al., Neuroscience 100:521-530; 2000), the TUNEL assay was performed by using an in situ apoptosis detection kit (sold under the commercial name ApopTag) according to the manufacturer's protocol (Intergen, Purchase , NY) to carry out. Briefly, 3-4 sections containing graft tissue were selected from each transplanted animal 4 days after transplantation. They were mounted on supercooled glass slides (Fisher Scientific, Pittsburgh, PA) for the following staining protocol. Sections were first quenched in 3% hydrogen peroxide in PBS to remove endogenous peroxidase, followed by incubation in working concentrations of TdT enzyme and peroxidase-conjugated anti-digoxigenin antibody solution. 3,3'-Diaminobenzidine (Sigma, St. Louis MO) was used as a chromophore to visualize the reaction product. After counterstaining with methyl green, sections were cleared in ethanol-dehydrated xylene and mounted with Permount mounting medium (Fisher Scientific, Fair Lown, NJ).

Morphological assessment

In order to avoid subjective bias in the interpretation of the results, all morphological assessments were performed in a blinded fashion under a microscope with brightfield illumination of stained sections (W.-M. Duan et al., Neuroscience 100:521-530; 2000).

cell counts

For cell culture experiments, TH was assessed at ×20 and ×40 magnifications with the aid of gridlines of 400 μm squares, respectively, as previously described (E.M. Grasbon-Frodl et al., BrainRes. Bull. 39:341-347; 1996). - Number of positive cells and total number of cells. In order to sample a representative area of each culture well in a systematic manner, eight fields of view were selected. The area sampled covers 1-3% of the total area of each well. For transplantation experiments, TH positive neurons in the grafts were counted on every fourth section under a Nikon light microscope (Nikon, Japan) using a 10× objective. Cell counts were performed only if they exhibited at least one axon or had distinct nuclei. After measuring the average diameter of TH-immunocompetent cells with an embedded grid using a 20× objective lens, the original Count to calculate the total number of TH-immunoreactive neurons.

TUNEL-positive cell count

For cell culture experiments, the number of TUNEL-positive cells was assessed at ×400 magnification by means of 400-μm square gridlines. Six fields per culture well were selected in a systematic manner. For transplantation experiments, TUNEL-positive cells were counted in the graft area of 3-4 sections per animal. The mean number of TUNEL-positive cells was calculated to represent individual animal values.

volume analysis

The volume of nerve grafts in the striatum was analyzed using a computer-aided image analysis system as previously described (W.-M. Duan et al., Eur. J. Neurosci. 10: 2595-2606; 1998). Briefly, all TH-immunostained grafts were imaged under a Nikon light microscope (Nikon, Japan) connected to a high-resolution digital camera (COOLPIX 950, Nikon, Japan) using a 1× objective. Slice digitization. Images are first collected and saved on a CompactFlash card. The CompactFlash card was then read by using a CompactFlash card reader connected to a Pentium III PC (Dell, Dimension XPS T700r, USA) and the images were analyzed using a software package (Scion Image, Version Beta4.0.2, Scion Corporation, Frederick, MD). In each section, the grafts were manually marked on the monitor and the surface area was determined. Pixel values are then converted to square millimeters. Graft volume was calculated from graft area, section thickness, and frequency.

Statistical Analysis

Data above and in figures are presented as mean ± SEM. Statistical comparisons of parameters were performed using two-way analysis of variance (ANOVA); post-hoc Scheffé's F test or Student's t-test were performed after one-way ANOVA for group-way comparisons. Statistical significance level was set at p<0.05.

result

Cell Culture Experiment

Dopamine neuron survival. At 2 days in vitro, transiently treated TH-positive cells showed early developmental stages. Some were processed by contact with other cells in their vicinity (data not shown). At 7 days in vitro, dopamine neurons exhibited a more mature morphology. It showed some long protuberances with pronounced varicoseness (data not shown). At this time point, some cell bodies and bulges appear granular in reversed phase, possibly indicating that they are degenerating. There was a significant cell loss of TH-positive neurons and total cells in 7DIV control cultures (p<0.01, one-way ANOVA with post hoc Scheffé's F test). In fact, the number of TH-immunopositive neurons was only 30% of the value in 2DIV cultures. This severe cell loss was not observed in 7 DIV or TUDCA-treated cultures (data not shown). No difference in the number of TH-positive cells was found between 2DIV cultures and TUDCA-treated cultures (p > 0.05) (Fig. 1). TUDCA exerts neuroprotective effect in a dose-dependent manner.

