U.S. patent application number 14/520889 was filed with the patent office on 2015-04-23 for deuterated bile acids.
This patent application is currently assigned to Metselex, Inc.. The applicant listed for this patent is Metselex, Inc.. Invention is credited to Susana Dias Lucas de Oliveira, Michael D. Finch, Walter Low, Cyrus B. Munshi, Cecilia Rodrigues, Clifford Steer.
Application Number | 20150112089 14/520889 |
Document ID | / |
Family ID | 52826744 |
Filed Date | 2015-04-23 |
United States Patent
Application |
20150112089 |
Kind Code |
A1 |
Finch; Michael D. ; et
al. |
April 23, 2015 |
DEUTERATED BILE ACIDS
Abstract
This disclosure relates to deuterated bile acid compositions. A
deuterated compound is selected from the disclosed groups of bile
acids and their derivatives, analogs and salts. At least one of the
hydrogen atoms in the compound is replaced with deuterium.
Inventors: |
Finch; Michael D.; (Apple
Valley, MN) ; Low; Walter; (Shorewood, MN) ;
Steer; Clifford; (Eagan, MN) ; Munshi; Cyrus B.;
(Blaine, MN) ; Rodrigues; Cecilia; (Lisbon,
PT) ; Dias Lucas de Oliveira; Susana; (Sao Joao da
Talha, PT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Metselex, Inc. |
Minneapolis |
MN |
US |
|
|
Assignee: |
Metselex, Inc.
Minneapolis
MN
|
Family ID: |
52826744 |
Appl. No.: |
14/520889 |
Filed: |
October 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61894012 |
Oct 22, 2013 |
|
|
|
Current U.S.
Class: |
552/553 |
Current CPC
Class: |
C07J 9/005 20130101;
A61K 31/575 20130101; A61P 25/00 20180101; A61P 25/16 20180101;
C07J 41/0061 20130101 |
Class at
Publication: |
552/553 |
International
Class: |
C07J 9/00 20060101
C07J009/00 |
Claims
1. A deuterated compound selected from the group consisting of:
##STR00008## ##STR00009## wherein at least one of elements
R.sub.1-R.sub.xx comprises deuterium.
2. The deuterated compound of claim 1, wherein the compound is
water-soluble.
3. The deuterated compound of claim 1, wherein the compound is a
salt.
4. The deuterated compound of claim 1, wherein the compound is
hydrophobic.
5. The deuterated compound of claim 1, wherein the compound has
pro-drugs, derivatives and conjugates used for the treatment or
prevention of any degenerative disorders in humans.
6. The deuterated compound of claim 1, wherein the compound has
deuterium enrichment of at least 5%.
7. The deuterated compound of claim 6, wherein the compound has
deuterium enrichment of at least 10%.
8. The deuterated compound of claim 7, wherein the compound has
deuterium enrichment of at least 30%.
9. The deuterated compound of claim 8, wherein the compound has
deuterium enrichment of at least 50%.
10. The deuterated compound of claim 9, wherein the compound has
deuterium enrichment of at least 70%.
11. The deuterated compound of claim 1, wherein the compound has
the structure of formula I, and wherein the compound has deuterium
enrichment of at least 90%.
12. The deuterated compound of claim 10, wherein in the compound
has deuterium enrichment of at least 98%.
13. The deuterated compound of claim 1, wherein the compound has a
structure selected from the group consisting of formulae I, II and
III, and wherein the compound comprises one or more PO.sub.4
groups.
14. The deuterated compound of claim 1, wherein the compound has a
structure selected from the group consisting of formulae I, II and
III, and wherein the compound is chemically conjugated to a
pro-drug of dopaminergic neurons.
15. The deuterated compound of claim 1, wherein the compound has a
structure selected from the group consisting of formulae I, II and
III, and wherein the compound is chemically conjugated to a
monoamine oxidase inhibitor.
