U.S. patent application number 12/429492 was filed with the patent office on 2009-10-29 for use of epothilone d in treating tau-associated diseases including alzheimer's disease.
This patent application is currently assigned to Bristol-Myers Squibb Company. Invention is credited to Charles F. Albright, Donna Marie Barten, Francis Y. Lee.
Application Number | 20090270465 12/429492 |
Document ID | / |
Family ID | 40887113 |
Filed Date | 2009-10-29 |
United States Patent
Application |
20090270465 |
Kind Code |
A1 |
Albright; Charles F. ; et
al. |
October 29, 2009 |
USE OF EPOTHILONE D IN TREATING TAU-ASSOCIATED DISEASES INCLUDING
ALZHEIMER'S DISEASE
Abstract
Methods of treating Tau-associated diseases, preferably
tauopathies, are described using epothilone D that exhibit good
brain penetration, long half-life, and high selective retention in
brain, and provides effective therapies in treating tauopathies
including Alzheimer's disease.
Inventors: |
Albright; Charles F.;
(Madison, CT) ; Barten; Donna Marie; (West
Suffield, CT) ; Lee; Francis Y.; (Yardley,
PA) |
Correspondence
Address: |
LOUIS J. WILLE;BRISTOL-MYERS SQUIBB COMPANY
PATENT DEPARTMENT, P O BOX 4000
PRINCETON
NJ
08543-4000
US
|
Assignee: |
Bristol-Myers Squibb
Company
|
Family ID: |
40887113 |
Appl. No.: |
12/429492 |
Filed: |
April 24, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61047729 |
Apr 24, 2008 |
|
|
|
Current U.S.
Class: |
514/365 |
Current CPC
Class: |
A61K 31/427 20130101;
A61P 9/10 20180101; A61P 31/12 20180101; A61P 27/02 20180101; A61P
25/16 20180101; A61P 27/00 20180101; A61P 25/00 20180101; A61P
25/02 20180101; A61P 27/06 20180101; A61P 43/00 20180101; A61P
25/28 20180101; A61P 21/00 20180101; A61P 3/10 20180101 |
Class at
Publication: |
514/365 |
International
Class: |
A61K 31/427 20060101
A61K031/427 |
Claims
1. A method of treating a Tau-associated disease comprising
administering a therapeutically-effective amount of epothilone D to
a patient in need of treatment thereof.
2. The method of claim 1, wherein the Tau-associated disease is
Alzheimer's disease.
3. The method of claim 1, wherein the Tau-associated disease is
selected from frontotemporal dementia, including the subtype of
frontotemporal dementia and Parkinsonism linked to chromosome 17
(FTDP-17), progressive supranuclear palsy, corticobasal
degeneration, Pick's disease, and agyrophilic grain disease,
Parkinson's disease, Down syndrome, post-encephalic Parkinsonism,
myotonic dystrophy, Niemann-Pick C disease, dementia pugilistica,
Blint disease, a prion disease, amyotrophic lateral sclerosis,
Parkinsonism-dementia complex of Guam, multiple sclerosis,
glaucoma, diabetic retinopathy, and traumatic brain injury.
4. The method of claim 2, wherein the epothilone D is administered
orally.
5. The method of claim 2, wherein the epothilone D is administered
intravenously.
6. The method of claim 2, wherein the epothilone D has good brain
penetrance, long brain half-life, and a high selective
brain-to-liver retention rate.
7. The method of claim 1 wherein the epothilone D has brain
penetrance of 0.5 or more measured at a period between 20 min. and
1 h post-dosing, and either or both of 1) a brain half-life of 24 h
or more, and 2) brain to liver selective retention rate of 2 or
more at 24 or more hours post-dosing.
8. The method of claim 6, wherein the epothilone D has brain
penetrance of 0.5 or more measured at a period between 20 min. and
1 h post-dosing, and either or both of 1) a brain half-life of 24 h
or more, and 2) brain to liver selective retention rate of 2 or
more at 24 or more hours post-dosing.
9. The method of claim 2, wherein the method is therapeutically
effecting in treating AD in the patient without causing
drug-induced side effects that would require that use of the
epothilone D treatment be discontinued.
10. The method of claim 9, wherein the epothilone D is administered
to the patient at a dose of between 0.0001-10 mg/m.sup.2,
administered daily, weekly, or on an intermittent basis.
11. The method of claim 10, wherein the dose of epothilone D is
between 0.0001-0.05 mg/m.sup.2, and the epothilone D is
administered as a daily oral dose to a human patient.
12. The method of claim 10, wherein the dose of epothilone D is
between 0.01-30 mg/m.sup.2, and the epothilone D is administered
intravenously to a human patient on a weekly, biweekly, monthly, or
intermittent dosage cycle.
13. The method of claim 12, wherein the cumulative monthly dose is
between 0.1-3 mg/m.sup.2.
14. A method of treating Alzheimer's Disease in a human patient,
comprising the step of administering a therapeutically effective
amount of epothilone D to the patient, wherein the epothilone D is
administered orally and wherein the dose of epothilone D calculated
on a daily basis (regardless of dosing schedule) is between 0.001-2
mg/m.sup.2, and wherein the epothilone D is therapeutically
effective in having an impact on underlying disease and/or
providing cognitive benefits to the patient without causing
drug-induced side effects that would require that use of the
epothilone D treatment be discontinued.
15. The method of claim 14, wherein the epothilones D is
administered orally on a dosing schedule selected from once daily
and once a week, and wherein the daily dose of epothilone D is
between 0.2 to 2 mg/m.sup.2 and the weekly dose is between 1.4 to
14 mg/m.sup.2.
16. A pharmaceutical formulation comprising epothilone D suitable
for administration to a human patient in need of treatment for a
Tau-associated disease, wherein the formulation is therapeutically
effective in treating the Tau-associated disease in the patient
without causing drug-induced side effects and/or drug-plasma
concentration levels that would require use of said epothilone D
formulation to be discontinued.
17. The pharmaceutical formulation according to claim 16, wherein
the Tau-associated disease is Alzheimer's Disease, and
administration of the formulation provides
statistically-significant cognitive benefits and/or impact on
underlying disease, without causing drug-induced side effects
and/or drug-plasma concentration levels that would require use of
said epothilone D formulation to be discontinued.
18. The formulation according to claim 16, wherein said formulation
comprises epothilone D in a pharmaceutically acceptable solvent
system comprising from about 0 to 50% propylene glycol, about 1 to
10% TPGS, about 0.5 to 10% ethanol, about 0-90% water, and/or about
5 to 85% PEG such as PEG-400.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to the treatment of
Tau-associated diseases using epothilone D, and more specifically,
to the treatment of Alzheimer's Disease using epothilone D.
BACKGROUND OF THE INVENTION
[0002] Alzheimer's disease (AD) is the most common form of
dementia, affecting an estimated 27 million people worldwide in
2006. Age is the greatest known risk factor for AD with an
incidence of 25-50% in people aged 85 years or older. As the
average age of the population increases, the number of patients
with AD is expected to rise exponentially. AD is the fifth leading
cause of death in people aged 65 and older, and most patients
eventually need nursing home care. Consequently, AD has an enormous
economic impact, e.g., estimated direct and indirect costs for 2005
in the US only were $148 billion. Besides the economic costs, AD
has a devastating impact upon patients and their family members,
causing severe emotional distress and turmoil.
[0003] Patients are diagnosed with probable AD based on the
presence of dementia with progressive worsening of memory and other
cognitive functions and with the exclusion of other causes of
dementia. A diagnosis of AD can only be confirmed post-mortem as
the clinical diagnosis is based on brain neuropathology,
specifically, the diagnosis requires an evaluation of brain tissue,
including the existence and concentration of extracellular plaques
in the brain, intracellular tangles, and brain neurodegeneration.
Dementia is also a required part of the diagnosis, since plaques
and tangles are observed in cognitively normal adults, although
usually to a lesser extent.
[0004] Two classes of medications, cholinesterase inhibitors and an
N-methyl-D-aspartic acid (NMDA) antagonist, are currently approved
for AD. Although these two classes of therapeutics show some
clinical benefit, many patients do not respond, and these drugs
only ameliorate the symptoms of AD (e.g., cognitive function) with
little or no modification of disease progression. For these
reasons, identification of disease-modifying therapeutics for this
devastating disease is a major focus of the pharmaceutical
industry.
[0005] Microtubule stabilizers have been suggested as therapies to
treat tauopathies including AD. See, e.g., Lee et al. (references
list, infra). In U.S. Pat. No. 5,580,898, filed May 1994 and
granted Dec. 3, 1996, Trojanowski et al. suggest use of paclitaxel
(TAXOL.RTM.) to treat AD patients by stabilizing microtubules.
Paclitaxel has proven highly effective as a microtubule-stabilizing
agent in treating cancer patients; however, it presents
brain-penetration and peripheral neuropathy issues when considered
for AD (further described below), and has not emerged as a viable
therapy to treat AD.
[0006] In 1995, epothilone B was reported to exert
microtubule-stabilizing effects similar to paclitaxel (Bollag et
al. 1995). Epothilone A and epothilone B are naturally-occurring
compounds that were isolated by Hofle et al. from fermentation
products of the microorganism Sorangium cellulosum (e.g., WO
93/10121). Hofle et al. also discovered 37 natural epothilone
variants and related compounds produced by S. cellulosum and
modified strains, including epothilones C, D, E, F and other
isomers and variants (e.g., U.S. Pat. No. 6,624,310).
[0007] Unique characteristics of the natural epothilones generated
much interest in their exploration as potential anti-cancer drugs.
Now, nearly twenty years have passed since the first discovery of
the natural epothilones A and B. Hundreds of epothilone analogs
have been discovered and described in various patent applications,
and abundant literature has published under the rubric,
"epothilones" (See, e.g., Altmann et al., references list, infra,
at 396-423).
[0008] The assignee of the current application has developed
ixabepilone, a semi-synthetic analog of epothilone B, for treatment
of cancer. Ixabepilone has the structural formula:
##STR00001##
[0009] The chemical name for ixabepilone is
(1S,3S,7S,10R,11S,12S,16R)-7,11-dihydroxy-8,8,10,12,16-pentamethyl-3-[(1E-
)-1-methyl-2-(2-methyl-4-thiazolyl)ethenyl]-17-oxa-4-azabicyclo[14.1.0]
heptadecane-5,9-dione. See also U.S. Pat. No. 6,605,599, assigned
to the current assignee, Bristol-Myers Squibb Company (BMS).
Ixabepilone is a microtubule-stabilizing agent that has been
approved by the FDA for treatment of metastatic breast cancer and
is sold by BMS under the tradename IXEMPRA.RTM.. Ixabepilone can be
prepared as described in U.S. Pat. No. 6,605,599 or 7,172,884,
incorporated herein by reference.
[0010] Other natural epothilones and analogs are in advanced
clinical trials for treatment of cancer including epothilone B
(a/k/a patupilone, or EPO-906), in Phase III trials by Novartis
Pharma AG, for treatment of ovarian cancer, and sagopilone (or
ZK-EPO), a benzothiazolyl-7-propenyl synthetic analog of epothilone
B, in Phase II trials by Bayer Schering AG for treatment of various
cancers including tumors of the ovary, breast, lung, prostate and
melanoma. In 2007, a Phase II trial with sagopilone was initiated
in the US for treatment of brain metastases from breast cancer.
Additionally, an epothilone D analog, KOS-1584, had advanced to
Phase II clinical trials by Kosan Biosciences, Inc. (now a
wholly-owned subsidiary of BMS) for treatment of non-small-cell
lung cancer and solid tumors, and epothilone D had advanced to
Phase II clinical trials for treatment of cancer by Kosan in
collaboration with Hoffmann-La Roche, Inc.; however, the clinical
trials with epothilone D for treating cancer were discontinued in
2007. The structure for epothilone D can be represented by the
following formula:
##STR00002##
[0011] The epothilone D compound is claimed, as composition of
matter, in U.S. patent application Ser. No. 09/313,524 to Hofle et
al., and described in U.S. Pat. Nos. 6,242,469 and 6,284,781 to
Danishefsky et al., which application and patents were the subject
of Interference No. 105,298, before the USPTO Board of Patent
Appeals and Interferences.
[0012] The assignee of the current application also has clinically
evaluated BMS-310705 (Compound II herein), for cancer therapy.
BMS-310705 was pursued through Phase I clinical trials for
treatment of ovarian cancer; it is an amino-epothilone F analog and
has the chemical structure:
##STR00003##
[0013] Compound II (BMS 310705) can be prepared as described in
U.S. Pat. No. 6,262,094, incorporated herein.
[0014] While certain of the epothilone compounds and analogs have
been clinically evaluated for treating cancers, it is highly
unpredictable whether a cancer drug may be effectively used to
treat neurodegenerative diseases including AD. There are various
factors affecting this unpredictability. One factor is the
substantial difficulty of achieving good brain penetration due to
the blood-brain barrier (BBB). For a compound to be useful in
treating neurodegenerative brain diseases, it is necessary that the
compound cross the BBB; however, since a function of the BBB is to
protect the brain from external substances and toxins, discovering
a useful drug that has good BBB penetration is challenging.
Additionally, BBB penetration is an undesirable feature for a
cancer drug (other than brain cancer drugs). With a cancer drug,
BBB penetration is usually sought to be avoided, whereas for a drug
designed to treat AD or other neurodegenerative brain diseases,
good BBB penetration is necessary for the compound to be effective.
Thus, for example, while paclitaxel is a highly-successful cancer
drug, it has not emerged as a useful therapy to treat brain
diseases such as AD, as it has a low rate of brain penetration
through the BBB.
[0015] Further factors affecting the unpredictability of evaluating
the usefulness of cancer drugs, particularly
microtubule-stabilizing drugs, in treating AD and other brain
diseases involve the ability of a drug to penetrate the brain, to
be retained in the brain for long periods, and to selectively
accumulate in the brain relative to peripheral tissues. These
parameters can be measured using brain-to plasma ratios, brain
half-life, and the ratio of the amount of drug retained in the
brain as compared with peripheral tissues (most particularly the
liver). Additionally, measuring brain penetration, retention and
selective brain accumulation with microtubule-stabilizers is
complex because these compounds are typically rapidly cleared from
plasma but more slowly cleared from microtubule-containing tissues,
making it important to set appropriate time windows for comparisons
of plasma and tissue levels. The brain-to-peripheral-tissue ratio
is a particularly important measurement given that
microtubule-stabilizing agents at certain doses are highly
cytotoxic to peripheral tissues: when microtubule-stabilizing
agents, such as paclitaxel, are administered at chemotherapeutic
doses, a peripheral neuropathy and other side effects often occur
(Postma et al. 1999). These side effects may be tolerable in
treating cancer patients but a different therapeutic window and
acceptable side-effect profile exists in treating patients
suffering from AD and other brain diseases.
[0016] Yet further challenges involved with looking to cancer drugs
for potential application to neurodegenerative diseases involve the
mode of administration and the bioavailability and cytotoxicity
associated therewith.
[0017] In WO 2005/075023 A1, published Jan. 30, 2004, to Andrieux
et al. of INSERM, it is suggested that certain epothilones and
analogs including epothilone A, B, C, D, E, and F, and
benzothiazolyl and pyridyl epothilone B and D analogs may be useful
in treating diseases involving a neuronal connectivity defect, such
as schizophrenia or autism. However, Andrieux et al. disclaimed and
thereby taught against use of these compounds for treating AD,
stating that diseases associated with neuronal connectivity defects
(i.e., those claimed in that application) "are different from
progressive dementing disorders like Alzheimer, which involve
neuronal degeneration."
[0018] In WO 03/074053 ('053), to Lichtner et al. of Schering AG
(published Sep. 12, 2003), there is a broad claim to use of a broad
genus of epothilone compounds and synthetic analogs for treating
brain cancer and other brain diseases, including primary brain
tumor, secondary brain tumor, multiple sclerosis, and AD. Lichtner
et al. report certain data on four compounds, namely, paclitaxel as
compared with the compounds named therein as compound 1:
4,8-dihydroxy-16-(1-methyl-2-(2-methyl-4-thiazolyl)-ethenyl)-1-oxa-7-(1-p-
ropyl)-5,5,9,13-tetramethyl-cyclohexadec-13-ene-2,6-dione; compound
2:
dihydroxy-3-(1-methyl-2-(2-methyl-4-thiazolyl)-ethenyl)-10-propyl-8,8,12,-
16-tetramethyl-4,17-dioxabicyclo[14.1.0]heptadecane-5,9-dione; and
compound 3:
7,11-dihydroxy-3-(2-methylbenzothioazol-5-yl)-10-(prop-2-en-1-yl)-8,8,12,-
16-tetramethyl-4,17-diooxabicyclo[14.1.0]heptadecane-5,9-dione (see
WO '053 publication at page 21).
[0019] Notably, Lichtner et al. report brain and plasma
concentration data for the above three epothilone analogs, but only
for periods of up to 40 minutes. Lichtner et al. are not able to
report comparative data against paclitaxel on brain-to-plasma
levels because their paclitaxel brain levels were below the level
of detection, and they do not report data relating to
brain-to-liver ratios, half-life, or brain retention for any of the
compounds (e.g., concentration of drug in brain tissue over
extended periods of time).