apoptosis. As illustrated in Figure 2, the number of TUNEL-positive cells was significantly higher in 7DIV control cultures than in 2DIV control and 7DIV TUDCA-treated cultures (p<0.01, one-way ANOVA with post-hoc Scheffé's F test).

transplant experiment

rotation behavior. Amphetamine-induced rotational asymmetry scoring is summarized in FIG. 3 . At 2 weeks post-transplantation, 4 of 6 TUDCA-treated rats showed at least a 50% reduction in net motor asymmetry compared to pre-transplantation values. In contrast, no control animals showed a 50% reduction in motor asymmetry. At 6 weeks post-transplantation, all rats in the control and TUDCA-treated groups showed a >50% reduction in motor asymmetry scores.

Two-way ANOVA revealed a significant group × time interaction (F _{(1, 2)} = 43.83, p < 0.001) for the net rotation score throughout the testing period, indicating that TUDCA-treated rats exhibited significantly higher Faster Behavior Recovery. In fact, the mean value of net rotation at 2 weeks after transplantation compared with the value before transplantation was significantly higher in the TUDCA-treated group (p<0.01, paired Student's t-test) but not in the control group (p>0.05). reduce. Furthermore, one-way ANOVA with post-hoc Scheffé's F test showed that the TUDCA-treated group exhibited lower net asymmetry values than the control group at 2 weeks post-transplantation (F _(1,10) =5.51, p<0.05). At 6 weeks after surgery, both groups had significantly reduced motor asymmetry scores (paired Student's t-test, p<0.05) and there was no longer any difference between the two groups (one-way ANOVA, p>0.05) .

Graft survival. Most grafts are visible in the center of the striatum and are oval in shape. It contains many TH-immunoreactive neurons and fibers (Fig. 4). Some grafts were misplaced in the overlying corpus callosum and the frontal cortex along the needle path. Figure 5 summarizes the mean number of TH-immunoreactive neurons in grafts in control and TUDCA-treated groups at 6 weeks post-transplantation. The mean number of TH-immunoreactive neurons in grafts in the TUDCA-treated group was significantly higher than in the control group. A more than three-fold increase in the mean number of TH-immunoreactive neurons in grafts was observed in the TUDCA-treated group compared to the control group (Post-hoc Scheffé's F test after one-way ANOVA, F _(1,10) = 6.65, p<0.05). Graft volumes were also significantly greater in the TUDCA-treated group than in the control group (p<0.05, one-way ANOVA after post-hoc Scheffe's F-test after one-way ANOVA, F _(1,10) =8.22 ). At 6 weeks post-transplantation, grafts in control animals exhibited typical morphology of substantia nigra grafts. The majority of TH-immunoreactive neurons were located at the periphery of the graft, whereas those in the center of the graft were relatively devoid of TH-immunoreactivity (Fig. 4D). However, it appeared that at this time point the grafts in TUDCA-treated animals lacked the typical morphology of substantia nigra grafts. Most TUDCA-treated animals exhibited an even distribution of TH-immunoreactive neurons in the transplanted area (Fig. 4C). TH-immunoreactive neurons had multipolar cell bodies with some clearly stained axons (Fig. 4E). The area of host striatum grafted with graft nerves was found to be larger in the TUDCA-treated group than in the control group (Fig. 4A and B).

apoptosis

Figure 6 summarizes the mean number of TUNEL-positive cells in the graft area in the control and TUDCA-treated groups 4 days after transplantation. The number of apoptotic cells was significantly less in the TUDCA-treated group than in the control group (p<0.01, one-way ANOVA after post-hoc Scheffe's F-test, F _(1,10) =20.06). In the control group, a large number of apoptotic cells with fragmented nuclear DNA aggregated and localized within the graft (data not shown). Only a few apoptotic cells were observed in the TUDCA-treated group in some pockets within the graft (data not shown). Occasionally, some scattered apoptotic cells were found around the host tissue of the graft in both groups.