16. The deuterated compound of claim 1, wherein the compound has a
structure selected from the group consisting of formulae I, II and
III, and wherein the compound is chemically conjugated to a
glutamate receptor antagonists.
17. The deuterated compound of claim 1, wherein the compound has a
structure selected from the group consisting of formulae I, II and
III, and wherein the compound is chemically modified to function as
an antioxidant.
18. The deuterated compound of claim 1, wherein the deuterated
compound is ##STR00010## or a structural analog thereof.
19. The deuterated compound of claim 18, wherein R.sub.6 is
deuterium.
20. The deuterated compound of claim 18, wherein R.sub.12 is
deuterium.
Description
BACKGROUND
[0001] Ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid
(TUDCA) are anti-apoptotic molecules with protective effects
against several neurodegenerative disorders such as Alzheimer's and
Parkinson's diseases as well as against acute kidney injury. Both
UDCA and TUDCA block the initiating event of the apoptotic process,
in part, through stabilizing the mitochondrial membrane potential,
a mechanism that enhances the integrity of the mitochondria.
Enhanced mitochondrial integrity abolishes the release of several
mitochondrial proteins such as cytochrome C into the cytosol,
thereby preventing the onset of apoptosis. Further, UDCA and TUDCA
upregulate several pathways that function synergistically with
their anti-apoptotic properties.
[0002] The kinetic deuterium isotope effect (KDIE) is a function of
enhanced carbon-deuterium (C-D) bond strength over the
carbon-hydrogen (C--H) bond, often several-fold. Substitution of
the C--H by the C-D bond significantly decreases breakage, thereby
increasing the resident time of the molecule in the body.
Therefore, substituting one or more of the carbons with deuterium
significantly increases the metabolic clearance time. An added
benefit of the KDIE is the possibility of using lower dosages to
derive the same pharmacological effect. The C-D bond is twice as
strong as the C--H bond by virtue of a two-fold higher mass of
deuterium over hydrogen. Hence, the reaction rate of the C-D bond
breakage is significantly slower than that of the C--H bond
breakage.
SUMMARY
[0003] This disclosure relates to deuterated bile acid
compositions. A deuterated compound is selected from the disclosed
groups of bile acids and their derivatives, analogs and salts. At
least one of the hydrogen atoms in the compound is replaced with
deuterium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIG. 1 illustrates the structure of UDCA.
[0005] FIG. 2 illustrates the structure of TUDCA.
[0006] FIG. 3 illustrates the structure of lithocholic acid
(LCA).
[0007] FIG. 4 illustrates the structure of dehydro(11,12)ursolic
acid lactone.
[0008] FIG. 5 illustrates the structure of ursolic acid.
[0009] FIG. 6 illustrates the structure of ursocholanic acid.
[0010] FIG. 7 illustrates the effect of deuterated UDCA on
DCA-induced cytotoxicity and apoptosis in primary rat
hepatocytes.
[0011] FIG. 8 illustrates the effect of deuterated UDCA on
TGF-.beta.1-induced cytotoxicity and apoptosis in primary rat
hepatocytes.
DETAILED DESCRIPTION
[0012] This patent application pertains to the field of
pharmaceutical molecules and specifically to deuterated versions of
bile acids, such as UDCA and TUDCA as well as their analogs and
derivatives, with enhanced resident time following administration
to a patient.
[0013] Deuteration of bile acids can significantly increase the
half-life of the drug in the bloodstream and, hence, decrease the
dosage needed to treat various degenerative disorders. Disclosed
herein are various compositions of deuterated bile acids and their
analogs and derivatives, as well as methods of preparation.
[0014] In some embodiments, UDCA, its analogs and salts have the
structure of Formula I as illustrated in FIG. 1 and reproduced
below:
##STR00001##
where R.sub.1-R.sub.40 are individual hydrogen or deuterium atoms.
Any analog or salt of UDCA can contain at least one deuterium atom
represented by any of the R.sub.1-R.sub.40 locations in any
combination.