[0020] In view of the foregoing, there remains a need in the art
for methods of treating tauopathies, particularly Alzheimer's
disease.
SUMMARY OF THE INVENTION
[0021] The present inventors have discovered based on multiple in
vivo studies including behavioral and neuropathological studies,
that epothilone D achieves a surprisingly advantageous profile in
treating Tau-associated diseases, including AD. The inventors have
discovered that epothilone D exhibits a remarkable combination of
advantageous properties, making the compound particularly
well-suited to treat such diseases. These properties include not
only a high level of brain penetration across the BBB, but also a
surprisingly long half-life in the brain and a surprisingly high
selective retention rate in the brain as compared with drug levels
found in peripheral tissues, most notably, the liver, over extended
periods of time. Additionally, the inventors have further
discovered that surprising, therapeutic advantages in treating
Tau-associated diseases, particularly, AD, can be achieved with low
dosages of epothilone D, e.g., with dosages that are approximately
100-fold less than those administered to achieve chemotherapeutic
effects. Consequently, the inventors have discovered methods that
allow for therapies in treating Tau-associated diseases with
epothilone D, particularly treatment of AD, without causing
drug-induced side effects and/or drug-plasma concentration levels
that would require use of the epothilone D to be discontinued.
Given the low dose as compared with chemotherapeutic treatments,
any side effects are greatly reduced as compared with side effects
that are induced upon administration of the epothilones and analogs
for treatment of cancer.
[0022] The present invention provides methods of treating
Tau-associated diseases including tauopathies, using epothilone D
that exhibit a surprisingly advantageous therapeutic profile, and
particularly, a method of treating Alzheimer's disease comprising
the step of administering a therapeutically effective amount of
epothilone D to a patient.
[0023] The present invention further provides a pharmaceutical
composition comprising epothilone D for treating Tau-associated
diseases in a patient, wherein the composition exhibits a treatment
profile comprising good brain penetrance, long half-life in the
brain, and selective brain retention (e.g., high brain-to-liver
ratio), as defined herein. Preferred embodiments comprise
pharmaceutical compositions for treating tauopathies, particularly,
AD, comprising a therapeutically-effective amount of epothilone D
and a pharmaceutically acceptable carrier. Further embodiments and
aspects of the invention are set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 shows the basic design of an experiment on Tg4510
mice using epothilone D (Compound I).
[0025] FIG. 2 shows the results of a Morris water maze (MWM) test
of the Tg4510 mice at 2.5 months, prior to dosing with epothilone D
(Compound I).
[0026] FIG. 3 shows the results of a MWM test of the Tg4510 mice at
4.5 months, after 2 months of dosing with epothilone D (Compound
I).
[0027] FIG. 4 shows probe data 18 hours after 5 days of training in
the 4.5 month-old Tg4510 mice dosed for 2 months with epothilone D
(Compound I). "TQ" stands for target quadrant, "AR" stands for
adjacent right, "AL" stands for adjacent left, and "OP" stands for
opposite quadrant. Two measures of performance, namely % pathlength
(A) and number of platform crossings (B) are described.
[0028] FIG. 5 shows neuronal counts in the CA1 and CA3 regions of
the hippocampus in Tg4510 mice at 5.5 months following treatment
with vehicle, 1 mpk epothilone D (Compound I), and 10 mpk
epothilone D (Compound I).
[0029] FIG. 6 shows phosphorylated Tau staining of the Tg4510 mice
treated with vehicle, 1 mpk epothilone D (Compound I), and 10 mpk
epothilone D (Compound I) in the hippocampus. Representative
sections from 3 mice per group are shown. AT8 positive staining is
dark grey and black.
[0030] FIGS. 7A-7B show Gallyas silver staining for neurofibrillary
tangles in Tg4510 mice treated with vehicle, 1 mpk epothilone D
(Compound 1), or 10 mpk epothilone D (Compound I). FIG. 7A shows
representative micrographs of cortical staining, where the black
silver stain is positive. Lighter background staining and some
staining of blood vessels were observed in non-transgenic mice.
FIG. 7B shows the quantitation of the silver stain in both cortex
and hippocampus.
[0031] FIGS. 8A-8D show the concentration of Compound II (FIG. 8A),
ixabepilone (FIG. 8B), paclitaxel (FIG. 8C) and epothilone D
(Compound I) (FIG. 8D) in the plasma, brain, and liver of mice
following intravenous administration at various intervals of up to
24 hours.
[0032] FIG. 9 shows the concentration of epothilone D (Compound 1)
and Compound III (as described in Example 7 herein) in the brain
after oral administration (35 mpk) up to 5 to 24 hours after
dosing.
[0033] FIG. 10 shows the concentration of epothilone D in the
plasma, brain and liver in mice after time intervals up to one week
after dosing.
ABBREVIATIONS
[0034] The following are abbreviations of various terms used in
this specification: [0035] 3R=three repeats [0036] 4R=four repeats
[0037] AD=Alzheimer's disease [0038] APP=.beta.-amyloid precursor
protein [0039] BBB=blood-brain barrier [0040] BMS=Bristol-Myers
Squibb, Co. [0041] CHCl.sub.3=chloroform [0042]
CH.sub.2Cl.sub.2=methylene chloride [0043]
DMAP=4-dimethylaminopyridine [0044] EtOAc=ethyl acetate [0045]
HPLC=high pressure liquid chromatography [0046] FDA=US Food and
Drug Administration [0047] FTDP-17=frontotemporal dementia with
Parkinsonism linked to chromosome 17 [0048] H, h, hr=hour/hours
[0049] IP=intraperitoneal [0050] IV=intravenous [0051] LDA=lithium
diisopropylamide [0052] LLQ=lower limit of quantification [0053]
<LLQ=below LLQ, not detectable [0054] MAP=microtubule-associated
protein [0055] MeOH=methanol [0056] min=minutes [0057]
MTs=microtubules [0058] mpk=milligram per kilogram [0059]
MWM=Morris water maze [0060] nM=nanomolar [0061] NQ=not
quantifiable due to one or more datapoints <LLQ [0062]
PEG=polyethylene glycol [0063] PGP=P-glycoprotein [0064] PO=per os
(oral administration) [0065] PVP=polyvinylpyrrolidone [0066]
RT=room temperature [0067] SiO.sub.2=silica gel [0068]
TBAF=tetrabutylammoniumfluoride [0069] TBS=Tris buffered saline
[0070] TEA=triethylamine [0071] TFA=trifluoroacetic acid [0072]
THF=tetrahydrofuran [0073] TPGS=d-.alpha.-Tocopheryl polyethlene
glycol 1000 succinate
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0074] "About" or "approximately" as used herein means within an
acceptable range of standard deviation for the particular value as
determined by one of ordinary skill in the art, considering the
measurement in question and the instrument used to make the
measurement (i.e., the limitations of the measurement system). For
example, "about" can mean within one or more standard deviations.
As applied to formulations and dosages, "about" can mean a
deviation within 10%, more preferably within 5%, and even more
preferably, within 2%, of the numbers reported.
[0075] The terms "at least x" and "x or more", or "x or greater",
wherein x denotes a numerical value, are used interchangeably
herein as they are intended to have the same meaning.
[0076] "Brain penetrance" refers to the ability of a compound to
cross the BBB. Because of the rapid peripheral clearance for most
microtubule stabilizing agents, it is important to measure
brain-to-plasma ratios at relatively short times post-dosing, e.g.,
at periods of about of 20 min to 1 h post-dosing, to assess brain
penetrance itself. A compound having good brain penetrance as
defined herein means a compound which at 20 min to 1 h post-dosing
will show a brain-to-plasma ratio of 0.5 or greater, more
preferably, 0.8 or greater, and most preferably, a ratio of 1 or
more (again, at a time between 20 min and 1 h post-dosing). In
assessing whether a compound or drug satisfies this standard of
high brain/plasma ratio (e.g., as recited in the claims herein), in
vivo non-human studies must be relied upon as human brain tissue
cannot be analyzed to assess drug concentration.
[0077] "Cognitive benefits" means that an improvement or lessening
in decline of cognitive function for at least one patient in need
of treatment is observed or reported, as characterized by cognition
tests, measures of global function, and activities of daily living
and behavior. Typically, cognitive benefits are measured with
cognition tests designed to measure cognitive decline in a patient
or group of patients. Examples of such tests include cognition
tests like ADAS-cog (Alzheimer's disease Assessment Scale,
cognitive subscale) and the MMSE (Mini-mental state exam); behavior
tests like the NPI (Neuropsychiatric Inventory); daily living
activity tests like the ADCS-ADL (Alzheimer's Disease Cooperative
Study-Activities of Daily Living); and global function tests such
as the CIBIC-plus (Clinician Interview Based Impression of Change),
and CDR sum of boxes (Clinical Dementia Rating).
[0078] "Extended periods of time" as used herein means period of 24
hours or more, typically 24 to 76 h.
[0079] "High selective retention rate" or "high selective
retention" as used herein means that the drug or compound is
retained in one tissue or organ, specifically the brain, at a much
higher level than is found in other tissues and organs, especially
the liver, as measured at an extended period of time post-dosing.
More particularly as defined herein, a high selective retention
rate means the concentration of drug in the brain is 4 or more
times that found in the liver at 24 or more h post-dosing, more
preferably, a factor of at least 6 or more, and most preferably, at
a factor of at least 8 or more at 24 h or more h post-dosing. In
assessing whether a compound or drug satisfies this standard of
high selective retention (e.g., as recited in the claims herein),
naturally non-human studies must be relied upon as human brain
tissue cannot be analyzed to assess drug concentration.
[0080] "Impact on underlying disease" means an improvement in a
measure of the biomarkers and other parameters associated with the
disease process, including biochemical markers in CSF or plasma,
changes in brain volume, changes in brain function as measured by
functional imaging, and changes in histopathology or biochemistry
that might be observed after autopsy. Typical biomarkers that may
be used for AD clinical trials include analytes measured in CSF
such as Tau, phosphoTau, beta-amyloid, and isoprostanes, as well as
brain imaging modalities such as fluorodeoxyglucose PET and
volumeteric MRI. Additional biomarkers that potentially may be
useful, particularly those examining synaptic activity, MT
integrity/function, and oxidative stress include, but are not
limited to: GABA, neuropeptide Y, alpha-synuclein, neurogranin and
vasoactive intestinal peptide, tubulin, Tau fragments,
ubiquitinated proteins, soluble forms of amyloid precursor protein,
chromogranin B, 4-hydroxy nonenal, nitrotyrosine, and
8-hydroxy-deoxyguanidine.
[0081] "Intermittent" when used with reference to a dosing schedule
means that there are breaks in the dosing schedule that are
irregular. For example, a daily, weekly, biweekly, or monthly
dosing schedule is not considered intermittent under this
definition, because the break between doses is in each instance
regular and defined by the dose cycle of administering the drug.
However, a more elaborate dosing schedule with one or more
irregular breaks would be considered intermittent, such as 5 days
on, followed by 2 days off; or a dose administered on days 1, 8 and
15, of a 30 day cycle, and so forth.
[0082] "Long half-life", or "long brain half-life" as used herein
means that a drug has a half-life of 20 or more h post-dosing
(which is considered dose-independent), and more preferably, for a
period 30 or more h post-dosing, and most preferably, for a period
of 40 or more h post-dosing. As with the selective retention rates,
in vivo non-human studies must be relied upon in assessing whether
the compound has a long brain half-life.
[0083] "Low dose" as used herein means a dose of the epothilone D
compound that is significantly less than that administered to
achieve chemotherapeutic effects (e.g., given a particular mode of
administration, clinical trial, and/or experiment), preferably a
dose that is 10-fold or less than the chemotherapeutic dose, more
preferably a dose that is 50-fold or more less, and even more
preferably a dose that is 100-fold or more fold less than the
chemotherapeutic dose, i.e., that previously assessed as
chemotherapeutically effective using the same administrative method
for the given experiment, study or trial. For example, in Phase II
clinical trials of epothilone D, a dose administered was 100
mg/m.sup.2 administered as a 90 min. infusion once a week for three
weeks every four weeks (3 weeks on, 1 week off), for a cumulative
total of 300 mg/m.sup.2 administered every 4 weeks. A low dose
relative to this clinical trial dose, as defined herein, would mean
a cumulative one-month dose following IV administration of 30
mg/m.sup.2 or less, more preferably a dose of 6 mg/m.sup.2 or less,
and even more preferably a dose of 3 mg/m.sup.2 or less. Thus, as
an alternative example, a low dose as compared with the above
clinical trial dose when administered once every 4 weeks would be a
dose of 30 mg/m.sup.2, more preferably a dose of 6 mg/m.sup.2, and
even more preferably a dose of 3 mg/m.sup.2. Since bioavailability
may change depending upon the mode of administration (e.g., oral v.
IV administration, with less bioavailability achieved upon oral
administration), the relative dosages (i.e., assessment whether a
given dose is a "low dose" as defined herein), should be based on a
comparison involving the same or similar modes of
administration.
[0084] "Patient in need of treatment" as used herein is intended to
include use of epothilone D for a patient 1) already diagnosed with
a Tau-associated disease (including a tauopathy, particularly AD)
at any clinical stage, including patients having mild cognitive
impairment to advanced dementia; and/or 2) who has early or
prodromal symptoms and signs of a Tau-associated disease (including
a tauopathy, particularly AD); and/or 3) who has been diagnosed as
susceptible to a Tau-associated disease (including a tauopathy,
particularly AD), due to age, hereditary, or other factors for whom
a course of treatment is medically recommended to delay the onset
or evolution or aggravation or deterioration of the symptoms or
signs of disease.
[0085] "Statistically significant cognitive benefits" means that
there are cognitive benefits (e.g., improvement or the lessening in
decline of cognitive function), following a period of 6 months to a
year of treatment for at least 10% or more of patients evaluated,
more preferably at least 25% or more patients, and even more
preferably, 50% or more of the patient group. Preferably,
improvement at a rate as compared with a control group is assessed
and reflects an at least 10% improvement (e.g., as evaluated based
on comparative test scores between placebo and control, wherein
"improvement" is intended to include reduction in decline in a
patient's condition), more preferably, improvement at a rate of
more than 25% or more is observed, and most preferably, at a rate
of 35% or more.
[0086] "Tau-associated disease" as defined herein means diseases
associated with abnormalities in Tau as well as diseases that are
"tauopathies." Tau-associated diseases include, but are not limited
to, frontotemporal dementia, including the subtype of
frontotemporal dementia and Parkinsonism linked to chromosome 17
(FTDP-17), progressive supranuclear palsy, corticobasal
degeneration, Pick's disease, agyrophilic grain disease, as well as
Parkinson's disease, Down syndrome, post-encephalic Parkinsonism,
myotonic dystrophy, Niemann-Pick C disease, dementia pugilistica,
Blint disease, prion diseases, amyotrophic lateral sclerosis,
Parkinsonism-dementia complex of Guam, multiple sclerosis,
glaucoma, diabetic retinopathy, and traumatic brain injury; as well
as Huntington's disease, Lewy body dementia, Charcot-Marie-Tooth
disease, hereditary spastic paraplegia, and multiple system
atrophy.
[0087] "Tauopathy" as defined herein means a neurodegenerative
disease associated with fibrillar forms of Tau protein (tangles) in
brain. These diseases include AD; however, other tauopathies
include, but are not limited to, frontotemporal dementia, including
the subtype of frontotemporal dementia and Parkinsonism linked to
chromosome 17 (FTDP-17), progressive supranuclear palsy,
corticobasal degeneration, Pick's disease, and agyrophilic grain
disease.
[0088] "Therapeutically-effective amount of epothilone D" is meant
an amount of epothilone D sufficient to:
[0089] (1) relieve or alleviate at least one symptom of a
Tau-associated disease (preferably, a tauopathy, and more
preferably, AD), including cognitive functions such as dementia,
memory loss, reduced comprehension, dexterity in performing daily
living activities, and/or centrally-mediated effects such as motor
deficits and vision; and/or
[0090] (2) reverse, reduce, prevent, inhibit, or delay the onset or
aggravation of the loss of cognitive function associated with a
Tau-associated disease (preferably, a tauopathy, and more
preferably, AD), and/or reverse, reduce, prevent, inhibit, or delay
the onset or aggravation of one or more centrally mediated effects
of said disease, including motor deficits, vision, and so on. In
preferred embodiments of the invention, the epothilone D
pharmaceutical compound is therapeutically effective in not only
relieving or alleviating the symptoms of the Tau-associated disease
(preferably, a tauopathy, and more preferably, AD), but also is
effective in having an impact on underlying disease (i.e., as
defined above).
Alternative Embodiments of the Invention
[0091] The present inventors have found that epothilone D,
administered for the treatment of a Tau-associated disease achieves
a surprising level of brain penetration, long brain half-life, and
selective retention, particularly as compared with other
microtubule stabilizers. The inventors further have discovered that
remarkably, increased therapeutic effects in treating
Tau-associated diseases (particularly tauopathies, and more
particularly, AD), are achieved with low doses of epothilone D. As
such, a relatively low dosage of epothilone D can be administered
for effective treatment of a Tau-associated disease, preferably AD.