The above examples show the use of TUDCA to promote the survival of dopamine neurons in vitro and in vivo. Furthermore, TUDCA could significantly reduce apoptosis in VM tissue cultures and in grafts, suggesting that TUDCA exerts beneficial effects on the survival of dopamine neurons mainly through an anti-apoptotic mechanism. The three-fold increase in graft survival in the TUDCA-treated group resulted in a faster recovery of behavioral asymmetry when compared to the control group. These results confirm and provide further support for the non-binding theory that apoptosis is a cause of dopamine neuron loss. This significant cell loss can be prevented if anti-apoptotic agents are used in tissue preparations or immediately after transplantation.

Culture systems in which neuronal death is induced by serum deprivation in midbrain cultures are well known as an in vitro model for induction of apoptosis. This in vitro model has been widely used to examine the effect of anti-apoptotic agents and neurotrophic factors on apoptosis and dopamine neuron survival in VM tissue culture (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.5: 97-100; 1999; W.M. Zawada et al., Exp. Neurol. 140:60-67; 1996). The data presented above indicate that there is a cell loss of dopamine neurons following the medium switch to serum-free conditions. The results of the TUNEL assay suggested that most of the cell loss may be the result of apoptosis. These data also show that the addition of TUDCA to cultures causes a decrease in the number of apoptotic cells and an increase in the number of dopamine neurons, suggesting that TUDCA exerts its neuroprotective effect on dopamine neurons primarily through an anti-apoptotic mechanism.

Earlier studies have shown that about 80-95% of grafted DA neurons die with transplantation (P.Brundin et al., Prog. Brain Res. 71:293-308, 1987; N.Nakao et al., Proc. .Acad.Sci.USA91:12408-12412,1994; H. et al., Restor.Neurol.Neurosci.2:123-135,1991; H.Sauer et al., Exp.Brain Res.90:54-62,1992 ; G.S. Schierle et al., Nat. Med. 5:97-100, 1999; A. Zuddas et al., Eur. J. Neurosci. 3:72-85, 1991). Cell death in neural grafts was recently found to occur at four stages during the transplantation process (P. Brundin et al., Cell Transplant. 9: 179-195; 2000.). However, evidence suggests that most cell loss occurs within the first few days after transplantation (M. Emgard et al.; Exp. Neurol. 160; 279-288; 1999; G.S. Schierle et al.; Nat. Med. 5:97-100 ; 1999; W.M. Zawada et al.; Brain Res. 786; 96-103; 1998).

Several factors have been shown to cause cell death. Mechanical injury, hypoxia and nutritional deficiencies during transplantation can cause subsequent cell death and production of reactive oxygen species. Various free radicals can subsequently cause transplanted dopamine neurons to undergo programmed cell death. A substantia nigra graft transplanted into the striatum is a heterotopic graft. The new environment in the striatum is not necessarily conducive to the survival of substantia nigra grafts and the neural grafts may lack adequate neurotrophic support. TUDCA-supplemented medium used during cell suspension preparation increased neural graft survival approximately three-fold and the graft's function was also improved. While not wishing to be bound by theory, these data provide evidence that most cell loss in grafts is closely related to apoptosis (G.S. Schierle et al., Nat. Med. 5:97-100, 1999; W.M. Zawada et al., Brain Res. 786, 96-103; 1998). In addition, antioxidant and anti-apoptotic agents such as TUDCA may be used in combination to provide the additional effect of preventing cell death in transplanted substantia nigra explants.