[0015] In another embodiment of formula I, the deuterated UDCA,
UDCA analog or UDCA salt contains one or more PO.sub.4 groups,
preferably in positions 3, 7, 24.
[0016] In other embodiments, TUDCA, its analogs and salts have the
structure of Formula II as illustrated in FIG. 2 and reproduced
below:
##STR00002##
where R.sub.1-R.sub.44 are individual hydrogen or deuterium atoms.
Any analog or salt of TUDCA can contain at least one deuterium atom
represented by any of the R.sub.1-R.sub.44 locations in any
combination.
[0017] In another embodiment of Formula II, the deuterated TUDCA,
TUDCA analog or TUDCA salt contains one or more PO.sub.4 groups,
preferably in positions 3, 7, 24.
[0018] In other embodiments, lithocholic acid (LCA), its salts,
derivatives and analogs have the structure of Formula III as
illustrated in FIG. 3 and reproduced below:
##STR00003##
[0019] where R.sub.1-R.sub.39 are individual hydrogen or deuterium
atoms. Any analog or salt of LCA can contain at least one deuterium
atom represented by any of the R.sub.1-R.sub.39 locations in any
combination.
[0020] In another embodiment of Formula III, the deuterated LCA,
LCA analog, LCA derivative or LCA salt contains one or more
PO.sub.4 groups.
[0021] In other embodiments, dehydro-(11,12)-ursolic acid lactone,
its salts, derivatives and analogs have the structure of Formula IV
as illustrated in FIG. 4 and reproduced below:
##STR00004##
where R.sub.1-R.sub.48 are individual hydrogen or deuterium atoms.
Any analog, derivative or salt of dehydro-(11,12)-ursolic acid
lactone can contain at least one deuterium atom represented by any
of the R.sub.1-R.sub.48 locations in any combination.
[0022] In another embodiment of Formula IV, the analog, derivative
or salt of dehydro-(11,12)-ursolic acid lactone contains one or
more PO.sub.4 groups.
[0023] In other embodiments, ursolic acid, its salts, derivatives
and analogs have the structure of Formula V as illustrated in FIG.
5 and reproduced below:
##STR00005##
where R.sub.1-R.sub.50 are individual hydrogen or deuterium atoms.
Any analog, derivative or salt of ursolic acid can contain at least
one deuterium atom represented by any of the R.sub.1-R.sub.50
locations in any combination.
[0024] In another embodiment of Formula V, the analog, derivative
or salt of ursolic acid contains one or more PO.sub.4 groups.
[0025] In other embodiments, ursocholanic acid, its salts,
derivatives and analogs have the structure of Formula VI as
illustrated in FIG. 6 and reproduced below:
##STR00006##
[0026] where R.sub.1-R.sub.50 are individual hydrogen or deuterium
atoms. Any analog, derivative or salt of ursocholanic acid can
contain at least one deuterium atom represented by any of the
R.sub.1-R.sub.50 locations in any combination.
[0027] In another embodiment of Formula VI, the analog, derivative
or salt of ursocholanic acid contains one or more PO.sub.4
groups.
[0028] In another embodiment, structures with formulae I, II, III,
IV, V, and VI, and all derivatives thereof are conjugated to any
anti-neurodegenerative pro-drug molecules involved in modulating
neuronal apoptosis.
[0029] In another embodiment, structures with formulae I, II, III,
IV, V, and VI, and all derivatives thereof are conjugated to
pro-drugs of dopaminergic neurons (DA) neurons such as L-DOPA
((S)-2-Amino-3-(3,4-dihydroxyphenyl)propanoic acid) and any analog
of L-DOPA.
[0030] In another embodiment, structures with formulae I, II and
III and all derivatives thereof are conjugated to glutamate
receptor antagonists.
[0031] In another embodiment, structures with formulae I, II, III,
IV, V, and VI, and all derivatives thereof are conjugated to
antioxidants.