The inventors have thus developed a method of treating Alzheimer's
disease employing the administration of epothilone D to a patient
having AD. The method is expected to be therapeutically effective
in treating AD in human patients while also posing significantly
less serious or fewer side effects as compared with the side
effects that typically occur when microtubule stabilizers are
administered to human patients for chemotherapy. Such side effects
that are reduced or eliminated may include one or more of
gastrointestinal distress (including, without limitation, nausea,
diarrhea, stomatitis/mucositis, vomiting, anorexia, constipation,
and/or abdominal pain), liver toxicity, neutropenia, leucopenia,
myelosuppression, alopecia, myalgia/arthralgia, fatigue,
musculoskeletal pain, nail disorder, pyrexia, headache, skin
exfoliation, and/or neurosensory effects at various grade
levels.
[0092] According to an alternative embodiment of the invention,
there is provided a method of treating Alzheimer's disease
comprising the step of administering a therapeutically effective
amount of epothilone D to a patient, wherein the epothilone D
compound has two or more properties selected from good brain
penetrance, a long brain half-life, and a high selective retention
rate, as defined herein, more preferably, where the epothilone D
demonstrates all three properties of good brain penetrance, long
brain half-life, and a selective retention rate, as these terms are
defined herein.
[0093] According to another embodiment of the invention, there is
provided a method of treating Alzheimer's disease comprising the
step of administering a therapeutically effective amount of
epothilone D to a patient, wherein the epothilone D compound upon
administration has properties selected from two or more of:
[0094] brain penetrance of 0.5 or greater, more preferably, 0.8 or
greater, most preferably, 1 or greater, as measured at 20 min. to 1
h post-dosing; and/or
[0095] a brain half-life of at least 24 h, and more preferably, of
at least 30 h, and most preferably of up to 40 h or more;
and/or
[0096] a brain-liver selective retention rate of at least 4 at 24 h
or more post-dosing, more preferably at a rate of 6 or more at 24 h
or more, and most preferably, at a factor of 8 or more at 24 or
more h post-dosing.
[0097] According to another embodiment of the invention, there is
provided a method of treating Alzheimer's disease comprising the
step of administering a therapeutically effective amount of
epothilone D to a patient, wherein the epothilone D compound upon
administration has properties selected from all three of:
[0098] brain penetrance of 0.5 or greater, more preferably, 0.8 or
greater, most preferably, 1 or greater, as measured at 20 min. to 1
h post-dosing; and/or
[0099] a brain half-life of at least 24 h, and more preferably, of
at least 30 h, and most preferably of up to 40 h or more;
and/or
[0100] a brain-liver selective retention rate of at least 4 at 24 h
or more post-dosing, more preferably, at a rate of 6 or more at 24
h or more, and most preferably at a factor of 8 or more at 24 or
more h post-dosing.
[0101] According to another embodiment of the invention, there is
provided a method of treating Alzheimer's disease comprising the
step of administering a therapeutically effective amount of
epothilone D to a patient, wherein the method is therapeutically
effective in treating AD in the patient without causing
drug-induced side effects and/or drug-plasma concentration levels
that would require use of said method to be discontinued.
[0102] According to another embodiment of the invention, there is
provided a method of treating Alzheimer's disease comprising the
step of administering a therapeutically effective amount of
epothilone D to a patient, wherein the method provides cognitive
benefits, more preferably, statistically-significant cognitive
benefits, in treating AD, without causing drug-induced side effects
and/or drug-plasma concentration levels that would require use of
said method to be discontinued.
[0103] According to another embodiment of the invention, there is
provided a method of treating Alzheimer's disease comprising the
step of administering a therapeutically effective amount of
epothilone D to a patient, wherein the method has an impact on
underlying disease, more preferably, a statistically-significant
impact on underlying disease, without causing drug-induced side
effects and/or drug-plasma concentration levels that would require
use of said method to be discontinued.
[0104] According to another embodiment of the invention, there is
provided a method of treating Alzheimer's disease comprising the
step of administering a therapeutically effective amount of
epothilone D to a patient, wherein method has an impact on
underlying diseases, provides cognitive benefits, and/or is
otherwise therapeutically effective, without causing side effects
such as gastrointestinal side effects, leucopenia, and/or
neurotoxicity, that would require use of said method to be
discontinued.
[0105] According to another embodiment of the invention, there is
provided a method of treating Alzheimer's disease comprising the
step of administering a therapeutically effective amount of
epothilone D to a patient, wherein the dose of epothilone D is a
low dose, as defined herein.
[0106] According to another embodiment of the invention, there is
provided a method of treating Alzheimer's disease comprising the
step of administering a therapeutically effective amount of
epothilone D to a patient, wherein the dose of epothilone D is
between 0.001-10 mg/m.sup.2, or alternatively, at a dose between
0.00003-0.3 mpk, administered on a daily, weekly, or intermittent
dosing cycle.
[0107] According to another embodiment of the invention, there is
provided a method of treating Alzheimer's disease comprising the
step of administering a therapeutically effective amount of
epothilone D to a patient, wherein the epothilone D is administered
via IV, and the dose of epothilone D over a cumulative monthly
dosing cycle (i.e., total dosage of compound administered over a
one month cycle, regardless of schedule, e.g., weekly, bi-weekly, 3
week on, 1 week off, etc.) is in the range between 0.001-5
mg/m.sup.2, more preferably between 0.01-5 mg/m.sup.2, even more
preferably between 0.01-3 mg/m.sup.2, yet even more preferably
between 0.1-3 mg/m.sup.2, and most preferably between 0.1-1
mg/m.sup.2.
[0108] According to another embodiment of the invention, there is
provided a method of treating Alzheimer's disease comprising the
step of administering a therapeutically effective amount of
epothilone D to a patient, wherein the epothilone D is administered
orally, and the dose of epothilone D calculated on a daily basis is
in the range between 0.001-2 mg/m.sup.2, more preferably between
0.01-2 mg/m.sup.2, even more preferably between 0.1-2 mg/m.sup.2,
yet even more preferably between 0.2-2 mg/m.sup.2.
[0109] According to another embodiment of the invention, there is
provided a method of treating Alzheimer's disease comprising the
step of administering a therapeutically effective amount of
epothilone D to a patient, wherein the epothilone D is administered
orally, and the dose of epothilone D for a cumulative monthly basis
(i.e., total dosage of compound administered over a one month
cycle, regardless of schedule, e.g., daily, weekly, bi-weekly,
etc.) is in the range between 0.03-60 mg/m.sup.2, more preferably
between 0.30-60 mg/m.sup.2, even more preferably between 3-60
mg/m.sup.2, yet even more preferably between 6-60 mg/m.sup.2.
[0110] According to another embodiment of the invention, there is
provided a method of treating Alzheimer's disease comprising the
step of administering a therapeutically effective amount of
epothilone D to a patient, wherein the epothilone D is administered
orally on a dosing schedule selected from once daily, once weekly,
once every two weeks, or once a month.
[0111] According to another embodiment of the invention, there is
provided a method of treating Alzheimer's disease comprising the
step of administering a therapeutically effective amount of
epothilone D to a patient, wherein the epothilone D is administered
orally on a dosing schedule selected from once daily, and wherein
the daily dose of epothilone D is between 0.2 to 2 mg/m.sup.2.
[0112] According to another aspect of the invention, there are
provided methods of treating other tauopathies, besides AD,
according to any one of the embodiments of the invention recited
above. For example, such other tauopathies may include one or more
of the diseases referenced in the definition of
"tauopathy-associated disease" herein. For example, one embodiment
of the invention comprises use of epothilone D, according to any of
the above embodiments, to treat not only AD but also a disease
selected from frontotemporal dementia, including the subtype of
frontotemporal dementia and Parkinsonism linked to chromosome 17
(FTDP-17), progressive supranuclear palsy, corticobasal
degeneration, Pick's disease, and agyrophilic grain disease,
Parkinson's disease, Down syndrome, post-encephalic Parkinsonism,
myotonic dystrophy, Niemann-Pick C disease, dementia pugilistica,
Blint disease, prion diseases, amyotrophic lateral sclerosis,
Parkinsonism-dementia complex of Guam, multiple sclerosis,
glaucoma, diabetic retinopathy and/or traumatic brain injury. A
preferred embodiment comprises use of epothilone D, according to
any of the embodiments described herein, to treat a tauopathy,
including, without limitation, a disease selected from AD,
frontotemporal dementia, including the subtype of frontotemporal
dementia and Parkinsonism linked to chromosome 17 (FTDP-17),
progressive supranuclear palsy, corticobasal degeneration, Pick's
disease, and agyrophilic grain disease.
[0113] According to another embodiment of the invention, there is
provided epothilone D, for use in treating a Tau-associated
disease, more preferably, a tauopathy, most preferably AD.
[0114] It is contemplated that each of the above inventive methods
also may be combined with one or more other inventive methods, and
all such various combinations of the above inventive methods are
contemplated herein. For example, one combination of the above
inventive methods may comprise a method of treating Alzheimer's
disease comprising the step of administering a therapeutically
effective amount of epothilone D to a patient, wherein the method
is therapeutically effective in treating AD in the patient without
causing drug-induced side effects and/or drug-plasma concentration
levels that would require use of said method to be discontinued;
and wherein the dose of epothilone D is a cumulative monthly dose
of between 0.001-5 mg/m.sup.2, administered via IV; and/or wherein
the dose of epothilone D is between 0.001 to 2 mg/m.sup.2,
administered PO daily; and/or wherein the dose of epothilone D is
selected from a dose within any one of the preferred ranges
expressed above for oral or IV administration.
[0115] It also is contemplated also that any of the recited methods
of treatment may by combined with the embodiment involving
epothilone D, for use in treating a tauopathy, preferably AD, in a
human patient. Thus, for example, one embodiment of the invention,
comprising a combination of the above alternative embodiments,
would comprise epothilone D for treating AD, wherein the use is
therapeutically effective in treating AD, and wherein the
epothilone D is administered to the patient at a dose between
0.001-10 mg/m.sup.2, or alternatively, at a dose between
0.00003-0.3 mpk, administered on a daily, weekly, or intermittent
dosing cycle. Yet another embodiment would comprise epothilone D,
for treating a tauopathy, particularly AD, wherein the epothilone D
is administered to a human patient at a low dose and is
therapeutically effective in having an impact on underlying disease
and/or providing cognitive benefits.
[0116] According to another embodiment of the invention, there is
provided a pharmaceutical formulation comprising epothilone D
suitable for administration to a human patient in need of treatment
for a Tau-associated disease, preferably a tauopathy, more
preferably, AD, wherein administration of the formulation is
therapeutically effective in treating the disease in the patient
without causing drug-induced side effects and/or drug-plasma
concentration levels that would require use of said epothilone D
formulation to be discontinued.
[0117] According to yet another embodiment of the invention, there
is provided a pharmaceutical formulation comprising epothilone D
suitable for administration to a human patient for treating a
Tau-associated disease, preferably a tauopathy, more preferably AD,
wherein administration of the formulation provides
statistically-significant cognitive benefits in treating the
disease, without causing drug-induced side effects and/or
drug-plasma concentration levels that would require use of said
epothilone D formulation to be discontinued.
[0118] According to yet another embodiment of the invention, there
is provided a pharmaceutical formulation comprising epothilone D
suitable for administration to a human patient for treating a
Tau-associated disease, preferably a tauopathy, more preferably,
AD, wherein the formulation is effective in providing an impact on
underlying disease, without causing drug-induced side effects
and/or drug-plasma concentration levels that would require use of
said epothilone D formulation to be discontinued.
[0119] According to yet another embodiment of the invention, there
is provided a pharmaceutical formulation suitable for
administration to a human patient for treating a Tau-associated
disease, preferably a tauopathy, more preferably, AD, wherein the
formulation comprises a dosage unit of epothilone D of between
0.0001-10 mg/m.sup.2, more preferably between 0.001-5 mg/m.sup.2,
more preferably between 0.001-3 mg/m.sup.2, even more preferably
between 0.001-1 mg/m.sup.2, and most preferably between 0.001-0.5
mg/m.sup.2.
[0120] According to yet another embodiment of the invention, there
is provided a pharmaceutical formulation for IV administration to a
human patient, wherein said formulation is suitable for delivery of
a cumulative monthly dose of epothilone D in the range between
0.001-5 mg/m.sup.2, more preferably between 0.01-5 mg/m.sup.2, even
more preferably between 0.01-3 mg/m.sup.2, yet even more preferably
between 0.1-3 mg/m.sup.2, and most preferably between 0.1-1
mg/m.sup.2.
[0121] According to yet another embodiment of the invention, there
is provided a pharmaceutical formulation for oral administration to
a human patient, wherein said formulation is suitable for delivery
of a cumulative monthly oral dose of epothilone D in the range
between 0.03-60 mg/m.sup.2, more preferably between 0.30-60
mg/m.sup.2, even more preferably between 3-60 mg/m.sup.2, yet even
more preferably between 6-60 mg/m.sup.2.
[0122] According to yet another embodiment of the invention, there
is provided a pharmaceutical formulation for administration to a
human patient, wherein said formulation comprises epothilone D in a
pharmaceutically acceptable solvent system comprising from about 0
to 50% propylene glycol, about 1 to 10% TPGS, about 0.5 to 10%
ethanol, about 0-90% water, and/or about 5 to 85% PEG such as
PEG-400.
[0123] Combinations of each of the above inventive pharmaceutical
formulations are also contemplated herein.
Utility
Tauopathies
[0124] Tauopathies are neurodegenerative diseases associated with
abnormal forms of Tau protein in brain tissue. Alzheimer's Disease
(AD) was the first neurodegenerative disease to be identified as
implicating Tau dysfunction. In particular, neurofibrillary
tangles--the presence of which is one of the hallmark pathologies
in AD--were found to contain fibrillar, hyperphosphorylated,
conformationally-altered forms of the Tau protein. Subsequently,
other tauopathies were identified including frontotemporal dementia
and Parkinsonism linked to chromosome 17 (FTDP-17), progressive
supranuclear palsy, corticobasal degeneration, Pick's disease, and
agyrophilic grain disease. In addition, a link with Tau
abnormalities (including hyperphosphorylated Tau, Tau aggregates,
and/or an association with the H1/H1 Tau haplotype) has been
associated with Parkinson's disease, Down syndrome, post-encephalic
Parkinsonism, myotonic dystrophy, Niemann-Pick C disease, dementia
pugilistica, Blint disease, prion diseases, amyotrophic lateral
sclerosis, Parkinsonism-dementia complex of Guam, multiple
sclerosis, glaucoma, diabetic retinopathy and traumatic brain
injury (Avila et al. 2004; Bartosik-Psujek et al. 2006; Dickey et
al. 2006; Wostyn et al. 2008).
[0125] Tau is a 50 to 75 kDa microtubule-associated protein (MAP)
that binds and stabilizes microtubules (MTs). There are six primary
sequence variants of Tau, and these variants are formed by
alternative splicing (Lace et al. 2007). The splice variants
contain zero, one, or two (0N, 1N, or 2N) N-terminal inserts in
combination with either three repeats (3R) or four repeats (4R) of
a microtubule-binding domain. The repeat domains are necessary for
microtubule stabilization, while proline-rich regions on either
side of the repeat domains are necessary for binding to the
microtubules (Preuss et al. 1997). The repeat domains and
proline-rich regions are phosphorylated by multiple kinases,
leading to dissociation of Tau from microtubules. The N-terminus of
Tau extends away from the microtubule surface, where it is believed
to assist in determining the spacing between microtubules and in
binding of the motor protein dynactin to the microtubules (Magnani
et al. 2007). In normal cells, there are roughly equal levels of 3R
Tau and 4R Tau present. 4R Tau binds more tightly to microtubules
than does 3R Tau.
[0126] Tau is most abundant in neurons where it is predominantly
localized to axons. Tau is the major microtubule-associated protein
in neuronal axons, while the MAP1 family is widely distributed in
neurons, and the MAP2 family is predominantly somatodendritic. Tau
stabilizes axonal microtubules, thereby facilitating transport of
proteins, organelles, lipids, cellular components targeted for
degradation, and cell signaling molecules bi-directionally between
the cell body and the synaptic terminals. Tau dysfunction could
interfere with axonal trafficking and thereby affect neuronal
function and survival. The Tau gene can be knocked out in mice with
mild consequences, but if both Tau and MAP 1B genes are removed,
the double knockout mice die as embryos. It appears that
alterations in expression of some MAPs can substitute for each
other in many cases, including development (Avila et al. 2004).
[0127] In some cases, mutations in the gene encoding Tau (sometimes
called MAPT) cause tauopathies, particularly in FTDP-17 and other
frontotemporal dementias. Many FTDP-17 mutations decrease binding
to microtubules in vitro and/or increase their propensity to form
fibrils (Lace et al. 2007). Other tauopathy-associated mutations
alter the splice pattern of Tau to generate predominantly 3R or 4R
Tau. Yet another class of Tau mutations on the N-terminus alters
the ability to bind to dynactin (Magnani et al. 2007). All of these
mutations have the potential to interfere with normal functions of
Tau. In the case of AD, it is thought that .beta.-amyloid (A.beta.)
leads to abnormalities in Tau.
[0128] Although tangles and other Tau aggregates are a pathologic
feature of tauopathies, several lines of evidence suggest that some
other, soluble, unidentified form of abnormal Tau is neurotoxic.