Early onset of behavioral effects occurred in the treated group and all rats showed >50% reduction in net motor asymmetry. Although the mechanisms behind the rapid onset of behavioral recovery require further evaluation, one possible but not limiting explanation is that administration of anti-apoptotic agents and neurotrophic factors promotes rapid maturation of transplanted dopamine neurons. Furthermore, previous studies have demonstrated that a certain number of intrastriatal TH-positive transplanted neurons are required to induce 50% behavioral recovery and found a plateau recovery of slightly more than 100% in the range of over 2000 intrastriatal TH-positive transplanted neurons. level. These phenomena reflect the fact that when a certain number of dopamine neurons survives at an early time point after transplantation, the same number of transplanted dopamine neurons will be maintained to a later time point (W.-M.Duan et al. , Exp. Brain Res. 104: 227-242, 1995; W.-M. Duan et al., Neuroscience 100: 521-530, 2000). These observations highlight the value of applying neuroprotective strategies to prevent cell loss in neural transplants at early post-transplant time points rather than later. Another possible explanation for the apparent difference between cell number and behavior is that a certain level of dopamine innervation is required to induce behavioral modification. This level was achieved at an earlier time point in grafts treated with TUDCA because of higher survival of transplanted cells. Compared to TUDCA-treated cells, non-TUDCA-treated cells initiate and innervate the host striatum with an additional process that will eventually also provide a level of innervation that achieves corrected motor asymmetry, but in a time-dependent manner compared to TUDCA-treated cells. cells later.

Cascade activity is thought to play a role in apoptosis (G.S. Salvesen et al., Cell 91:443-446, 1997). One pathway of cascade activation involves the mitochondria (D.R. Green et al., Science 281:1309-1312, 1998; G. Kroemer et al., Nat. Med. 6:513-519, 2000). TUDCA has been shown to prevent mitochondrial swelling and rupture of the mitochondrial outer membrane. Membrane stabilization can inhibit the release of the pro-apoptotic molecule, cytochrome c and cause cytochrome c-mediated downstream events such as altered cascade activity. TUDCA is now believed to significantly reduce 3-nitropropionic acid (3-NP)-mediated neuronal cell death in striatal tissue culture. Furthermore, in the 3-NP-lesioned Huntington's disease rat model (C.D. Keene et al., Exp. Neurol. 171; 351-360, 2001), the systemic administration of TUDCA can be reduced mainly by anti-apoptotic mechanisms. Degeneration of the striatum and improvement of neurological deficits.

Since most transplanted dopamine neurons die immediately after transplantation, attempts have focused on improving graft survival for optimal graft outcomes. In fact, many experimental studies have shown that the degree of behavioral rehabilitation is related to graft survival and its correlation (P.Brundin et al., In: S.B.Dunnett and A.Björklund, eds., Functional neural transplantation, New York Raven Press; 1994: 9-46; N. Nakao et al., Pfoc. Natl. Acad. Sci. USA 91: 12408-12412, 1994; H. Sauer et al., Exp. Brain Res. 90: 54-62, 1992). In addition, clinical studies of nerve transplantation in Parkinson's disease have also confirmed that the degree of symptom improvement after transplantation is closely related to the survival of nerve grafts and the degree of nerve redistribution of grafts (P.Brundin et al., Brain123:1380-1390, 2000 ; J. H. Kordower et al., J. Comp. Neurol. 370:203-230, 1996; P. Piccini et al., Nat. Neufosci. 2: 1137-1140, 1999). Larger numbers of transplanted dopamine neurons usually lead to more significant clinical improvement. However, recent work by Freed et al. has led to controversy regarding the number of transplanted dopamine neurons required for clinical improvement (P. Brundin et al., Nat. Med. 7:512-513, 2001; S.B. Dunnett et al., Nat . Rev. Neurosci. 2: 365-369, 2001; C. R. Freed et al., N. Engl. J. Med. 344: 710-719, 2001). In its double-blind placebo-controlled clinical trial of embryogenic tissue grafts for Parkinson's disease, a small proportion of transplant recipients eventually developed dystonia and dyskinesias were reported and it was determined that this side effect may be due to Continued fiber derivation at the site, resulting in a relative excess of dopamine production in the recipient brain. They therefore recommend transplanting less tissue to address this issue in future clinical trials. However, only a small number of dopamine neurons survived in the two postmortem cases reported in this study. In addition, other factors such as tissue preparation, grafting technique, and grafting site can contribute to the development of this transplant-related side effect.