[0032] In another embodiment, structures with formulae I, II, III,
IV, V, and VI, and all derivatives thereof are combined, without
conjugation, to any anti-neurodegenerative pro-drug molecules
involved in modulating neuronal apoptosis.
[0033] In another embodiment, structures with formulae I, II, III,
IV, V, and VI, and derivatives thereof are combined, without
conjugation, to any anti-neurodegenerative pro-drugs of DA neurons
such as L-DOPA and any analog of L-DOPA.
[0034] In another embodiment, structures with formulae I, II, III,
IV, V, and VI, and derivatives thereof are combined, without
conjugation, to glutamate receptor antagonists.
[0035] In another embodiment, structures with formulae I, II, III,
IV, V, and VI, and derivatives thereof are combined, without
conjugation, to antioxidants.
[0036] Typically, for some embodiments, the compound described
herein can be formulated in pharmaceutical compositions. A
pharmaceutical composition containing a compound of the present
disclosure 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.
[0037] The formulations can be conveniently presented in unit
dosage form and can 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 carrier, a finely
divided solid carrier, or both, and then, if necessary, shaping the
product into a desired formulation.
[0038] Formulations of the present disclosure suitable for oral
administration can be presented as discrete units such as tablets,
troches, capsules, lozenges, wafers, or cachets, each containing a
predetermined amount of the described 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.
[0039] The tablets, troches, pills, capsules, and the like can 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.
When the unit dosage form is a capsule, it can further contain a
liquid carrier, such as a vegetable oil or a polyethylene glycol.
Various other materials can be present as coatings or to otherwise
modify the physical form of the solid unit dosage form. For
instance, tablets, pills, or capsules can be coated with gelatin,
wax, shellac, sugar, and the like. A syrup or elixir can 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 can be incorporated into sustained-release preparations
and devices, if desired.
[0040] A compound suitable for use in the methods of the disclosure
can also be incorporated directly into the food of a subject's
diet, as an additive, supplement, or the like. Thus, the disclosure
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.
[0041] Formulations suitable for parenteral administration
conveniently include a sterile aqueous preparation of the desired
compound, or dispersions of sterile powders having 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.
Preferred 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, thiomersal (Ethyl(2-mercaptobenzoato-(2-)-O,S)mercurate(1-)
sodium), 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.
[0042] 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 can be presented as a suppository with a
suitable carrier such as cocoa butter, or hydrogenated fats or
hydrogenated fatty carboxylic acids.
[0043] In addition, a compound of the present disclosure 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. In addition, a compound can 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. Some examples of pro-drug
forms include ketal, acetal, oxime, and hydrazone forms of a
compound that contains ketone or aldehyde groups.
[0044] Preferably, when a compound of the present disclosure can be
delivered in vivo, the dosage level of the compound is on the order
of about 10 milligrams to about 15 milligrams per kilogram of body
weight per day. Preferably, the effective amount is on the order of
about 500 milligrams to about 1000 milligrams per subject per day.
When a compound of the present disclosure is delivered to a
subject, the compound can be delivered in one or multiple dosages
for injection, infusion, and/or ingestion.
[0045] Deuterated compounds according to the present disclosure can
have various levels of deuterium enrichment. Deuterium enrichment
is defined as the percentage of R.sub.1-R.sub.xx groups having
deuterium atoms. In one embodiment, the deuterated compound has
deuterium enrichment of at least 5%. In another embodiment, the
deuterated compound has deuterium enrichment of at least 10%. In
another embodiment, the deuterated compound has deuterium
enrichment of at least 30%. In another embodiment, the deuterated
compound has deuterium enrichment of at least 50%. In another
embodiment, the deuterated compound has deuterium enrichment of at
least 70%. In another embodiment, the deuterated compound has
deuterium enrichment of at least 98%. In another embodiment, the
deuterated compound has the structure of formula I and has
deuterium enrichment of at least 90%. Determination of the level of
deuterium enrichment can be performed by mass spectrometry or
nuclear magnetic resonance evaluation.