The data suggesting that Tau aggregated into neurofibrillary
tangles is not directly pathogenic include observations of human
brains and mouse models. For instance, examination of different
regions and disease stages of Alzheimer's disease brains has led to
the conclusion that neurons can survive and function with
neurofibrillary tangles for decades (Morsch et al. 1999). Likewise,
human Tau (hTau) transgenic mice have tangles and severe
neurodegeneration, but the neurons with tangles do not show
selective signs of distress and are too few in number to account
for the dramatic loss in neurons observed in this model (Andorfer
et al. 2005). Tg4510, an inducible Tau transgenic line, shows
dramatic and rapid tangle formation, neurodegeneration, and
behavioral deficits when Tau-P301L is induced (Santacruz et al.,
2005). When Tau-P301L expression is repressed, neurodegeneration
and cognitive deficits are greatly reduced, but tangle formation
continues. Further studies using these mice show that soluble Tau
multimers correlate with cognitive deficits. Similar Tau multimers
are also observed in FTDP-17 and AD brain tissue (Berger et al.
2007). Finally, evidence for non-fibrillar Tau being involved in
behavioral deficits in AD was obtained using transgenic mice
overexpressing a mutant form of .beta.-amyloid precursor protein
(APP) (Roberson et al., 2007). When these APP mice were crossed
with Tau knockout mice, amyloid plaques were formed, but behavioral
deficits and synaptic abnormalities were prevented. In this APP
line, Tau abnormalities could not be detected in the presence of
synaptic and behavioral deficits. Taken together, these studies
show that a soluble, unidentified form of abnormal Tau is likely
the neurotoxic species.
Microtubule Stabilization for Treatment of Tauopathies
[0129] There are two major hypotheses for the role of Tau in
neurodegenerative disease. One hypothesis posits that abnormal
forms of Tau disrupt cellular function, while the other hypothesis
posits that the loss of functional Tau leads to microtubule
destabilization (Avila et al. 2004; Lace et al. 2007). It is based
on the second hypothesis that microtubule stabilizers have been
suggested as therapies to treat tauopathies (Lee et al. 1994; U.S.
Pat. No. 5,580,898). Inappropriate disruptions in axonal
trafficking have been implicated in a number of diseases in
addition to those identified with abnormalities in Tau. These
include Huntington's disease, Lewy body Dementia,
Charcot-Marie-Tooth disease, hereditary spastic paraplegia, and
multiple system atrophy (Roy et al. 2005).
[0130] To test if microtubule stabilizers could benefit mice that
overexpress Tau, PrP T44 Tau transgenic mice were treated with
paclitaxel (Zhang et al., 2005). PrP T44 mice overexpress normal
human 0N3R Tau in spinal cord neurons and consequently develop
motor deficits due to Tau overexpression. Paclitaxel treatment for
3 months reduced motor dysfunction and increased microtubule
numbers and axonal transport in the ventral roots of the spinal
cord. Although paclitaxel is poorly CNS-penetrant, it was able to
influence the efferent axons from neurons in the ventral horn of
the spinal cord which are outside the blood-brain barrier.
Interestingly, Tau pathology in this model (spheroids) was
unaffected. Since this model does not show neuronal loss, the
effect of microtubule stabilizers on neuronal survival could not be
assessed. Additionally, it is unclear whether the motor benefits
observed in the spinal cord tauopathy would translate into
cognitive benefits in a cortical-hippocampal tauopathy. For
example, opposite results were observed with the cross of two
different tauopathy transgenic mouse lines to transgenic mice that
overexpress glycogen synthase kinase 3 (Gsk3). In the spinal cord
tauopathy model, the Tau-Gsk3 bigenic animals had reduced
pathology, while in the forebrain tauopathy model, the Tau-Gsk3
bigenic animals showed increased pathology (Spittaels et al. 2000;
Terwel et al. 2008).
[0131] Microtubule stabilization has been offered as an explanation
for the effects of NAP, a peptide of sequence NAPVSIPQ, in several
animal models. In particular, NAP has neurotrophic,
anti-inflammatory, anti-apoptotic, and neuroprotective activities
in many cellular and in vivo models, including middle cerebral
artery occlusion (stroke model), head trauma, cholinotoxic lesions,
aging, and developmental defects in fetal alcohol syndrome and
apolipoprotein E deficient mice (Gozes et al. 2006; Gozes 2007). As
for tauopathies, NAP administration for 3 or 6 months is reported
to reduce A.beta. levels, hyperphosphorylated Tau, and sarcosyl
insoluble Tau while increasing soluble Tau in 3.times.Tg mice
(Matsuoka et al. 2007; Matsuoka et al. 2008). 3.times.Tg mice
overexpress APP and Tau-P301L (Oddo et al. 2003). The mechanism of
NAP activity is not fully defined, but there is evidence, based on
binding of tubulin to a NAP affinity column and effects on
microtubule formation and/or stabilization in cultured neurons,
that NAP binds to microtubules (Divinski et al. 2006). NAP is also
known to inhibit A.beta. aggregation, so it may be acting upstream
of Tau in the 3.times.Tg model. NAP is not likely to act as a
typical microtubule-stabilizing agent, as it is able to protect
against paclitaxel-induced peripheral neuropathy in rats (U.S.
Patent Application Publication No. 2006/0247168 A1). When
microtubule-stabilizing agents, such as paclitaxel, are
administered at high, chemotherapeutic doses, a peripheral
neuropathy often occurs (Postma et al. 1999) that is believed to
result from the over-stabilization and bundling of microtubules in
peripheral nerves. Since NAP prevents paclitaxel-induced peripheral
neuropathy in rats, paclitaxel and NAP are unlikely to act through
identical mechanisms.
[0132] There are also suggestions that microtubule stabilizers
could have neuroprotective effects unrelated to obvious Tau
dysfunction. Microtubule-stabilizing compounds protect cultured
neurons from multiple toxic insults, including A.beta.42, oxidative
stress from soluble A.beta.40, lysosomal disruption,
calcium-induced toxicity, and glutamate-induced toxicity (Burke et
al. 1994; Furukawa 1995; Sponne et al. 2003; Michaelis et al. 2005;
Butler et al. 2007). It is hypothesized that microtubules play a
key role not only in transport mechanisms, but also in regulation
of cell signaling, particularly calcium signaling, possibly through
anchoring of macromolecular signaling complexes in the vicinity of
the plasma membrane (Michaelis et al. 2005). Microtubule
stabilizing agents have also been shown to enhance mitochondrial
function by reducing reactive oxygen species generation and
increasing expression of the oxidative phosphorylation genes
involved in ATP production (Wagner et al. 2008).
Microtubule-stabilizing agents are also known to broadly influence
cell signaling during disruption of the mitotic spindle in cancer
cells (Bergstralh et al. 2006).
Brain-Penetrant Microtubule Stabilizers
[0133] The therapeutic target of microtubule stabilizers for
tauopathies and other neurodegenerative diseases is microtubules in
the brain. However, microtubule stabilizers can cause toxicity to
peripheral tissues, such as inhibition of cell proliferation,
particularly in the gastrointestinal tract and hematopoietic cells,
and peripheral neuropathy. It is thus highly desired to identify
microtubule stabilizers with excellent brain penetration and
selective retention in the brain as compared with peripheral
tissues, so as to maximize the therapeutic index for tauopathies
and other neurodegenerative diseases. The ability of compounds to
bind with a longer half life to brain tissue relative to peripheral
tissues is a highly desired property.
[0134] The taxane series of microtubule stabilizers are substrates
of multiple multi-drug resistance transporters, such as
P-glycoprotein (POP), ATP-binding cassette, multidrug resistance
protein, and breast cancer resistance protein. These multi-drug
resistance transporters prevent compounds from accumulating in
tumor and brain tissue. Multiple labs have worked to synthesize
taxanes that are not substrates for multi-drug resistance
transporters, particularly PGP, with limited success (Minderman et
al. 2004; Rice et al. 2005; Ballatore et al. 2007).
Co-administration of a PGP inhibitor with paclitaxel has also been
attempted (Fellner et al. 2002). These efforts have shown results
of some taxane entry into the brain, achieving, for example,
approximately 1/30th the levels of paclitaxel in the brain as in
the kidney with a PGP inhibitor, or brain levels in the .mu.M range
with KU-237, but only for 4 h after administration (Michaelis
2006). Hence, use of PGP inhibitors are not an attractive method to
increase the brain penetration of taxanes.
Methods of Preparation and Formulations
[0135] Epothilone D is a known compound which has been chemically
synthesized de novo and also has been isolated from fermentations
of Sorangium cellulosum strains as minor products in the
fermentation of S. cellulosum. Total synthesis of epothilone D is
reported in U.S. Pat. No. 6,242,469 to Danishefsky et al., and
additional methods for preparing epothilone D and other epothilone
compounds can be found at U.S. Pat. Nos. 6,204,388, 6,288,237,
6,303,342; WO 03/072730, U.S. Pat. No. 6,410,301; U.S. Patent
Application Publication No. 2002/0137152A1; U.S. Pat. No.
6,867,333, U.S. Patent Application Publication No. 2006/004065,
each of which is incorporated herein by reference. Synthetic
methods for manufacturing epothilone D have been characterized as
impractical for full-scale pharmaceutical development. One
alternative method of preparation is to engage in large-scale
fermentation of epothilone B, for example, as described in U.S.
Pat. No. 7,172,884 B2, with use of improved strains designed to
provide relatively large yields of epothilone B, and the epothilone
B can be de-epoxidized to provide epothilone D. Methods of
de-epoxidation are well known but also can be found in U.S. Pat.
No. 6,965,034 (WO 99/43653), to Danishefsky et al., particularly as
applied to epothilone D.
[0136] Further methods for making epothilone D are set forth in
U.S. Pat. Nos. 6,998,256 B2 and 7,067,286, "Methods of Obtaining
Epothilone D using Crystallization and/or By the Culture of Cells
in the Presence of Methyl Oleate," which describe the biosynthetic
production of epothilone D using Myxococcus xanthus strains
K111-40-1 and K111-72.4.4, and/or other recombinant strains that
have been developed by Kosan Biosciences Inc. (now BMS), to improve
production of epothilone D. Fermentation and purification
conditions for making epothilone D are also set forth in U.S. Pat.
Nos. 6,998,256 B2 and 7,067,286, as well as U.S. Pat. Nos.
6,583,290, 6,858,411, 6,921,650, and 7,129,071, each of which is
assigned to Kosan (now BMS, the current assignee), and incorporated
herein by reference. See also, Lau et al., Kosan Biosciences,
"Optimizing the Heterologous Production of Epothilone D in
Myxococcus xanthus," Biotechnology & Bioengineering,
78(3):280-288 (May 5, 2002).
[0137] Yet further methods that may be used in making epothilone D
are illustrated in U.S. patent application Ser. No. 12/118,432.
This application discloses a combination of chemical and
biosynthetic steps to prepare epothilones such as epothilone D. For
example, methods are provided in which one or more intermediates
that may be used for epothilone synthesis are obtained through
fermentation of recombinant cells, and then the biosynthesized
intermediates with use of recombinant cells, disclosed therein, are
converted to the final epothilone compounds via chemical
synthesis.
[0138] The epothilone D used in methods of the present invention
can be administered to a patient in various ways known in the art,
typically by intravenous (IV) administration, subcutaneous
administration, oral administration, and so on. For example,
epothilone D can be formulated with a pharmaceutically acceptable
vehicle or diluent. A pharmaceutical composition comprising
epothilone D can be formulated in a classical manner using solid or
liquid vehicles, diluents, and additives appropriate to the desired
mode of administration.
[0139] Exemplary compositions for parenteral administration include
injectable solutions or suspensions which can contain, for example,
suitable non-toxic, parenterally acceptable diluents or solvents,
such as mannitol, 1,3-butanediol, water, Ringer's solution, an
isotonic sodium chloride solution (0.9% Sodium Chloride Injection
[Normal Saline] or 5% Dextrose Injection), or other suitable
dispersing or wetting and suspending agents, including synthetic
mono- or diglycerides, and fatty acids. Pharmaceutically acceptable
compositions and/or methods of administering compounds of the
invention may include use of co-solvents including, but not limited
to ethanol, N,N dimethylacetamide, propylene glycol, glycerol and
polyethylene glycols, e.g., polyethylene glycol 300 and/or
polyethylene glycol 400. Surfactants (pharmaceutically-acceptable
surface active agent) may be used to increase a compound's
spreading or wetting properties by reducing its surface tension,
including without limitation, d-.alpha.-Tocopheryl polyethlene
glycol 1000 succinate (TPGS), Cremophor, Solutol HS 15, polysorbate
80, polysorbate 20, poloxamer, pyrrolidones such as
N-alkylpyrrolidone (e.g., N-methylpyrrolidone) and/or
polyvinylpyrrolidone; however, use of Cremophor has disadvantages
and is not preferred. The formulation may also comprise use of one
or more "buffers" (e.g., an ingredient which imparts an ability to
resist change in the effective acidity or alkalinity of a medium
upon the addition of increments of an acid or base), including,
without limitation, sodium phosphate, sodium citrate,
diethanolamine, triethanolamine, L-arginine, L-lysine, L-histidine,
L-alanine, glycine, sodium carbonate, tromethamine (a/k/a
tris[hydroxymethyl]aminomethane or Tris), and/or mixtures
thereof.
[0140] Formulations for administering epothilone compounds,
including formulations that avoid use of non-ionic surfactants such
as Cremophor, are described in the prior art. For example, a
formulation for use in IV administration that comprises a mixture
of propylene glycol and ethanol is described in U.S. Pat. No.
6,683,100. Further formulations may comprise mixtures of
polyethylene glycol/dehydrated alcohol, or propylene glycol or
glycerol/dehydrated alcohol. For example, WO 2006/105399
(PCT/US2006/011920) to BMS, discloses formulations that include
mixtures of about 30 to 70 percent by volume dehydrated alcohol for
each 30 to 70 percent by volume PEG 300 and/or PEG 400, which can
be diluted with saline or dextrose infusion fluids for IV
administration, and may be applied for use in administering
epothilone D to patients via IV administration. In such
formulations, it is preferred that the amount of ethanol be
minimized to avoid side effects associated with ethanol
administration. Optimal ratios of solvents may be readily obtained
by one skilled in the field.
[0141] Further preferred formulations specifically designed for
administering epothilone D and analogs are disclosed in U.S. Pat.
No. 7,091,193 (also published as U.S. Patent Application
Publication No. 2005/0148543), to Kosan (now BMS). This patent
describes a formulation wherein epothilone D and a
hydroxypropyl-beta-cyclodextrin are combined in an alcohol-water
solution that is then lyophilized. Embodiments involve use of about
10 mg epothilone D and about 0.4 g of
hydroxypropyl-beta-cyclodextrin combined in a 60%
tert-butanol-water solution that is then lyophilized (ingredients
can be reduced proportionately for preparation of individual, lower
dosages units, according to the current invention). The lyophilized
active ingredient "cake" can then be reconstituted for IV
administration with use of water, ethanol, and/or glycol, which may
include propylene glycol, polyethylene glycol 400, polyoxyethylene
sorbitan monooleate (sold under the trade name TWEEN 80), and
related oxygenated hydrocarbons. It is understood that glycols of
various chain lengths and molecular weights (e.g., polyethylene
glycol 1000, other TWEEN compounds) may be used.
[0142] As a more specific example, a formulation that may be used
to deliver epothilone D to a patient according to the invention may
comprise about 0 to 50% propylene glycol, about 1 to 10% TPGS,
about 0.5 to 10% ethanol, about 0-90% water, and/or about 5 to 85%
PEG such as PEG-400. More specifically, a formulation may
comprise:
[0143] 50% propylene glycol, 10% TPGS, 10% ethanol, 30% water;
or
[0144] 10% propylene glycol, 40% PEG-400, 5% TPGS, 5% ethanol, 40%
water; or
[0145] 85% PEG-400, 10% TPGS, 5% ethanol; or
[0146] 8.5% PEG-400, 1% TPGS, 0.5% ethanol, 90% water.
[0147] One preferred method of administering epothilone D according
to the invention involves oral administration. U.S. Pat. No.
6,576,651 discloses methods for oral administration of epothilones
with use of one or more pharmaceutically acceptable
acid-neutralizing buffers. However, a preferred method of
administration would involve use of a tablet or capsule, including
a solid tablet or capsule or fluid or gelatin-filled capsule. A
solid tablet or capsule of epothilone D may be prepared with one or
more enteric coatings. Enteric coatings have been used for many
years to arrest the release of the drug from orally ingestible
dosage forms. Depending upon the composition and/or thickness, the
enteric coatings are resistant to stomach acid for required periods
of time before they begin to disintegrate and permit slow release
of the drug in the lower stomach or upper part of the small
intestines. Examples of some enteric coatings are disclosed in U.S.
Pat. Nos. 6,224,910, 5,225,202, 2,809,918, 3,835,221, 4,728,512 and
4,794,001, each of which is incorporated herein by reference.