As another example, TUDCA was also effective in reducing apoptosis of islet cells after 2 days in vitro. The number of TUNEL-positive cells was approximately 29 in the no-treatment control, but only 12 in the cells exposed to TUDCA. A reduction in apoptosis of more than 50% was achieved during exposure of cells to TUDCA during isolation followed by incubation with bile acids during in vitro culture.

The entire disclosures of all patents, patent documents, and publications cited herein are incorporated by reference in their entirety. The foregoing detailed description and examples are provided for clearer understanding only. No unnecessary limitations should be understood therefrom. The invention is not limited to the details shown and described, for variations obvious to a person skilled in the art will be encompassed within the scope of the invention defined by the claims.

Claims (65)

1. one kind is improved the method that the transplanted cells all living creatures deposits ability, comprise with this transplanted cells group be selected from hydrophilic bile acid, its salt, its analog contacts with the compound of the effective dose of its combination.

2. the process of claim 1 wherein that transplanted cells group's cell is noble cells.

3. the process of claim 1 wherein that transplanted cells group's cell is a precursor.

4. the process of claim 1 wherein to contact and carry out external.

5. the process of claim 1 wherein to contact and carry out in vivo.

6. the method for claim 5 wherein contacts in transplanted cells group's donor and carries out.

7. the process of claim 1 wherein the transplanted cells group contacted with compound to be included in and before the transplanted cells group is implanted into acceptor this transplanted cells group is contacted with compound.

8. the method for claim 7, wherein acceptor is behaved.

9. the method for claim 7, it further comprises uses the compound treatment acceptor.

10. the method for claim 9 wherein is included in the compound treatment acceptor and gives acceptor with compound before the transplanted cells group is implanted into acceptor.

11. the method for claim 10 wherein is included in the compound treatment acceptor and gives acceptor with compound after the transplanted cells group is implanted into acceptor.

12. the method for claim 9 wherein is included in the compound treatment acceptor and gives acceptor with compound after the transplanted cells group is implanted into acceptor.

13. the method for claim 9 is wherein handled acceptor and is comprised with compound and handle acceptor through parenteral.

14. the method for claim 9 is wherein handled acceptor and is comprised with compound oral administration processing acceptor.

15. the process of claim 1 wherein the transplanted cells group contacted with compound and comprise combined with transplanted cells group and pharmaceutical acceptable carrier.

16. the process of claim 1 wherein that transplanted cells group's cell comprises autogenous cell, heterogenous cell or variant cell.

17. the process of claim 1 wherein that transplanted cells group's cell comprises at least a portion of autologous tissue, heteroplasm or allosome tissue.

18. the process of claim 1 wherein that this transplanted cells group is organ.

19. the method for claim 18, wherein organ is liver, kidney, heart, lung or pancreas.

20. the method for claim 18, it uses the compound treatment acceptor after further being included in the transplanted cells group being implanted into acceptor.

21. the method for claim 1, it uses the compound treatment acceptor after further being included in the transplanted cells group being implanted into acceptor.

22. a treatment suffers from the people's of Parkinson's disease method, this method comprises and external the transplanted cells group being contacted with Tauro ursodesoxy cholic acid with the combined effective dose of pharmaceutical acceptable carrier, and wherein this transplanted cells group is noble cells; And transplant this transplanted cells group and go into philtrum.

23. a treatment suffers from the method for the acceptor of Parkinson's disease, this method comprises the transplanted cells group contacted with the compound of the effective dose that is selected from urso, its salt, its analog and its combination and improves this transplanted cells group's survival ability; And transplanting this transplanted cells group goes in the acceptor.

24. the method for claim 23, wherein this ursodeoxycholic acid-like substance comprises conjugated derivatives.

25. the method for claim 24, wherein conjugated derivatives is a Tauro ursodesoxy cholic acid.

26. the method for claim 23, wherein acceptor is behaved.

27. the method for claim 23, wherein cell is noble cells.

28. the method for claim 23, wherein cell is a precursor.

29. the method for claim 23 wherein contacts external and carries out.

30. the method for claim 23, wherein contact is carried out in vivo.

31. the method for claim 30 wherein contacts in transplanted cells group's donor and carries out.