Examples
Synthesis of
7,8-[.sup.2H]-3.alpha.,7.alpha.-dihydroxy-5.beta.-cholan-24-oic
acid/7-[.sup.2H]-3.alpha.,7.alpha.-dihydroxy-5.beta.-cholan-24-oic
acid (5a,b)
##STR00007##
[0047] General Methods:
[0048] All reagents were purchased from Sigma-Aldrich Corp. (St.
Louis, Mo., USA) and used without further purification unless
otherwise noted. Reactions were followed by thin layer
chromatography (TLC), carried out using Merck aluminum backed
sheets coated with 60 F254 silica gel, using EtOAc or
EtOAc/n-Hexane mixtures as eluent. Visualization of the TLC spots
was achieved by spraying with a 10% solution of sulfuric acid in
methanol followed by heating. Silica gel was acquired from Merck
& Co. (White House Station, N.J., USA; 60 G, 0.040-0.063
mm).
[0049] .sup.1H-- and spectra were recorded on a Bruker Avance III
(300 and 100 MHz, respectively). All chemical shifts are quoted on
the .delta. scale in ppm using residual solvent peaks as the
internal standard. Coupling constants (J) are reported in Hz with
the following splitting abbreviations: s=singlet, d=doublet,
t=triplet, m=multiplet.
Synthesis of Methyl-3.alpha.,7.alpha.-dihydroxy-5.beta.-cholanoate
(Compound 2)
[0050] To a solution of comercial
3.alpha.,7.alpha.-dihydroxy-5.beta.-cholanic acid (500 mg, 1.27
mmol) in MeOH (40 mL) was added p-toluenosulfonic acid and the
mixture was refluxed under nitrogen atmosphere for 2 hours. The
reaction mixture was poured into water (100 mL) and the product was
extracted with EtOAc (3.times.50 mL). The organics were combined,
dried over anhydrous sodium sulfate, filtered and concentrated in
the rotatory evaporator to yield a colorless oil (516 mg, 1.27
mmol, quantitative yield). The residue was pure as judged by TLC
and was reacted without further purification.
Synthesis of Methyl-3.alpha.-hydroxy-7-oxo-5.beta.-cholanoate
(Compound 3)
[0051] To a solution of
methyl-3.alpha.,7.alpha.-dihydroxy-5.beta.-cholanoate (Compound 2;
500 mg, 1.23 mmol) in CHCl.sub.3 (30 mL, anhydrous) was added
silica-gel (2 g) and to this suspension was then added pyridinium
chlorochromate (300 mg, 1.38 mmol). The reaction was stirred at
room temperature for 5 hours under nitrogen atmosphere. Et.sub.2O
was added (100 mL) and the suspension was filtered over a
silica-gel column eluting with Et.sub.2O/DCM (7/3) to afford the
desired product as a colorless solid (309 mg, 62%). MS (ESI+): m/z
427.1[M+Na].sup.+. By-product methyl-3,7-di-oxo-5.beta.-cholanoate
was obtained as a colorless solid (154 mg, 31%). MS (ESI+): m/z
425.2[M+Na].sup.+.
Synthesis of 3.alpha.-hydroxy-7-oxo-5.beta.-cholan-24-oic acid
(Compound 4)
[0052] To a solution of
methyl-3.alpha.-hydroxy-7-oxo-5.beta.-cholanoate (Compound 3; 269
mg, 0.66 mmol) in THF was added LiOH (12 mL, 0.3M aqueous soln) and
the mixture was stirred at room temperature for 4 hours. The
mixture was acidified with 1M HCl until pH 1 and the product
extracted with EtOAc (3.times.30 mL). The organics were combined,
dried over anhydrous sodium sulfate, filtered and concentrated in
the rotatory evaporator to yield a crystalline solid (192 mg, 0.49
mmol, 74%). MS (ESI+): m/z 413.2 [M+Na].sup.+. .sup.1H NMR (300
MHz, MeOD) .delta. 3.60-3.46 (m, 1H), 2.99 (m, 1H), 2.54 (t, J=11.2
Hz, 1H), 2.34 (m, 1H), 2.26-2.10 (m, 2H), 2.09-1.71 (m, 7H),
1.71-1.25 (m, 9H), 1.25-0.99 (m, 8H), 0.96 (d, J=6.5 Hz, 3H), 0.71
(s, 3H).