[0148] An enteric coated tablet directed to use of epothilone D is
described in U.S. patent application Ser. No. 11/281,834,
incorporated herein by reference, which may be used to formulate
tablets of capsules of epothilone to practice the invention. This
formulation involves use of an inactive base particle, such as a
sugar bead, to which the active ingredient (i.e., epothilone D), is
applied, which is then encapsulated by an enteric coating polymer,
and/or one or more subcoat layers. The beads are then included
within a capsule. Enteric coatings for use in formulating
epothilone D tablets or capsules may include enteric coating
polymers, such as, for example, hydroxypropyl methylcellulose
phthalate, polyvinyl acetate phthalate, cellulose acetate
phthalate, acrylic acid copolymers, and methacrylic acid
copolymers. One example of a methacrylic acid copolymer that may be
used to form an enteric coating is EUDRAGIT.RTM. L-30-D 55 aqueous
copolymer dispersion, which comprises an anionic copolymer derived
from methacrylic acid and ethyl acrylate with a ratio of free
carboxyl groups to the ethyl ester groups of approximately 1:1, and
a mean molecular weight of approximately 250,000, which is supplied
as an aqueous dispersion containing 30 weight % solids.
EUDRAGIT.RTM. L-30-D 55 aqueous copolymer dispersion is supplied by
Rohm-Pharma Co., Germany.
[0149] In preparing enteric coated beads to form capsules of
epothilone D, it may be desirable to include one or more subcoat
layers that are situated between the epothilone D core and the
enteric coating to minimize contact between those layers. For
example, suitable materials to form the subcoat layer include
starch; gelatin; sugars such as sucrose, glucose, dextrose,
molasses, and lactose; natural and synthetic gums such as acacia,
sodium alginate, methyl cellulose, carboxymethylcellulose, and
polyvinylpyrrolidone (PVP) polymers and copolymers such as PVP-PVA
copolymers; celluloses such as ethylcellulose, hydroxypropyl
cellulose, and hydroxypropyl methyl cellulose; polyethylene glycol;
and waxes. The subcoat layer may further comprise one or more
plasticizers, such as polyethylene glycol, propylene glycol,
triethyl citrate, triacitin, diethyl phthalate, tributyl sebecate,
or combinations thereof.
[0150] The tablet or capsule of epothilone D optionally may
comprise other materials such as flavoring agents, preservatives,
or coloring agents as may be necessary or desired.
[0151] An appropriate dosage of epothilone D can be determined by
one of skill in the art, taking into consideration the findings
described herein together with typical factors such as the body
mass of the patient, the physical condition of the patient, and so
on. The dosage should contain epothilone D in an amount that is
effective for treating Tau-associated diseases, including
tauopathies such as AD. Generally, a range for the dosage of
epothilone D administered for the treatment of Tau-associated
diseases (including tauopathies such as AD) is considered to be
between 0.0001-10 mg/m.sup.2, more preferably between 0.001-5
mg/m.sup.2. Other, more preferred dosage ranges for PO and IV
administration are set forth above in the alternative embodiments
section. The units mg/m.sup.2, are used herein, for purposes of
comparison with the chemotherapeutic dosages previously
administered with epothilones and their analogs. However, the units
mg/m.sup.2 can be readily converted to mpk, considering the animal
species receiving (or having received) the drug and the patient's
bodyweight and/or height. For example, for a human patient weighing
about 70 kg, the dose range of 0.0001-10 mg/m.sup.2 converts to
about 0.00003-0.3 mpk. Further information concerning dose
conversions can be found at www.rphworld.com/viewlink-25045.html,
and in Freireich et al., Cancer Chemother. Reports, 50(4):219
(1966).
[0152] The drug can be administered daily, weekly, or on an
intermittent basis. For example, the drug can be administered for
three weeks on, followed by one week off, or for two weeks on,
followed by one week off, or under other dosing schedules as can be
determined by one skilled in the field. The particular dose
selected will depend upon the mode of administration and dosing
regime selected. One preferred schedule is a once daily oral dosing
schedule. When longer periods of time are prescribed between each
application (typically the case for IV administration), each unit
dose may be larger than when daily dosages are provided.
[0153] Notably, the dose of epothilone D that was administered to
patients for treatment of cancer in certain Phase II clinical
trials was 100 mg/m.sup.2 administered as a 90 minute infusion
given weekly for 3 of 4 weeks (i.e., on days 1, 8, and 15, every 4
weeks), following Phase I trials involving dose escalations of from
9 to 150 mg/m.sup.2 for each dose. The dose of drug contemplated
for treatment of AD is about ten-fold less, and more likely, about
100-fold less, and in another contemplated embodiment, even more
than 1000-fold less, than the therapeutic dose of epothilone D that
was administered for treatment of cancer patients in clinical Phase
II trials, although the dosing schedule and mode of administration
will influence the dose.
[0154] The present invention will be explained in further detail by
way of non-limiting examples below, which make reference to the
appended drawings. The following methods were used in the
experiments described in the examples that follow the description
of the methods.
Methods for Experimentals (Examples 1 Through 5)
[0155] The creation of Tg4510, an aggressive Tau transgenic mouse
line, was recently described (Santacruz et al., 2005; Berger et
al., 2007). The Tg4510 line expressed Tau-P301L, a Tau mutant found
in FTDP-17, using the calmodulin kinase II promoter. The Tg4510
line was unique in several respects:
[0156] 1. High level of Tau expression (13-fold relative to mouse
Tau);
[0157] 2. Restriction of Tau expression to the frontal-temporal
lobes (thereby avoiding the motoric deficits that had characterized
previous Tau lines that expressed Tau in the spinal cord); and
[0158] 3. Rapid and extensive neurodegeneration (60% CA1 neurons
were lost by 5.5 months) preceded by cognitive deficits measurable
at 4.5 months.
Drug Preparation for Tg4510 Study
[0159] Epothilone D (Compound I) was dosed intraperitoneally with a
26-gauge needle, in 10% ethanol, 90% water, 10 ml/kg at 0
(vehicle), 1 mpk, and 10 mpk. A 10.times. stock solution was made
in 100% ethanol, and diluted just before dosing. Mice were dosed in
3 cohorts and data were combined to give a final N of 12, 9, and 15
for the vehicle, 1 mpk, and 10 mpk groups, respectively. Mice were
dosed in a chemical fume hood.
Injection and Behavioral Testing Schedule for Tg4510 Study
[0160] Tg4510 mice were used in this study. These mice are a
well-characterized, aggressive model of tauopathy that overexpress
human P301L mutant Tau in the forebrain (Santacruz et al., 2005;
Berger et al., 2007). The mice are characterized by accumulations
of abnormal forms of Tau, including tangles similar to those
observed in AD brain, behavioral deficits, and eventually neuronal
loss. At 9 weeks (+/-15 days) of age, mice were acclimated to
handling with a single mock injection of phosphate buffered saline,
performed within a chemical fume hood. The mice were then housed in
cages kept within the chemical fume hood for 48 hours. Following
the 48-hour period, the mice were transferred to clean cages and
brought to a behavioral suite for testing.
[0161] The mice were then tested in a Morris water maze (MWM) for
six days. The mice were distributed into treatment groups based on
the results of the behavioral analysis using the rank scores for
probe trial 2 annulus crossing index. Mice were 11 weeks (+/-15
days) of age at the start of dosing and were dosed once weekly. A
panel of neurological and physical propensity tests (Modified
SHIRPA) were performed following the first week of dosing,
including analysis of body position, tremor, coat appearance, gait,
touch escape, positional passivity, limb grasping, and righting
reflex. Mice were additionally examined 48 hours after each weekly
dose for coat appearance, limb grasping, righting reflex and for
any overt stereotyped behavior. No signs of overt toxicity, weight
loss, or motor deficits were observed in the course of the
study.
[0162] Mice were again tested in the MWM after the eighth dose (19
weeks of age +/-15 days) for six days. After behavioral testing,
dosing resumed until the animals were 5.5 months of age at the time
of harvest. Animals were housed and treated according to
Institutional Care and Animal Use Committee and National Institutes
of Health standards.
Morris Water Maze Protocol
[0163] Mice were tested in Morris water maze (MWM) on two
occasions, once prior to dosing, and once two months after dosing
began. The second round of water maze testing was performed in
another testing room. Mice were acclimated to the experimental room
for 2-3 days prior to testing. The mice were placed in a water maze
of 1.5 m diameter, with a 16 cm diameter platform placed 0.5-1.0 cm
under the surface of the water. The water was made opaque with
non-toxic white paint and the water temperature was regulated
between 22-25.degree. C.
[0164] The mice were given 4 trials per day of up to 90 seconds
each with a 10 second rest period on the platform after each trial.
If the mouse did not find the platform within 90 seconds, the mouse
was gently guided to the platform and allowed to remain there for
10 seconds. The testing room rooms each had large external cues to
allow the mice to orient as they learned the location of the
platform. Mice were placed under a heat lamp to prevent hypothermia
after each trial. The interval between trials ranged from 25 to 45
minutes. The mice were tracked using HVS Image Advanced Tracker
VP200 software (Buckingham, UK) and the total distance traveled
until reaching of the platform was determined.
[0165] Statistical analysis for acquisition path length from the
five trials involved a repeated measures analysis of variance. The
statistical model included "treatment" (0, 1 mpk, or 10 mpk of
epothilone D (Compound I)) as a between animal term, and the 5
trials as repeated measures on each animal. If the analysis
indicated a significant effect of treatment, or a
treatment-by-trial interaction, differences between the 1 mpk and
10 mpk groups were compared to the vehicle group using Dunnett's
test. The probe pathlengths in each quadrant, and number of
platform crossings in each quadrant, were analyzed using Dunnett's
test. In all cases, 1 mpk and 10 mpk groups were compared to the
vehicle group. All calculations were done in SAS, version 9.1,
under the Windows XP Professional operating system.
[0166] Acquisition training was performed for 5 consecutive days. A
Probe trial was performed 18 h after the last acquisition training
on day 6. During these 60 second trials, the platform was removed,
and the distance that the mouse spent in the target quadrant and
the number of crossings of a region where the platform was
previously located were measured. Swim speed was monitored for all
animals; drug treatment did not cause any changes in swim speed
consistent with the drug not affecting motor behavior. Float time
(swim speeds of <5 cm/sec) also did not vary between treatment
groups.
Tissue Harvesting
[0167] Mice were euthanized by cervical dislocation at 5.5 months
followed by decapitation. Brains were immediately removed and
divided down the midline into two hemispheres. The right hemisphere
was placed into 20 mL of 4% paraformaldehyde (prepared fresh on the
day of sacrifice) and stored overnight at 4.degree. C. The
following day, the brains were transferred to a tube containing 20
mL TBS (pH 7.4, 20 mM TRIS, 100 mM NaCl) and then stored at
4.degree. C. until processing. Right hemispheres were embedded in
paraffin, sectioned at 5 microns, and mounted on positively charged
glass slides. The slides were dried overnight in a 60.degree. C.
oven and stored at room temperature until stained. The left
hemispheres were frozen (within 2 minutes) on dry ice.
Gallyas Method
[0168] The Gallyas staining method was used to detect
silver-positive neurofibrillary tangles and dystrophic neurites.
Paraffin-embedded thin sections (5 microns) mounted on glass slides
were deparaffinized and rehydrated via serial incubation in xylene
(two times for 10 minutes each), 100% ethanol (two times for 10
minutes each), 95% MeOH 15% H.sub.2O.sub.2 (30 minutes), 95%
ethanol (two times for 5 minutes each), 80% ethanol (two times for
5 minutes each), 50% ethanol (two times for 5 minutes each), and
water (two times for 5 minutes each). The sections were then placed
into 5% periodic acid for 5 minutes, washed in dH.sub.2O two times
for 5 minutes each time, and placed in alkaline silver iodide
solution (containing 1% silver nitrate) for 1 minute.
[0169] The sections were washed in 0.5% acetic acid for 10 minutes,
placed in freshly prepared developer solution for 15 minutes, and
washed again in 0.5% acetic acid for 5 minutes. Following a rinse
in deionized water, the sections were placed in 0.1% gold chloride
for 5 minutes and rinsed again in deionized water. The sections
were incubated in 1% sodium thiosulphate (hypo) for 5 minutes and
then rinsed in tap water. Counterstain was performed in 0.1%
nuclear fast red for 2 minutes. The sections were then rinsed in
tap water, dehydrated in graded series of alcohol (95%, 100%, 100%)
for 2 minutes, and cleared in 3 changes of xylene, 10 dips each.
Finally, Cytoseal 60 mounting medium and cover slips were added to
the slides (Richard-Allan Scientific of Kalamazoo, Mich.).
Statistics was performed using the non-parametric Kruskal-Wallis
test, followed by Dunn's multiple comparison test using Graphpad
Prism 4. The same results were obtained with the parametric ANOVA
followed by Dunnett's post-hoc test.
Immunohistochemistry
[0170] Paraffin-embedded tin sections (5 microns) were
deparaffinized and rehydrated to water in 3 changes of xylene, two
changes of 100% ethanol, and 1 change of 95% ethanol, followed by
rinsing in water. Antigen retrieval was performed by steaming the
slides in 10 mM sodium citrate buffer, pH 6.0 for 30 minutes in a
Black and Decker Steamer (Model # HS900) and then cooled for 30
minutes. Endogenous peroxidase activity is removed by incubation in
0.6% hydrogen peroxide in 90% MeOH for 15 minutes. After washing in
TBS, slides are blocked in 10% normal goat serum in TBS for one
hour. This is followed by incubation of the AT8 phosphoTau antibody
(Pierce Biotechnology Inc., Rockford, Ill., Goedert et al., 1995)
diluted in the blocking solution overnight at 4.degree. C. After 3
washes in TBS, the slides are incubated with an anti-mouse IgG
antibody for 1 hour at room temperature. After washing in TBS, the
signal is detected using a Vectastain ABC Elite Kit (Vector Labs
Burlingame, Calif.) for 1 hour followed by detection using the
diaminobenzadine reagent from Vector labs. Nuclei were
counterstained blue with hematoxylin, followed by dipping slides 2
times in Scott's tap water substitute (Surgipath # 02900, Richmond,
Ill.) and then rinsing in tap water. The sections were then
dehydrated in graded series of alcohol (95%, 100%, 100%) then
cleared in 3 changes of xylene. Cover slips and Cytoseal 60
mounting medium were then added.
Stereology
[0171] Nissl stained slides were scanned and digitized using the
Aperio ScanScope (Aperio Technologies, Inc., Vista, Calif.). Images
of the entire brain section were captured at high resolution and
stored as files within Spectrum (Aperio software). To process
images, a region of 4,000.times.4,000 pixels including the entire
hippocampus was captured using the extract tool and saved as a JPEG
file for importing into Metamorph (Molecular Devices, Sunnyvale,
Calif.) for quantification of cell loss within the CA1 and CA3
regions of the hippocampus. A modified version of the single
section dissector method (Moller et al. 1990) was utilized to
quantify cell loss because of its suitability for thin,
paraffin-embedded tissue sections. To obtain relative numbers of
cells, every fifth section was collected as the paraffin-embedded
brains were cut sagitally between the Bregma and approximately 0.75
mm laterally. Three regions were drawn and counted per section
using Metamorph software. The same regions were used for every
image and 5 sections were counted per animal, 5 slides apart.
Statistics were performed using ANOVA followed by Dunnett's
post-hoc test.
Example 1
[0172] The design of the Tg4510 experiment with epothilone D
(Compound I) as described above is depicted in FIG. 1. In this
experiment, mice were tested at 2.5 months in the MWM and assigned
to one of three groups (N=12, 13, 16) such that the pre-treatment
performance of each group was determined to be similar. Starting at
2.5 months, mice were administered a weekly intraperitoneal (IP)
injection of either vehicle alone or vehicle with 1 mpk or 10 mpk
of epothilone D (Compound I). At 4.5 months, the mice were again
tested in the MWM to determine the effect of treatment on cognitive
performance. After 5.5 months, mice were euthanized and brains were
collected for subsequent analysis.
[0173] In tumor xenograft experiments, investigators typically
administer epothilone D (Compound I) intraperitoneally at 30 mpk
every other day for 5 days, yielding a cumulative dose of 150 mpk.
(Chou et al., 1998) Hence, treatment with 1 mpk epothilone D
(Compound I) for 12 weeks, as described herein, is considered to be
about 100-fold below the oncology dose, with the treatment at 10
mpk being about 10-fold below the typical oncology dose
administered in this type of experiment. When mice were dosed once
weekly intraperitoneally with 1 mpk and 10 mpk epothilone D
(Compound I) for 2 or 6 months, no histopathological abnormalities
were observed in multiple tissues, including liver, kidney, heart,
testes, adrenal gland, bone marrow, peripheral nerve, stomach, and
small and large intestines.
[0174] FIG. 2 shows the results of a MWM test of the Tg4510 mice at
2.5 months, prior to dosing with epothilone D (Compound I) or with
vehicle. There were no statistically significant differences
between the groups prior to dosing in acquisition or during probe
trials, which was the basis for separating animals into groups. In
other words, FIG. 2 operates as a control in showing the
pre-treatment performance of each group was similar.