32. the method for claim 23 wherein contacts the transplanted cells group to be included in and before the transplanted cells group is implanted into acceptor the transplanted cells group is contacted with compound with compound.

33. the method for claim 32, it further comprises uses the compound treatment acceptor.

34. the method for claim 33 wherein is included in the compound treatment acceptor and gives acceptor with compound before cell transplantation gone into acceptor.

35. the method for claim 34 wherein is included in the compound treatment acceptor and gives acceptor with compound after cell transplantation gone into acceptor.

36. the method for claim 33 wherein is included in the compound treatment acceptor and gives acceptor with compound after cell transplantation gone into acceptor.

37. the method for claim 23 is wherein handled acceptor and is comprised with compound and handle acceptor through parenteral.

38. the method for claim 23 is wherein handled acceptor and is comprised with compound oral administration processing acceptor.

39. the method for claim 23, wherein the transplanted cells group is contacted with compound comprise transplanted cells group and pharmaceutical acceptable carrier combined.

40. the method for claim 23, wherein cell comprises autogenous cell, heterogenous cell or variant cell.

41. the method for claim 23, wherein cell comprises at least a portion of autologous tissue, heteroplasm or allosome tissue.

42. a method, it comprises: with the compound treatment transplanted cells group's who is selected from hydrophilic bile acid, its salt, its analog and its combination donor.

43. the method for claim 42, wherein hydrophilic bile acid comprises urso, and the processing donor comprises with ursodeoxycholic acid treatment donor.

44. the method for claim 42, wherein hydrophilic bile acid comprises Tauro ursodesoxy cholic acid, and handles donor and comprise with Tauro ursodesoxy cholic acid and handle donor.

45. the method for claim 42, wherein transplanted cells group's donor is alive.

46. the method for claim 42 is wherein handled donor and is included in the transplanted cells group uses the compound treatment donor before donor takes out.

47. the method for claim 42 is wherein handled donor and is included in the transplanted cells group uses the compound treatment donor during donor takes out.

48. the method for claim 42 is wherein handled donor and is included in the transplanted cells group from donor taking-up back compound treatment donor.

49. the method for claim 42, wherein the transplanted cells group is at least a portion of donor organ.

50. the method for claim 49, wherein at least a portion of organ is the complete organ of donor.

51. the method for claim 42 is wherein handled donor and is comprised with compound and handle donor through parenteral.

52. the method for claim 42 is wherein handled donor and is comprised with compound oral administration processing donor.

53. the method for claim 42 wherein comprises transplanted cells group and pharmaceutical acceptable carrier combined with the compound treatment donor.

54. a method, it comprises: with the compound treatment transplanted cells group's who is selected from hydrophilic bile acid, its salt, its analog and its combination acceptor.

55. the method for claim 54, wherein hydrophilic bile acid comprises urso, and the processing acceptor comprises with ursodeoxycholic acid treatment acceptor.

56. the method for claim 54, wherein hydrophilic bile acid comprises Tauro ursodesoxy cholic acid, and handles acceptor and comprise with Tauro ursodesoxy cholic acid and handle acceptor.

57. the method for claim 54 is wherein handled acceptor and is comprised and use the compound treatment acceptor before the transplanted cells group is implanted into acceptor.

58. the method for claim 54 is wherein handled acceptor and is comprised during the transplanted cells group is implanted into acceptor and use the compound treatment acceptor.

59. the method for claim 58 is wherein handled acceptor and is included in processing transplanted cells group in the acceptor.

60. the method for claim 54 is wherein handled acceptor and is comprised and use the compound treatment acceptor after the transplanted cells group is implanted into acceptor.

61. the method for claim 54, wherein the transplanted cells group is at least a portion of organ.

62. the method for claim 61, wherein at least a portion of organ is complete organ.

63. the method for claim 54 is wherein handled acceptor and is comprised with compound and handle acceptor through parenteral.

64. the method for claim 54 is wherein handled acceptor and is comprised with compound oral administration processing acceptor.

65. the method for claim 54 wherein comprises transplanted cells group and pharmaceutical acceptable carrier combined with the compound treatment acceptor.

Images