[0053] Synthesis of
7,8-[.sup.2H]-3.alpha.,7.alpha.-dihydroxy-5.beta.-cholan-24-oic
acid (Compound
5a)/7-[.sup.2H]-3.alpha.,7.alpha.-dihydroxy-5.beta.-cholan-24-o- ic
acid (Compound 5b) (65/35 isotopic ratio)
[0054] To a refluxing solution of
3.alpha.-hydroxy-7-oxo-5.beta.-cholic acid (Compound 4; 59 mg, 0.15
mmol) in .sup.ipropan(ol-d) (6 mL) in nitrogen atmosphere, was
added potassium metal (ca. 100 mg) in small pieces and the mixture
was refluxed for 45 minutes. A 1M HCl soln was carefully added (up
to 20 mL) and the product was extracted with EtOAc (3.times.10 mL).
The organics were combined, dried over anhydrous sodium sulfate,
filtered and concentrated in the rotatory evaporator. The product
was recrystallized with EtOAc/n-Hexane to yield a colorless solid
(Compound 5a,b; 46 mg, 78%). MS (ESI-): m/z 393.0 [M(1D)-1].sup.-
(35%); 393.0 [M(2D)-1].sup.- (65%). .sup.1H NMR (300 MHz, MeOD)
.delta. 3.44-3.57 (m, 1H), 2.15-2.40 (m, 2.4H), 2.02-2.07 (m, 1H),
1.79-1.92 (m, 5H), 1.56-1.63 (m, 3H), 1.08-1,53 (m, 15H), 0.87-1.03
(m, 5H), 0.72 (s, 3H). Compound 5 is a mixture of Compound 5a
(7,8-[.sup.2H]-3.alpha.,7.alpha.-dihydroxy-5(3-cholan-24-oic acid)
and Compound 5b
(7-[.sup.2H]-3.alpha.,7.alpha.-dihydroxy-5.beta.-cholan-24-oic
acid).
[0055] Cell Culture and Treatments
[0056] Primary rat hepatocytes were isolated from male rats
(100-150 grams) by collagenase perfusion (as described by Sola S,
et al., 2003, Journal of Biological Chemistry 278: 48831-48838)).
Briefly, rats were anesthetized with phenobarbital sodium (100
mg/kg body weight) injected into the peritoneal cavity. After
administration of heparin (200 units/kg body weight) in the tail
vein, the animal's abdomen was opened and the portal vein exposed
and cannulated. The liver was then perfused at 37.degree. C. in
situ with a calcium-free Hanks' Balanced Salt Solution (HBSS) for
about 10 minutes, and then with 0.05% collagenase type IV in
calcium-present HBSS for another 10 minutes. Hepatocyte suspensions
were obtained by passing collagenase-digested livers through 125
.mu.m gauze and washing cells in Complete William's E medium
(William's E medium, Sigma-Aldrich Corp., St Louis, Mo., USA)
supplemented with 26 mM sodium bicarbonate, 23 mM HEPES, 0.01
units/mL insulin, 2 mM L-glutamine, 10 nM dexamethasone, 100
units/mL penicillin, and 10% heat-inactivated fetal bovine serum
(Invitrogen Corp., Carlsbad, Calif., USA). Viable primary rat
hepatocytes were enriched by low-speed centrifugation at 200 g for
3 minutes. Cell viability was determined by trypan blue exclusion
and was typically 80-85%. After isolation, hepatocytes were
resuspended in Complete William's E medium and plated on
Primaria.TM. tissue culture dishes (BD Biosciences, San Jose,
Calif., USA) at 5.times.10.sup.4 cells/cm.sup.2. Cells were
maintained at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2 for 6 hours to allow attachment. Plates were then washed
with medium to remove dead cells and incubated in Complete
William's E medium supplemented with either 100-400 .mu.M UDCA
(Sigma-Aldrich Corp.), 100-400 .mu.M deuterated UDCA (compound 5),
or no addition (control). Eight hours after pre-incubation, cells
were exposed to 1 nM recombinant human TGF-.beta.1 (R&D Systems
Inc., Minneapolis, Minn., USA) for 36 hours, or to 50 or 100 .mu.M
DCA for 16 hours before processing for cell viability, cytotoxicity
and apoptosis assays.