[0175] The Tg4510 mice were then administered epothilone D
(Compound I)) once weekly intraperitoneally at 1 mpk, 10 mpk, and
with vehicle, and the MWM test was performed at 4.5 months,
following this weekly dosing over 12 weeks. The results which are
reported in FIG. 3, revealed that mice treated with 1 mpk
epothilone D (Compound I) were able to locate the hidden platform
in the MWM more quickly (i.e., in a statistically significant
manner (p<0.01)), than could mice that were treated with the
vehicle. The 10 mpk treatment group showed a trend toward
improvement as compared with the vehicle group. These findings show
that treatment of Tg4510 mice with epothilone D (Compound I) led to
statistically significant improved cognitive function relative to
vehicle treatment, and additionally, that the lower dose of 1 mpk
generated improved results as compared with the higher dose (10
mpk). Notably, the inventors herein further confirmed that the
exposure using this paradigm was dose dependent based on separate
experiments comparing 1 mpk and 10 mpk doses in mice. For this
reason, the reduced behavioral improvement in the 10 mpk group,
relative to the 1 mpk group, was not due to unanticipated, reduced
drug levels in the mpk treated animals.
Example 2
[0176] FIG. 4 shows probe data 18 h after 5 days of training in the
4.5 month-old Tg4510 mice dosed for 2 months with epothilone D
(Compound I) at 1 mpk, 10 mpk, and with vehicle. In FIG. 4, "TQ"
stands for target quadrant, "AR" stands for adjacent right, "AL"
stands for adjacent left, and "OP" stands for opposite quadrant.
Two measures of performance, namely % pathlength (A) and number of
platform crossings (B) in each quadrant, are indicated in FIG. 4. A
preference for the target quadrant indicates that the mouse
remembered the location where the platform was located during the
acquisition phase of the study. As can be seen from the data, the
vehicle-treated mice performed at chance with similar results for
each of TQ, AR, AL, and OP, for both the pathlength (A) and
platform crossing (B) measures, and they did not show a quadrant
preference. However, the mice treated with 1 mpk (Compound I)
showed statistically significant differences in both measures as
compared with the vehicle group in memory, e.g., in recalling that
the platform had been located at the TQ. Additionally, the 10 mpk
group showed significantly greater performance compared to the
vehicle group in the % pathlength measure (A) but not when using
the number of platform crossings measure (B).
Example 3
[0177] To determine the effect of epothilone D (Compound I) on
brain pathology, brain tissue was examined from a subset (N=5) of
the Tg4510 mice from the preceding experiment. Previous studies had
shown that Tg4510 mice lost about 60% of their neurons in the CA1
region of the hippocampus at 5.5 months (Santacruz et al. 2005).
Thus, the present inventors first examined the number of the
neurons in the CA1 region of the hippocampus, followed by
examination of the CA3 region. FIG. 5 depicts neuronal counts in
the CA1 and CA3 regions of hippocampus in the mice at 5.5 months
following treatment with vehicle, 1 mpk of epothilone D (Compound
I), and 10 mpk of epothilone D (Compound I).
[0178] Surprisingly, as can be seen in FIG. 5, the Tg4510 mice
treated with 1 mpk epothilone D (Compound I) had substantially more
CA1 neurons than vehicle-treated animals. In fact, the difference
between the mice treated with vehicle and the mice treated with 1
mpk of epothilone D (Compound I) shows that the 1 mpk of epothilone
D (Compound I) prevented neuronal loss with a statistically
significant difference from vehicle (p<0.01). The mice treated
with 10 mpk of epothilone D (Compound I) had CA1 neuronal levels
that were intermediate between the vehicle-treated mice and the
mice treated with 1 mpk of epothilone D (Compound I). These results
are consistent with and reinforce the findings from the behavioral
studies of Examples 1 and 2, i.e., showing that the 100-fold lower
dose (i.e., than the chemotherapeutic dosages administered in tumor
xenograft experiments) consistently produced significantly improved
results in treating tauopathy.
[0179] A similar trend was also observed for the total cell counts
of CA3 regions of the hippocampus, with significant differences
between the 1 mpk and non-transgenic mice compared to vehicle
treated mice. The elevation in cell count at the CA3 region in the
treated group was less pronounced than in the CA1 region where
there is more neurodegeneration at this age; however, these results
show an impact on underlying disease in multiple regions of the
brain.
Example 4
[0180] The effect of treatment on phosphorylated Tau staining in
the CA1 region was also examined. The AT8 antibody recognizes Tau
that is phosphorylated on both the 202 and 205 residues. This form
of hyperphosphorylated Tau is greatly enriched in AD and other
Tauopathy patient brains. (Goedert et al. 1995).
[0181] FIG. 6 shows AT8 phosphoTau staining of the Tg4510 mice
treated with vehicle, 1 mpk epothilone D (Compound I), and 10 mpk
epothilone D (Compound I) as described above. PhosphoTau staining
is indicated in dark black. Surprisingly, the mice treated with 1
mpk of epothilone D (Compound I), showed much less phosphoTau
staining, particularly in comparison to the vehicle-treated mice.
Mice treated with 10 mpk of epothilone D (Compound I) showed
intermediate levels of phosphoTau staining.
Example 5
[0182] The effect of treatment with epothilone D (Compound I) on
neurofibrillary tangle formation in the cortex was examined by
Gallyas silver staining. FIG. 7A shows Gallyas silver staining for
neurofibrillary tangles in the frontal cortex of the Tg4510 mice
treated with vehicle, 1 mpk of epothilone D (Compound I), and 10
mpk of epothilone D (Compound I) as described above. In FIG. 7A,
silver staining is in black (positive), and "NT" stands for
non-transgenic, demonstrating some non-specific staining associated
with blood vessels. As can be seen in FIG. 7A, the mice treated
with 1 mpk of epothilone D (Compound I) had much lower levels of
neurofibrillary tangles than did vehicle-treated mice; this is
quantitated for all animals in the study in FIG. 7B. A significant
impact on underlying disease in both cortex and hippocampous was
observed at the 1 mpk dose, with the 10 mpk dose again showing a
trend toward improvement.
[0183] As described in the preceding Examples, treatment of Tg4510
mice with epothilone D (Compound I) prevented cognitive decline and
improved cognitive function over time as compared with the
untreated Tg4510 mice. Furthermore, neuropathological tests as
measures of impact on underlying disease (i.e., cell count,
phosphoTau staining, and silver staining tests), demonstrate that
treatment with epothilone D prevents neuronal loss, reduces
accumulation of abnormal Tau, and prevents the formation of
neurofibrillary tangles at statistically significant levels as
compared with untreated Tg4510 mice. Thus, the inventors herein
believe they are the first to discover and demonstrate the
prevention of cognitive loss, Tau pathology, and neurodegeneration
upon treatment with a microtubule-stabilizing compound, namely,
epothilone D.
[0184] Additionally, the inventors herein have discovered that the
therapeutic effects achievable upon treatment with epothilone D is
likely non-linearly dose dependent. Specifically, consistent
dose-dependent results were repeatedly obtained in each of the
behavioral and neuropathological studies reported, wherein at the
lower dose (1 mpk) (about 100-fold less than the chemotherapeutic
dose in tumor xenograft experiments), a significantly-enhanced
beneficial effect was obtained in all measures as compared with the
vehicle, while the higher dose (10 mpk), showed a trend toward
effect with most measures and a statistically significant
difference over vehicle in one measure of the MWM probe test.
Example 6
Epothilone D Performance Compared with Other
Microtubule-Stabilizers in Bolus IV Experiments
[0185] In one group of experiments, ixabepilone (aza-epothilone B
analog), Compound II (BMS 310705, 21-amino epothilone F), and
epothilone D (Compound I) were evaluated and compared to paclitaxel
after bolus IV administration into the tail veins of nude mice at
dosages of 1 to 12 mpk with 3 mice/group. Each of the four
compounds were dosed at 5 ml/kg using 10% Cremophor, 10% ethanol,
and 80% water containing 5% dextrose. To determine the relative
brain penetrance of each compound, the plasma, brain, and liver
levels of the compounds were measured at various times after a
single dose using liquid chromatography with tandem mass
spectrometry (LC/MS/MS) after an organic phase extraction, as
reported in FIGS. 8A-8D and Table 1. Liver levels were not measured
in the paclitaxel treated mice.
[0186] FIG. 8A shows the concentration of Compound II in the
plasma, brain, and liver of mice following IV administration at 1
mpk at various times.
[0187] FIG. 8B shows the concentration of ixabepilone in the
plasma, brain, and liver of mice following IV administration at 12
mpk at various times.
[0188] FIG. 8C shows the concentration of paclitaxel in the plasma
and brain of mice following IV administration at 4 mpk at various
times.
[0189] FIG. 8D shows the concentration of epothilone D (Compound I)
in the plasma, brain, and liver of mice following IV administration
at 5 mpk at various times.
[0190] The data showed that Compound II and ixabepilone had modest
brain levels relative to peripheral tissue as measured by the ratio
of the brain-to-liver compound levels, particularly at later times
after the initial distribution and clearance of plasma drug. As
expected, paclitaxel brain levels were low. In particular,
paclitaxel brain levels did not exceed plasma drug levels for at
least 24 h after dosing. Unexpectedly, epothilone D (Compound I)
had the combined properties of remarkably better brain penetration
and selective retention than the compounds tested in this
experiment, as evidenced by high brain levels that exceeded liver
levels at 6 and 24 h after dosing. This demonstrates unexpected
retention of epothilone D (Compound I) in the target organ (brain)
relative to the periphery, including the plasma and tissues, most
notably the liver, which is a potential site of toxicity.
[0191] More specifically, Table 1 reports comparative brain
penetration data for four microtubule stabilizers--paclitaxel,
Compound II (BMS 310705), ixabepilone, and epothilone D--after
bolus IV dosing (varied mpk, as reported in the table) using nude
mice, which data is also reflected in FIGS. 8A-8D. The
brain-to-plasma ratio generally increases with time after dosing
for each compound due to the rapid loss from the plasma and
retention of the drug in the brain by binding to microtubules. The
brain-to-plasma ratio may then fall for compounds where there is
less retention in the brain, such as is observed for Compound II
showing a decrease between 6 and 24 h. Despite the change in
brain-to-plasma ratio with time, this ratio provides a measure of
the intrinsic brain penetration for a compound when data from short
times after dosing (e.g., between 20-60 minutes following dosing)
are compared. In the Tables herein, brain-to-plasma and
brain-to-liver ratios were calculated by first calculating the
ratios for individual animals, and then determining the mean of the
ratios; the Tables herein report the mean values thus obtained.
TABLE-US-00001 TABLE 1 Plasma Brain Brain-to- Brain-to- Dose Time
conc conc plasma Liver Compound (mpk) (hr) (nM) (nM) Ratio Ratio
Paclitaxel 4 1 447 47 0.10 NQ 6 43 16 0.37 NQ 24 12 15 1.25 NQ
Compound II 1 6 3 6.8 2.1 0.01 24 1 1.3 1.2 0.02 Ixabepilone 12
0.12 11,236 579 0.05 0.05 0.33 3057 495 0.16 0.09 1 390 284 0.73
0.07 2 171 360 2.1 0.11 6 53 371 7.0 0.30 24 8 236 30 1.2
Epothilone D 5 6 6 2794 470 149 24 1 2046 2046 1204
[0192] Looking at Table 1, paclitaxel is poorly brain penetrant as
evidenced by a brain-to-plasma ratio of 0.1 at 1 hour after dosing;
ixabepilone is more brain penetrant than paclitaxel with a
brain-to-plasma ratio of 0.73 at 1 hour after dosing (Table 1). At
times from 6-24 h after dosing, the brain-to-plasma ratio is a
reflection of both intrinsic brain penetration and retention
(half-life) in the brain. The data at 6 and 24 h after dosing of
epothilone D shows at least a 60-fold increase in brain-to-plasma
ratio above ixabepilone, the compound with the next highest
brain-to-plasma ratio in this group.
[0193] The brain-to-liver ratios not only provide a more singular
measure of brain retention and half life, but also selective
retention compared to peripheral tissues This is valuable because
the liver, chosen largely because it is well perfused and tends to
have higher levels than many other peripheral tissues, contains
microtubules where the compound can be retained, unlike the
non-cellular plasma. In contrast to the brain-to-plasma ratio where
the optimal measurement time is in the 20-60 minute range, it is
preferable to compare the brain-to-liver ratios at later times
after dosing (e.g., 24 h or more), when the plasma levels have
significantly decreased, thereby allowing a more accurate measure
of the drug that is specifically retained within brain and liver
cells. A comparison of the brain-to-liver ratios shows that
epothilone D is highly, selectively retained in the brain relative
to the liver. For instance, the 24 hour brain-to-liver ratio of
epothilone D is 1204, a remarkably, much higher ratio as compared
with the lower ratios for ixabepilone (1.2) and Compound II (0.02)
in the same set of experiments.
[0194] In a separate experiment, epothilone D plasma and brain
concentrations were evaluated for longer periods of time, i.e., up
to 168 h, following bolus W administration, using a similar
protocol as described above, but with middle-aged triple transgenic
mice (Oddo et al. 2003), in the hands of different scientists. The
results of this experiment are reported below in Table 2 and in
FIG. 10.
TABLE-US-00002 TABLE 2 (EPOTHLONE D ONLY) Brain Brain/Plasma
Brain/Liver Time (hr) Plasma conc (nM) conc (nM) Ratio Ratio 0.05
25,100 1127 0.04 0.42 0.17 2003 595 0.28 1.1 .33 549 529 0.86 0.38
1 325 422 1.2 0.42 3 70 468 6.1 0.30 6 43 141 3.3 0.24 16 0.8 210
265 NQ 24 0.9 82.7 89 NQ 96 <LLQ (0.6 nM) 25.2 NQ NQ 168 <LLQ
(0.6 nM) 16.3 NQ NQ
[0195] These data demonstrate the extended retention of epothilone
D in brain tissue to at least 168 h (7 days) after a single dose.
The absolute brain levels and ratios in Tables 1 and 2 for
epothilone D vary; it is important to note that the experiments
described in Tables 1 and 2 were separately performed at different
times by different scientists with different strains of mice. The
inventors have observed that small differences in IV injection time
can alter the exact exposure profile, particularly the maximal
plasma concentration, which will influence the brain concentration,
and further, that the IV injection time can differ between
scientists. For this reason, it is best to compare the results
within a single experiment. Despite this issue, the overall trends
and relative characteristics of the microtubule-stabilizers as
compared with each other are consistent, and these results show
that epothilone D is highly brain penetrant with substantially
improved brain penetration and retention as compared with Compound
II, ixabepilone, and paclitaxel. For example, even when engaging in
a comparison of data obtained from two separate experiments, the
brain-to-plasma ratio of epothilone D at 6 h after dosing was 2046
in Table and 89 in Table 2, still markedly greater than the ratios
at the same times for paclitaxel (0.37), and Compound II (2.1) in
Table 1. Because the liver levels in the study described in Table 2
fell below the lower level of quantitation (LLQ of 49 nM in this
study) at 24 hr, a brain-to-liver ratio was not quantifiable (NQ)
at this time.
[0196] WO 03/074053 A1 broadly discloses the use of certain
epothilones for the treatment of brain diseases. According to that
publication, plasma and brain levels for three epothilones (not
including epothilone D) were measured during the first 40 minutes
following bolus IV administration at 5 mpk. The brain and plasma
concentration data reported in WO 03/074053 A1 for what is
identified therein as compound 1:
4,8-dihydroxy-16-(1-methyl-2-(2-methyl-4-thiazolyl)-ethenyl)-1-oxa-7-(1-p-
ropyl)-5,5,9,13-tetramethyl-cyclohexadec-13-ene-2,6-dione, and
paclitaxel, are reproduced in Table 1 below. Data was reported in
WO 03/074053 according to the units, .mu.g/ml, and in minutes; this
data was converted to nM and is reported in Table 3 in nM and hr,
for purposes of comparison. This data (per Table 3) was not
independently confirmed by the inventors herein but rather, it is
reproduced based on the values presented in that publication (as
converted to hr and nM). Additionally, it is noted stereoisomerism
and/or a method of preparation are not reported for compound 1
within WO 03/074053, and a 13 E/Z mixture is referenced (see page
13, line 15).
TABLE-US-00003 TABLE 3 Time Plasma conc. Brain conc.
Brain-to-plasma Compound (hr) (nM) (nM) Ratio Compound III 0.17
1540 580 0.4 0.33 1150 1540 1.3 0.67 580 1150 2 Paclitaxel 0.17 940
<LLQ NQ 0.33 700 <LLQ NQ 0.67 230 <LLQ NQ
[0197] Because the data was reported to only 40 minutes, only brain
penetration can be assessed from this study by examining the
brain-to-plasma ratio. Paclitaxel is presumed to have poor brain
penetrance (consistent with the data in Table 1) because the brain
levels are below LLQ, although the level of detection was not
disclosed. Measures of brain retention which need to be measured at
least 24 h post-dosing, and selective brain penetration by
comparison with peripheral tissues were not discussed in WO
03/074053 A1.
Example 7
Epothilone D Performance Following Oral Administration
[0198] To further analyze epothilone D's performance in treating
tauopathies, the compound was evaluated in two experiments
involving administration to C57BL/6 mice at 10 mpk and 35 mpk by
oral gavage, respectively. Additionally, in these experiments, an
isomer of
4,8-dihydroxy-16-(1-methyl-2-(2-methyl-4-thiazolyl)-ethenyl)-1-oxa-7-(1-p-
ropyl)-5,5,9,13-tetramethyl-cyclohexadec-13-ene-2,6-dione (Compound
III herein), was also evaluated and a side-by-side comparison made
as between epothilone D and Compound III. Below, we first report
the experimental detail for preparation and isolation of Compound
III, and then the biological, in vivo data is described.
General Experimental Information:
[0199] In the following procedures, all temperatures are given in
degrees Celsius. .sup.1H-NMR spectra were run on a Bruker 500, 400,
or 300 MHz instrument and chemical shifts were reported in ppm
(.delta.) with reference to tetramethylsilane (.delta.=0.0). All
evaporations were carried out under reduced pressure. Unless
otherwise stated, LC/MS analyses were carried out on a Waters
instrument using a Phenomenex-Luna 3.0.times.50 mm S 10 reverse
phase column employing a flow rate of 4 mL/min using a 0.1% TFA in
MeOH/water gradient [0-100% in 3 min, with 4 min run time], and a
UV detector set at 220 nm or Phenomenex-Luna 3.0.times.50 mm 10 u
reverse phase column employing a flow rate of 5 mL/min using a 10
mM ammonium acetate acetonitrile/water gradient [5-95% in 3 min,
with 4 min run time] and a UV detector set at 220 nm. Unless
otherwise stated, purifications were done on 40-63 mesh silica gel
columns, or using a BIOTAGE.RTM. Horizon system, or using specified
HPLC equipment and conditions.
Step 1:
##STR00004##
[0201] To a solution of phosphonium salt A (prepared according to
Nicolaou et al., J. Am. Chem. Soc., 119:7974-7991 (1997); 40.5 g,
57.9 mmol) in 300 mL THE at 0.degree. was added sodium
bis(trimethylsilyl)amide (63.7 mL, 63.7 mmol), and the solution was
stirred for 5 min. A solution of
(6S)-6-methyl-7-(tetrahydro-2H-pyran-2-yloxy)heptan-2-one prepared
according to U.S. Pat. No. 7,326,798; 14.54 g, 63.7 mmol) in 50 mL
of THF was added rapidly, and the mixture was allowed to warn to RT
over 16 h. The reaction mixture was poured into saturated
NH.sub.4Cl, and extracted with EtOAc (300 mL). The organic layer
was washed with brine, dried over magnesium sulfate, filtered, and
evaporated in vacuo. The crude product was purified on a silica gel
column (EtOAc/hexane 0-10%) to yield 16 g (30.7 mmol, 53%) of
Synthesis Intermediate-1. .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.
ppm 6.90 (s, 1H), 6.43 (s, 1H). 5.20-5.05 (m, 1H), 4.6-4.5 (m, 1H),
4.15-4.00 (m, 1H), 3.9-3.8 (m, 1H), 3.6-3.3 (m, 2H), 3.25-3.05 (m,
1H), 2.70 (s, 3H), 2.35-2.15 (m, 2H), 2.05-1.90 (m, 5H), 1.90-1.75
(m, 1H), 1.75-1.65 (m, 3H), 1.65-1.45 (m, 6H), 1.45-1.25 (m, 3H),
1.15-1.00 (m, 1H), 1.00-0.80 (m, 12H), 0.05-0.05 (dd, 6H). MS
(LCMS) [M+H]=522.44, [M+Na]=544.42.
Step 2:
##STR00005##
[0203] To a solution of Synthesis Intermediate-1 (16 g, 30.7 mmol)
in 300 mL of ethanol at RT was added p-toluenesulfonic acid
monohydrate (5.83 g, 30.7 mmol). The mixture was stirred for 7 h,
poured into saturated NaHCO.sub.3 and extracted twice with
methylene chloride (300 mL). The combined organic layers were
washed with brine and dried over magnesium sulfate. After
filtration and removal of the solvent, the crude material was
purified using a BIOTAGE.RTM. system (EtOAc/hexane, 10-45%) to
yield 9.8 g (22.4 mmol, 73%) of Synthesis Intermediate-2. .sup.1H
NMR (500 MHz, CDCl.sub.3) .delta. ppm 6.92-6.90 (m, 1H), 6.45-6.40
(m, 1H), 5.15-5.00 (m, 1H), 4.10-4.00 (m, 1H), 3.50-3.30 (m, 2H),
2.69 (s, 3H), 2.35-2.15 (m, 2H), 2.1-1.9 (m, 5H), 1.75-1.50 (m,
5H), 1.50-1.25 (m, 3H), 1.10-0.75 (m, 13H), 0.05-0.05 (dd, 6H). MS
(LCMS) [M+H]=438.29, [M+Na]=460.24.
Step 3:
##STR00006##
[0205] To a solution of oxalyl chloride (2.94 mL, 33.6 mmol) in 100
mL of methylene chloride was added DMSO (4.88 mL, 68.8 mmol) slowly
at -78.degree.. After stirring 10 minutes, Synthesis Intermediate-2
(7 g, 15.99 mmol) in 100 mL methylene chloride was added and
stirring was continued for 30 min. TEA (11.14 mL, 80 mmol) was
added, and the mixture was allowed to warm to -10.degree.. After
saturated NaHCO.sub.3 was added, the reaction mixture was extracted
twice with methylene chloride (100 mL). The combined organic layers
were washed with brine, dried over Na.sub.2SO.sub.4, filtered and
evaporated to give the crude product as a yellow oil. Filtration
through a short SiO.sub.2 column, eluting with 15% EtOAc/hexane
solvent, and concentration in vacuo provided 7 g (16 mmol, 100%) of
Synthesis Intermediate-3 as a colorless oil. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. ppm 9.6-9.5 (d, 1H), 6.9 (m, 1H). 6.4 (m, 1H),
5.2-5.1 (m, 1H), 4.1-4.0 (m, 1H), 2.7 (s, 3H), 2.3-2.2 (m, 3H),
2.2-1.8 (m, 5H), 1.7-1.5 (m, 4H), 1.4-1.2 (m, 3H), 1.1-1.0 (m, 3H),
0.87 (s, 9H), 0.03 (s, 3H), 0.01 (s, 3H).
Step 4:
##STR00007##
[0207] The method of Klar et al. (Angew. Chem. Int. Ed.,
45:7942-7948 (2006)) was followed. To 200 mL of THF at -78.degree.
was added 70 mL of 0.5M freshly prepared LDA (35 mmol), followed by
8.48 g (35 mmol) of
(S)-2-(2,2-dimethyl-1,3-dioxan-4-yl)-2-methylheptan-3-one (Klar et
al., Synthesis, 2:301-305 (2005)). Stirring was continued for 30
min at -30.degree.. After cooling to -78.degree., a solution of
1.0M ZnCl.sub.2 (35.0 mL, 35 mmol) was added, and the resulting
solution was stirred for 20 min. A solution of Synthesis
Intermediate-3 (7 g, 16.06 mmol) in 50 mL of THF was added over 20
min. The mixture was stirred for an additional 8 h at -78.degree..
The mixture was poured into saturated NH.sub.4Cl and extracted
twice with EtOAc (300 mL). The organic layers were washed with
brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The
residue was purified using a SiO.sub.2 column (EtOAc/hexane, 0-10%)
to provide 7.5 g (11 mmol, 69%) of Synthesis Intermediate-4 as the
first eluting and major aldol isomer. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. ppm 6.90 (m, 1H), 6.43 (m, 1H), 5.15-5.05 (m,
1H), 4.15-400 (m, 2H), 4.00-3.80 (m, 2H), 3.50-3.40 (m, 1H),
3.30-3.20 (m, 1H), 2.85-2.75 (m 1H), 2.69 (s, 3H), 2.35-2.15 (m,
2H), 2.10-1.90 (m, 5H), 1.75-0.75 (m, 41H), 0.05-0.05 (d, 6H). MS
(LCMS) [M+H]=678.47, [M+Na]=700.44.
Step 5:
##STR00008##
[0209] To a solution of Synthesis Intermediate-4 (8 g, 11.8 mmol)
in 200 mL of methylene chloride at 0.degree. was added 2,6-lutidine
(6.87 mL, 59 mmol), followed by tert-butyldimethylsilyl
trifluoromethanesulfonate (8-13 mL, 35.4 mmol). The mixture was
allowed to warm to RT over 16 h, poured into saturated NaHCO.sub.3
and extracted with methylene chloride. The organic layers were
washed with brine, dried over Na.sub.2SO.sub.4 and the solvents
were removed in vacuo. The residue was purified on a SiO.sub.2
column (EtOAc/hexane, 5-10%) to yield Synthesis Intermediate-5 (7.8
g, 9.84 mmol, 83%). .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. ppm
6.90 (s, 1H), 6.44 (s, 1H), 5.15-5.05 (m, 1H), 4.25-4.20 (m, 1H),
4.10-4.00 (m, 1H), 4.00-3.90 (m, 1H), 3.90-3.75 (m, 1H), 3.75-3.70
(m, 1H), 3.10-3.00 (m, 1H), 2.70 (s, 3H), 2.35-2.15 (m, 2H),
2.00-1.85 (m, 5H), 1.70-0.75 (m, 50H), 0.10-0.05 (m, 12H). MS
(LCMS) [M+H]=792.48, [M+Na]=814.44.
Step 6:
##STR00009##
[0211] To a solution of Synthesis Intermediate-5 (6.8 g, 8.58 mmol)
in 100 mL of ethanol at RT was added p-toluenesulfonic acid
monohydrate (1.8 g, 9.44 mmol). After stirring for 6 h, saturated
NaHCO.sub.3 was added and the mixture was extracted with EtOAc. The
organic layers were washed with brine, dried over Na.sub.2SO.sub.4
and concentrated in vacuo. The reaction was repeated using 1 g of
Synthesis Intermediate-5. The combined crude products were purified
using a BIOTAGE.RTM. system (EtOAc/hexane, 10-40%) to yield
Synthesis Intermediate-6 (5 g, 6.65 mmol, 68%). .sup.1H NMR (500
MHz, CDCl.sub.3) .delta. ppm 6.9 (s, 1H), 6.44 (s, 1H), 5.15-5.05
(m, 1H), 4.15-4.00 (m, 1H), 4.00-3.90 (m, 1H), 3.90-3.80 (m, 1H),
3.75-3.65 (m, 1H), 3.65-3.55 (m, 1H), 3.10-2.90 (m, 2H), 2.69 (s,
3H), 2.25-2.15 (m, 2H), 2.00-0.75 (m, 50H), 0.10-0.05 (m, 12H). MS
(LCMS) [M+H]=752.42.
Step 7:
##STR00010##
[0213] To a solution of Synthesis Intermediate-6 (5 g, 6.65 mmol)
in 200 ml of methylene chloride at 0.degree. was added 2,6-lutidine
(7.7 mL, 66.5 mmol), followed by tert-butyldimethylsilyl
trifluoromethanesulfonate (9.16 mL, 39.9 mmol). The mixture was
allowed to warm to RT over 16 h, then poured into saturated
NaHCO.sub.3 and extracted with methylene chloride. The organic
solvent was evaporated and the crude mixture was filtered through a
layer of SiO.sub.2 with EtOAc/hexane (10-20%) to provide Synthesis
Intermediate-7 as an oil (6.9 g, 100%). .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. ppm 6.9 (s, 1H), 6.44 (s, 1H), 5.20-5.05 (m,
10H), 4.10-4.00 (m, 1H), 3.95-3.85 (m, 1H), 3.80-3.75 (m, 1H),
3.70-3.60 (m, 1H), 3.60-3.50 (m, 1H), 3.10-3.00 (m, 1H), 2.69 (s,
3H), 2.25-2.15 (m, 2H), 2.00-0.75 (m, 67H), 0.10-0.05 (m, 24H).
Step 8:
##STR00011##
[0215] To a solution of Synthesis Intermediate-7 (5 g, 5.1 mmol) in
80 mL of methylene chloride and 40 mL of MeOH at 0.degree. was
added (+/-)-camphor-10-sulfonic acid (1.18 g, 5.1 mmol). The
mixture was stirred for 6 h at 0.degree., poured into saturated
NaHCO.sub.3 and extracted with methylene chloride. The organic
layers were washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated to yield Synthesis Intermediate-8 as an oil (3.6 g,
4.15 mmol, 81%). .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. ppm 6.90
(s, 1H), 6.44 (s, 1H), 5.2-5.1 (m, 1H), 4.1-4.0 (m, 2H), 3.85-3.75
(m, 1H), 3.7-3.6 (m, 2H), 3.1-3.0 (m, 1H), 2.7 (s, 3H), 2.3-2.2 (m,
2H), 2.0-1.9 (m, 6H), 1.7-1.5 (m, 5H), 1.5-1.3 (m, 3H), 1.3-1.1 (m,
6H), 1.1-1.0 (m, 4H), 1.0-0.8 (m, 35H), 0.1-0.1 (m, 18H). MS (LCMS)
[M+H]=866.49.
Step 9:
##STR00012##
[0217] To a solution of oxalyl chloride (0.8 mL, 9.14 mmol) in 40
mL of methylene chloride at -78.degree. was added DMSO (1.24 mL,
17.5 mmol). After stirring for 10 min, a solution of Synthesis
Intermediate-8 (3.6 g, 4.15 mmol) in 40 mL of methylene chloride
was added. After 30 min, TEA (3.76 mL, 27.0 mmol) was added and the
reaction mixture was allowed to warm to 0.degree. over 2 h.
Saturated NaHCO.sub.3 was added and the mixture was extracted with
methylene chloride. The organic layers were washed with brine,
dried over Na.sub.2SO.sub.4 and concentrated in vacuo to provide
Synthesis Intermediate-9 as an oil (3.6 g, 4.16 mmol, 100%).
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. ppm 9.77 (m, 1H), 6.9 (s,
1H), 6.44 (s, 1H), 5.3 (s, 1H), 5.2-5.1 (m, 1H), 4.5-4.4 (m, 1H),
4.1-4.0 (m, 1H), 3.8-3.7 (m, 1H), 3.1-3.0 (m, 1H), 2.7 (s, 3H),
2.6-2.1 (m, 4H), 2.0-1.9 (m, 4H), 1.7-1.5 (m, 4H), 1.5-1.3 (m, 5H),
1.3-1.1 (m, 5H), 1.1-1.0 (m, 3H), 1.0-0.8 (m, 35H), 0.1-0.1 (m,
18H).
Step 10:
##STR00013##
[0219] To a solution of Synthesis Intermediate-9 (3.6 g, 4.16 mmol)
in 120 mL of t-BuOH and 85 mL of THF at 0.degree. was added 30 mL
of water, 2-methylbut-1-ene (18.5 g, 264 mmol), sodium
dihydrogenphosphate (1.6 g, 13.2 mmol) and sodium chlorite (2.98 g,
26.4 mmol). After stirring 2 h at 0.degree., the mixture was poured
into saturated Na.sub.2S.sub.2O.sub.3 solution (100 mL) and
extracted three times with EtOAc (300 mL). The combined organic
layers were washed with brine, dried over Na.sub.2SO.sub.4, and
concentrated in vacuo. The residue was purified using a
BIOTAGE.RTM. system (EtOAc/hexane, 10-50%) to give Synthesis
Intermediate-10 as a colorless oil (2.8 g, 3.18 mmol, 72%). .sup.1H
NMR (500 MHz, CDCl.sub.3) .delta. ppm 6.93 (s, 1H), 6.66 (s, 0.5H),
6.46 (s, 0.5H), 5.25-5.0 (m, 1H), 4.4-4.3 (m, 1H), 4.2-4.0 (m, 1H),
3.9-3.7 (m, 1H), 3.2-3.0 (m, 1H), 2.7 (d, 3H), 2.6-2.4 (m, 1H),
2.4-2.0 (m, 3H), 2.0-1.0 (m, 23H), 1.0-0.8 (m, 35H), 0.1-0.1 (m,
18H). MS (LCMS) [M+H]=881.53.
Step 11:
##STR00014##
[0221] To a solution of Synthesis Intermediate-10 (2.8 g, 3.18
mmol) in 5 mL of THF at RT was added TBAF (42 mL, 11.0M). The
mixture was stirred for 6 h, then poured into saturated NH.sub.4Cl,
and extracted twice with EtOAc (300 mL). The combined organic
layers were washed with HCl (1.0 N, 200 mL), saturated NaHCO.sub.3
and brine, and dried over Na.sub.2SO.sub.4 to give Synthesis
Intermediate-11 as a viscous oil (2.6 g, 3.3 mmol, 100%). .sup.1H
NMR (500 MHz, CDCl.sub.3) .delta. ppm 6.9 (s, 1H), 6.6-6.5 (m, 1H),
5.2-5.1 (m, 1H), 4.4-4.3 (m, 1H), 4.15-4.05 (m, 1H), 3.8-3.7 (m,
1H), 3.4-3.2 (m, 1H), 3.1-3.0 (m, 1H), 2.8-2.7 (m, 2H), 2.66 (d,
3H), 2.5-2.4 (m, 1H), 2.4-2.3 (m, 2H), 2.3-2.2 (m, 1H), 2.0-0.8 (m,
46H), 0.1-0.0 (m, 12H). MS (LCMS) [M+H)=766.3,
[M-H.sub.2O]=748.3.