[0057] Cell Viability, Cytotoxicity, and Caspase Activity
Assays
[0058] The ApoTox-Glo.TM. Triplex Assay (Promega Corp., Madison,
Wis., USA) was used to evaluate cell viability, cytotoxicity and
caspase-3/7 activity, according to the manufacturer's protocol,
using a GloMax+ Multi Detection System (Promega Corp.). General
cell death was also evaluated using the lactate dehydrogenase (LDH)
Cytotoxicity Detection Kit.sup.PLUS (Roche Diagnostics GmbH,
Mannheim, Germany), following the manufacturer's instructions.
[0059] Morphologic Evaluation of Apoptosis
[0060] Hoechst labeling of cells was used to detect apoptotic
nuclei by morphological analysis. Briefly, culture medium was
gently removed to prevent detachment of cells. Attached primary rat
hepatocytes were fixed with 4% paraformaldehyde in
phosphate-buffered saline (PBS), pH 7.4, for 10 minutes at room
temperature, washed with PBS, incubated with Hoechst dye 33258
(Sigma-Aldrich Corp.) at 5 .mu.g/mL in PBS for 5 minutes, washed
with PBS, and mounted using Fluoromount-GTM (SouthernBiotech,
Birmingham, Ala., USA). Fluorescence was visualized using an
Axioskop fluorescence microscope (Carl Zeiss GmbH).
Blue-fluorescent nuclei were scored blindly and categorized
according to the condensation and staining characteristics of
chromatin. Normal nuclei showed non-condensed chromatin dispersed
over the entire nucleus. Apoptotic nuclei were identified by
condensed chromatin, contiguous to the nuclear membrane, as well as
by nuclear fragmentation of condensed chromatin. Five random
microscopic fields per sample containing approximately 150 nuclei
were counted, and mean values expressed as the percentage of
apoptotic nuclei.
[0061] Statistical Analysis
[0062] Statistical analysis was performed using GraphPad InStat
version 3.00 (GraphPad Software, San Diego, Calif., USA) for the
analysis of variance and Bonferroni's multiple comparison tests.
Values of p<0.05 were considered significant.