Step 12:
##STR00015##
[0223] To a solution of Synthesis Intermediate-11 (2.6 g, 3.3 mmol)
in 27 mL of THF at RT was added TEA (2.36 mL, 17 mmol), followed by
2,4,6-trichlorobenzoyl chloride (3.31 g, 13.57 mmol). The reaction
mixture was stirred for 20 min, then diluted with 260 mL of
toluene. The toluene solution was added slowly to a stirred mixture
of DMAP (3.86 g, 31.6 mmol) in 1400 mL toluene over 4 h, after
which TLC indicated completion. HCl (4.0N, 12.5 mL) was added, and
the solvent was removed in vacuo. The residue was partially
purified using a BIOTAGE.RTM. system (EtOAc/hexane, 0-10%),
providing a mixture which contained a mono-silyl product and the
above mixture (13 E/Z isomers) including Compound III (1.1 g). MS
(LCMS) (520.2, 634.2). This material was subjected to deprotection
without further purification.
Step 13: Compound III
[0224] To a solution of the reaction mixture above (102 mg) at
-20.degree. was added 1 mL of TFA/CH.sub.2Cl.sub.2 (20% v/v). The
reaction mixture was transferred to an ice bath and stirred for 1
h. The solvent was removed in vacuo, adding small portions of
toluene then re-evaporating, which provided a white solid. The same
reaction was repeated with 125 mg of the partially purified
mixture. The two reaction residues were combined and purified on a
SiO.sub.2 column (EtOAc/hexane, 20-35%) which provided a white
solid (180 mg). The white solid was taken up in 5 mL of MeOH, and
purified by HPLC (Varian, Dynamax PDA-2 detector; Waters C18
column; A: water with 0.05% TFA; B: acetonitrile with 0.05% TFA,
isocratic). Two major peaks were collected (Peak 1, 73.4 mg, 38%;
and Peak 2, 41.8 mg, 22%).
[0225] Peak 1 was determined to be the 13-Z (1-oxa numbering)
isomer by observation of NOE between the C-14 olefinic proton and
the C-13 methyl. .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. ppm 7.14
(s, 1H), 6.75 (s, 1H), 5.15-5.05 (m, 1H), 5.05-5.00 (m, 1H),
4.45-4.35 (m, 1H), 3.65-3.55 (m, 1H), 3.35-3.25 (m, 1H), 2.92 (s,
3H), 2.55-2.45 (m, 2H), 2.35-2.25 (m, 2H), 2.25-2.15 (m, 1H), 2.00
(s, 3H), 1.90-1.80 (m, 1H), 1.80-1.65 (m, 5H), 1.60-1.45 (m, 2H),
1.45-1.30 (m, 5H), 1.25-1.15 (m, 3H), 1.05-0.95 (m, 6H), 0.90-0.85
(t, 3H). MS (LCMS) [M+H]=520.3.
[0226] Peak 2 was determined to be the 13-E isomer (Compound III
for Example 7 experiment, below) by absence of NOF between the C-14
olefinic proton and the C-13 methyl. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. ppm 7.03 (s, 1H), 6.65 (s, 1H), 5.3-5.2 (m,
1H), 5.05-5.00 (m, 1H), 4.45-4.40 (m, 1H), 3.65-3.60 (m, 1H),
3.4-3.3 (m, 1H), 2.77 (s, 3H), 2.6-2.3 (m, 4H), 2.15-2.05 (m, 1H),
1.99 (s, 3H), 1.95-1.85 (m, 1H), 1.8-1.7 (m, 2H), 1.57 (s, 3H),
1.50-1.35 (m, 4H), 1.29 (s, 3H), 1.25-1.10 (m, 3H), 1.0-0.9 (m,
6H), 0.9-0.8 (t, 3H). MS (LCMS) [M+H]=520.3.
Oral In-Vivo Studies with Epothilone D and Compound III
[0227] For each compound (epothilone D and Compound III, prepared
and described as per the experiment immediately above), three mice
per group (10 mpk and 35 mpk) were dosed at 10 ml/kg using 85%
PEG-400, 10% TPGS, and 5.0% ethanol. At various intervals after
dosing, the plasma, brain, and liver compound levels were measured
following tissue homogenization, extraction with acetonitrile, and
liquid chromatography with tandem mass spectrometry (LC/MS/MS).
Results from the studies are summarized in Tables 4 and 5 and also,
the results of the 35 mpk study are reported in FIG. 9.
Specifically, Table 4 reports the concentration of epothilone D
(Compound 1) and Compound III in the brain after oral
administration (10 mpk) up to 24 h after dosing (for Compound III,
to the extent still detectible given LLQ), and Table 5 reports and
FIG. 9 plots, the concentration of epothilone D (Compound 1) and
Compound III in the brain after oral administration (35 mpk) up to
5 to 24 h after dosing (again, for Compound III, to the extent
detectible). (A plot was not prepared for the Table 4 data as only
one brain concentration value was detectible for Compound III.) In
Tables 4 and 5, below, where the values were <LLQ, the LLQ value
is noted in the parenthetical.
TABLE-US-00004 TABLE 4 Plasma Brain Brain/ Time conc conc Plasma
Brain/Liver Compound (hr) (nM) (nM) Ratio Ratio Compound III 1 12.3
9.6 0.8 0.1 5 1.3 <LLQ NQ NQ (3.7 nM) 7 1.2 <LLQ NQ NQ (3.7
nM) 24 <LLQ <LLQ NQ NQ (0.2 nM) (3.7 nM) Epothilone D 1 15.1
10.6 0.7 0.4 3 3.9 7.0 1.8 1.3 4 2.5 9.9 4.0 3.4 8 1.4 6.4 4.6 1.8
13 0.1 6.3 47.3 5.1 24 0.2 9.1 44.5 8.0 48 <LLQ 5.4 NQ NQ (0.1
nM) 96 <LLQ 3.0 NQ NQ (0.1 nM)
TABLE-US-00005 TABLE 5 Plasma Brain Time conc conc Brain/Plasma
Brain/Liver Compound (hr) (nM) (nM) Ratio Ratio Compound III 1 47.7
67.7 2.3 0.4 5 3.0 4.6 1.4 NQ 24 0.7 <LLQ NQ NQ Epothilone D 1
52.7 61.3 1.1 0.5 5 3.6 76.9 25.2 11.3 24 <LLQ 118 NQ 18.7 (0.5
nM)
[0228] As can be seen, for Compound III, tissue levels from later
times (i.e., after 1 h or more) show that Compound III levels
decreased rapidly in brain tissue. Hence, Compound III has poor
brain retention as observed in the lack of measurable brain levels
at 24 h in both experiments. Oral dosing with epothilone D revealed
brain-to-plasma ratios of 0.7 and 1.1 at 1 hour, reflecting good
brain penetrance. Unlike Compound III, brain levels of epothilone D
were maintained for more than 24 h (Tables 2, 4 and 5, FIGS. 9-10).
The brain-to-liver ratio for oral dosing of epothilone D indicates
that epothilone D is selectively retained in the brain, consistent
with the data in Tables 1 and 2 after IV dosing. In particular, the
brain-to-liver ratio for epothilone D was 8 and 19 at 24 h after
dosing at 10 mpk and 35 mpk, respectively (Tables 4 and 5). These
values reflect remarkably high selective brain-to-liver retention
rates for epothilone D.
Example 8
Epothilone D Half-Life Data Following IV, Oral, and IP
Administration
[0229] To further evaluate epothilone D's properties for treating
tauopathies, the brain half-life of epothilone D was calculated
from multiple studies, and the results are reported in Table 6. To
calculate an accurate brain half-life for a long half-life
compound, measurements need to be taken for several half lives
after a single dose. From the study described in Table 2, where
brain concentrations were measured through 7 days after a single
dose, the brain half life of epothilone D (Compound I) after IV
dosing is 61 h (Table 6). The brain half life in mice after
multiple routes of administration and dosages averaged 46.0+/-7 h
(Table 6). Similarly, the brain half-life after IV dosing in rats
was 31 h (Table 6). In contrast, the brain half life of Compound
III was clearly significantly shorter than epothilone D, as
reflected in FIG. 9. As a further illustration of the epothilone D
brain half-life, FIG. 10 is provided which plots the results of a
study (data reported in Table 2, above), showing brain
concentration levels at time periods of up to 175 h post-dosing,
following a 5 mpk bolus IV administration.
TABLE-US-00006 TABLE 6 Species Route Dose (mpk) Half life (hours)
Mouse IV 5 61 Mouse Oral 10 46 Mouse IP 1 44 IP 10 41, 37, 45, 46
Rat IV 1 31
LIST OF CITATIONS
[0230] Altmann et al., "The Chemistry and Biology of
Epothilones--The Wheel Keeps Turning", Chem. Med. Chem., Vol. 2
(2007). [0231] Andorfer, C. et al., "Cell-cycle reentry and cell
death in transgenic mice expressing nonmutant human tau isoforms",
J. Neurosci., 25(22):5446-5454 (2005). [0232] Andrieux, A. et al.,
"Microtubule stabilizer ameliorates synaptic function and behavior
in a mouse model for schizophrenia", Biol. Psychiatry,
60(11):1224-1230 (2006). [0233] Avila, J. et al., "Role of tau
protein in both physiological and pathological conditions",
Physiol. Rev., 84(2):361-384 (2004). [0234] Ballatore, C. et al.,
"Paclitaxel C-10 carbamates: potential candidates for the treatment
of neurodegenerative tauopathies", Bioorg. Med. Chem. Lett.,
17(13):3642-3646 (2007). [0235] Bartosik-Psujek, H. et al., "The
CSF levels of total-tau and phosphotau in patients with
relapsing-remitting multiple sclerosis", J. Neural. Transm.,
113(3); 339-345 (2006). [0236] Berger, Z. et al., "Accumulation of
pathological tau species and memory loss in a conditional model of
tauopathy", J. Neurosci., 27(14):3650-3662 (2007). [0237]
Bergstralh, D. T. et al., "Microtubule stabilizing agents: their
molecular signaling consequences and the potential for enhancement
by drug combination", Cancer Treat. Rev., 32(3):166-179 (2006).
[0238] Bollag, D. M. et al., "Epothilones, a new class of
microtubule-stabilizing agents with a taxol-like mechanism of
action", Cancer Res., 55(11):2325-2333 (1995). [0239] Burke, W. J.
et al., "Taxol protects against calcium-mediated death of
differentiated rat pheochromocytoma cells", Life Sci.,
55(16):313-319 (1994). [0240] Butler, D. et al.,
"Microtubule-stabilizing agent prevents protein
accumulation-induced loss of synaptic markers", Eur. J. Pharmacol.,
562(1-2):20-27 (2007). [0241] Chou, T. C. et al., "Desoxyepothilone
B: An efficacious microtubule-targeted antitumor agent with a
promising in vivo profile relative to epothilone B", Proc. Natl.
Acad. Sci. USA, 95:9642-9647 (1998). [0242] Dickey, C. A. et al.,
"Current strategies for the treatment of Alzheimer's disease and
other tauopathies", Expert Opin. Ther. Targets, 10(5):665-676
(2006). [0243] Divinski, I. et al., "Peptide neuroprotection
through specific interaction with brain tubulin", J. Neurochem.,
98(3):973-984 (2006). [0244] Fellner, S. et al., "Transport of
paclitaxel (Taxol) across the blood-brain barrier in vitro and in
vivo", J. Chin. Invest., 110(9):1309-1318 (2002). [0245] Furukawa,
K. et al., "Taxol stabilizes [Ca2+]i and protects hippocampal
neurons against excitotoxicity", Brain Res., 689(1):141-146 (1995).
[0246] Gerth et al. "Epothilons A and B: Antifungal and Cytotoxic
Compounds from Sorangium cellulosum (Myxobacteria)," J.
Antibiotics, 49(6):560-563 (June 1996), [0247] Goedert, M. et al.,
"Monoclonal antibody AT8 recognises tau protein phosphorylated at
both serine 202 and threonine 205", Neurosci. Lett., 189:167-170
(1995). [0248] Gozes, I., "Activity-dependent neuroprotective
protein: from gene to drug candidate", Pharmacol. Ther.,
114(2):146-154 (2007). [0249] Gozes, I. et al., "Neurotrophic
effects of the peptide NAP: a novel neuroprotective drug
candidate", Curr. Alzheimer Res., 3(3):197-199 (2006). [0250]
Kolman, A., "Epothilone D (Kosan/Roche)", Curr. Opin. Investig.
Drugs, 5(6):657-667 (2004). [0251] Lace, G. L. et al., "A brief
history of tau: the evolving view of the microtubule-associated
protein tau in neurodegenerative diseases", Clin. Neuropathol.,
26(2):43-58 (2007). [0252] Lee et al., "Microtubule stabilizing
drugs for the treatment of Alzheimer's disease", Neurobiol. Aging,
15(Supp. 2):S87-S89 (1994). [0253] Magnani, E. et al., "Interaction
of tau protein with the dynactin complex", EMBO J.,
26(21):4546-4554 (2007). [0254] Matsuoka, Y. et al., "Intranasal
NAP administration reduces accumulation of amyloid peptide and tau
hyperphosphorylation in a transgenic mouse model of Alzheimer's
disease at early pathological stage", J. Mol. Neurosci.,
31(2):165-170 (2007). [0255] Matsuoka, Y. et al., "A neuronal
microtubule-interacting agent, NAPVSIPQ, reduces tau pathology and
enhances cognitive function in a mouse model of Alzheimer's
disease", J. Pharmacol. Exp. Ther., 325(1):146-153 (2008). [0256]
Michaelis, M. L., "Ongoing in vivo studies with cytoskeletal drugs
in tau transgenic mice", Curr. Alzheimer Res., 3(3):215-219 (2006).
[0257] Michaelis, M. L. et al., "{beta}-Amyloid-induced
neurodegeneration and protection by structurally diverse
microtubule-stabilizing agents", J. Pharmacol. Exp. Ther.,
312(2):659-668 (2005). [0258] Michaelis, M. L. et al.,
"Cytoskeletal integrity as a drug target", Curr. Alzheimer Res.,
2(2):227-229 (2005). [0259] Minderman, H. et al., "Broad-spectrum
modulation of ATP-binding cassette transport proteins by the taxane
derivatives ortataxel (IDN-5109, BAY 59-8862) and tRA96023", Cancer
Chemother. Pharmacol., 53(5):363-369 (2004). [0260] Moller, A. et
al., "Efficient estimation of cell volume and number using the
nucleator and the disector", J. Microsc., 159(Pt. 1):61-71 (1990).
[0261] Morsch, R. et al., "Neurons may live for decades with
neurofibrillary tangles", J. Neuropathol. Exp. Neurol.,
58(2):188-197 (1999). [0262] Oddo, S. et al., "Triple-transgenic
model of Alzheimer's disease with plaques and tangles:
intracellular Abeta and synaptic dysfunction", Neuron,
39(3):409-421 (2003). [0263] Postma, T. J. et al., "Peripheral
neuropathy due to biweekly paclitaxel, epirubicin and cisplatin in
patients with advanced ovarian cancer", J. Neuro-Oncology,
45:241-246 (1999). [0264] Preuss, U. et al., "The `jaws` model of
tau-microtubule interaction examined in CHO cells", J. Cell Sci.,
110(Pt. 6):789-800 (1997). [0265] Rice, A. et al., "Chemical
modification of paclitaxel (Taxol) reduces P-glycoprotein
interactions and increases permeation across the blood-brain
barrier in vitro and in situ", J. Med. Chem., 48(3):832-838 (2005).
[0266] Roberson, E. D. et al., "Reducing endogenous tau ameliorates
amyloid beta-induced deficits in an Alzheimer's disease mouse
model." Science, 316(5825):750-754 (2007). [0267] Roy, S. et al.,
"Axonal transport defects; a common theme in neurodegenerative
diseases", Acta Neuropathol, 109(1):5-13 (2005). [0268] Santacruz,
K. et al., "Tau suppression in a neurodegenerative mouse model
improves memory function", Science, 309(5733):476-481 (2005).
[0269] Spittaels, K. et al., "Glycogen synthase kinase-3beta
phosphorylates protein tau and rescues the axonopathy in the
central nervous system of human four-repeat tau transgenic mice",
J. Biol. Chem., 275(52):41340-41349 (2000). [0270] Sponne, I. et
al., "Apoptotic neuronal cell death induced by the non-fibrillar
amyloid-beta peptide proceeds through an early reactive oxygen
species-dependent cytoskeleton perturbation", J. Biol. Chem.,
278(5):3437-3445 (2003). [0271] Terwel, D. et al., "Amyloid
activates GSK-3beta to aggravate neuronal tauopathy in bigenic
mice", Am. J. Pathol., 172(3):786-798 (2008). [0272] Wagner, B. K
et al., "Large-scale chemical dissection of mitochondrial
function", Nat. Biotech., 26(3):343-351 (2008). [0273] Wostyn, P.
et al., "Alzheimer's disease-related changes in diseases
characterized by elevation of intracranial or intraocular
pressure", Clin. Neurol. Neurosurg., 10(2):101-109 (2008). [0274]
Zhang, B. et al., "Microtubule-binding drugs offset tau
sequestration by stabilizing microtubules and reversing fast axonal
transport deficits in a tauopathy model", Proc. Natl. Acad. Sci.
USA, 102(1):227-231 (2005).
* * * * *
References