RESULTS
[0063] We tested the cytoprotective and anti-apoptotic effects of
newly synthesized deuterated UDCA (Compound 5), using
well-established cellular models of apoptosis. It has been
previously shown that non-deuterated UDCA significantly inhibits
DCA-induced apoptosis by .about.60% (Castro et al., 2007, American
Journal of Physiology Gastrointestinal Liver Physiology 293:
G327-G334) and TGF-.beta.1-induced apoptosis by .about.50% (Sola et
al., 2003) in primary rat hepatocytes. Cells exposed to 100 and 200
.mu.M concentrations of Compound 5 alone showed no relevant signs
of cytotoxicity. Nevertheless, a .about.2% and 15% increase in
cytotoxicity was observed in cells incubated with the highest
concentration of 400 .mu.M Compound 5 for 24 and 44 hours,
respectively. This was comparable to UDCA cytotoxicity. In
dissecting the cytoprotective effects of Compound 5, we tested its
ability to prevent DCA-induced cytotoxicity and apoptosis, as
compared with UDCA. UDCA and Compound 5 inhibited 50 .mu.M
DCA-induced LDH release by 70 and 90%, respectively (FIG. 7A). This
protective effect was more than 10% greater for Compound 5 as
compared to UDCA. Slightly reduced protection was obtained for both
UDCA and Compound 5 in cells exposed to 100 .mu.M DCA. Notably,
Compound 5 pretreatment was more effective than UDCA at markedly
inhibiting caspase activity in cells exposed to both 50 and 100
.mu.M DCA (FIG. 7B). Similar results were obtained after evaluating
nuclear morphology and counting apoptotic nuclei. Nuclear
fragmentation induced by 50 .mu.M DCA was prevented by .about.50%
and 65% in cells pretreated with UDCA and Compound 5, respectively
(FIG. 7C). Compound 5 was .about.15% more effective than UDCA at
reducing the percentage of apoptotic cells. Finally, in cells
exposed to a stimulus unrelated to bile acids, Compound 5 was about
10% more effective than UDCA at inhibiting TGF-.beta.1-induced LDH
release, which in turn was very significantly abrogated (FIG. 8A).
In addition, the percentage of apoptotic cells was reduced by
almost 40% after UDCA and Compound 5 pre-treatment (FIG. 8B).
Altogether, these results show that Compound 5 displays powerful
cytoprotective and anti-apoptotic properties in vitro that may even
exceed those of UDCA.
[0064] FIG. 7 illustrates that Compound 5 prevents DCA-induced
cytotoxicity and apoptosis in primary rat hepatocytes. Primary rat
hepatocytes were incubated with 100 .mu.M UDCA, Compound 5, or no
addition (control) for 8 hours. Cells were then exposed to 50 or
100 .mu.M DCA for 16 hours before processing for LDH activity (A),
caspase-3/7 activity (B) and nuclear fragmentation assays (C).
Fluorescent microscopy of Hoechst staining in cells exposed to
either DCA 50 .mu.M (a), DCA 50 .mu.M+Compound 5 (b), DCA 100 .mu.M
(c), or DCA 100 .mu.M+Compound 5 (d). Normal nuclei showed
non-condensed chromatin dispersed over the entire nucleus.
Apoptotic nuclei were identified by condensed chromatin, contiguous
to the nuclear membrane, as well as nuclear fragmentation of
condensed chromatin. Results are expressed as means.+-.SEM of at
least three experiments. *p<0.01 from control; .sctn.p<0.05
and .dagger.p<0.01 from respective DCA.
[0065] FIG. 8 illustrates that Compound 5 prevents
TGF-.beta.1-induced cytotoxicity and apoptosis in primary rat
hepatocytes. Primary rat hepatocytes were incubated with 100 .mu.M
UDCA, Compound 5, or no addition (control) for 8 hours. Cells were
then exposed to 1 nM TGF-.beta.1 for 36 hours before processing for
LDH activity (A), caspase-3/7 activity (B) and nuclear
fragmentation assays (C). Fluorescent microscopy of Hoechst
staining in cells exposed to either TGF-.beta.1 (a) or
TGF-.beta.1+Compound 5 (b). Normal nuclei showed noncondensed
chromatin dispersed over the entire nucleus. Apoptotic nuclei were
identified by condensed chromatin, contiguous to the nuclear
membrane, as well as nuclear fragmentation of condensed chromatin.
Results are expressed as means.+-.SEM of at least three
experiments. *p<0.01 from control; .sctn.p<0.05 and
tp<0.01 from TGF-.beta.1.
[0066] Although the present invention has been described with
reference to preferred embodiments, workers skilled in the art will
recognize that changes may be made in form and detail without
departing from the spirit and scope of the invention.
* * * * *