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Bexarotene Alzheimers drug scam
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With
only the poorest of studies showing a modest reduction in the progression of
Alzheimer’s disease, the use of drugs as a treatment is a major scam not in the
patients’ interest. Three articles on
tobacco science for bexarotene. The rate
of the disease has increase 71% in the 55 to 74 age group from 1999 to 2010,
placing the U.S. as having the second highest rate behind Finland. Recent report
for the UK, carried in the BMJ,
placing them as a contender for first place--see. Five medications are
currently used to treat the cognitive problems of AD: four areacetylcholinesterase inhibitors (tacrine, rivastigmine, galantamine and donepezil) and the other (memantine) is an NMDA receptor antagonist.[164] The benefit from their use
is small.[165][166] No medication has been clearly
shown
to delay or halt the progression of the disease” Wiki,
and this positive finding is based on very biased studies. Bexarotene (brand
name: Targretin) is
an antineoplastic (anti-cancer) agent approved by the U.S.
Food
and Drug Administration (FDA) (in late 1999) and the European
Medicines Agency (EMA) (early 2001) for use as a treatment for cutaneous
T cell lymphoma (CTCL).[1] It
is a third-generation retinoid…. In 2012 and 2013, bexarotene
researchers reported that bexarotene reduced amyloid plaque and improved mental functioning in a small sample
of mice
engineered to exhibit Alzheimer's-like
symptoms and the findings were promoted in the media.[16][17] In
2013, several research groups reported on their attempts to reproduce these
findings. The results were mixed: none of the studies found a reduction in
amyloid plaques, but several of the studies found that soluble forms of
β-amyloid were reduced.[18][19][20][21][22] https://en.wikipedia.org/wiki/Bexarotene
As
is the norm |
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http://www.sciencemag.org/content/340/6135/924.6.full Science 24 May
2013: Vol. 340 no. 6135 p. 924 DOI: 10.1126/science.1235505
TECHNICAL
COMMENTS Comment
on “ApoE-Directed Therapeutics Rapidly Clear β-Amyloid and Reverse Deficits in
AD Mouse Models” Cramer et al. (Reports,
23 March 2012, p. 1503; published online 9 February 2012) reported that
bexarotene rapidly reduces β-amyloid (Aβ) levels and plaque burden in two mouse
models of Aβ deposition in Alzheimer’s disease (AD). We now report that,
although bexarotene reduces soluble Aβ40 levels in
one of the mouse models, the drug has no impact on plaque burden in three
strains that exhibit Aβ amyloidosis. Alzheimer’s disease
(AD), the major
cause of adult-onset dementia, is characterized by progressive memory loss and
severe cognitive decline that are associated with cerebral deposition of
β-amyloid (Aβ) peptides. Familial, autosomal dominant AD (FAD) is caused by
expression of mutant variants of Aβ precursor proteins (APP) and presenilins
(PS), and expression of these mutant genes in mice leads to cerebral deposition
of Aβ peptides (1). Cramer et al. (2) reported
that bexarotene
(Targretin), a retinoid X receptor (RXR) agonist approved by the U.S. Food and
Drug Administration (FDA), rapidly reduces Aβ levels in the interstitial fluid,
reduces amyloid plaque burden, and rescues behavioral deficits in transgenic
mouse models of Aβ amyloidosis. For example, 6-month-old mice expressing
FAD-linked APPswe and PS1∆E9 polypeptides [APP/PS1 mice (3)] that received
daily oral doses of Targretin
exhibited a reduction of Aβ plaques by ~75% within 7 days and significantly
reduced levels of soluble and insoluble Aβ peptides that accompanied increases
in brain levels of apoE, ABCA1, ABCG1, encoded by RXR-target genes, and
elevated levels of highly lipidated HDL. Similarly, Cramer et al. (2) reported
that 8-month-old mice
expressing APPswe and PS1L166P polypeptides [APPPS1-21 mice (4)], treated
with Targretin for 20
days showed lowered Aβ levels and amyloid plaques. Finally, Cramer et al. (2) reported the presence of Aβ-laden
microglia in mice treated with Targretin for 3 days, although a similar
analysis was not reported for vehicle (H2O)–treated
animals. In view of the important
implications
of these findings for the development of novel AD therapeutics, we have
attempted to replicate these findings. We noted that Cramer et al. (2) used a limited
number of mice (N = 5 for APP/PS1 mice), and of mixed gender, the latter a confound
in the author’s interpretation of results, because female APP/PS1 mice exhibit
accelerated amyloid deposition, elevated Aβ deposition, and elevated amyloid
burden, particularly at 6 months of age, compared with their male counterparts
(5). We performed studies on
three mouse
models of Aβ amyloidogenesis. In the first, cohorts of 6-month-old male APP/PS1
mice (3) [the same
strain used by Cramer et al. (2)], were treated
orally with 100 mg
per kg of weight (mg/kg) of Targretin or vehicle (6.6% dimethyl sulfoxide; 4%
ethanol; 89.6% sunflower oil) for seven consecutive days. Fixed hemibrains were
sectioned and stained with 3D6, an Aβ N-terminal–specific antibody (6), and bound
primary antibodies were
visualized using fluorescently labeled secondary antibodies; representative
images from individual animals treated with vehicle or Targretin are shown in Fig. 1, A and D, respectively.
Colabeling studies with 3D6 and
microglia-specific Iba1 antibodies revealed that Aβ was abundantly present
within microglia that surrounded plaques in both vehicle- (Fig. 1, B and C) and Targretin-treated
mice (Fig. 1, E and F). Amyloid
plaque area (Fig. 1G) and plaque
numbers (Fig. 1H), in the cortex
and hippocampus of seven animals per group were
quantified and revealed no significant differences between groups in these
measures. Pieces of tissue from corresponding hemibrains were homogenized, then
subjected to sandwich enzyme-linked immunosorbent assay (ELISA) analysis to
assess levels of tris-buffered saline –soluble (s) and formic acid–extractable
insoluble (ins) Aβ peptides. We observed a medium effect size [Cohen’s d (7)] for soluble and insoluble Aβ40 and soluble Aβ42, and a small
effect size for insoluble Aβ42 between vehicle and Targretin
treatment groups (Fig. 1I). Finally,
Western blot analysis using antibodies to ABCA1
revealed an ~2.32-fold increase in ABCA1 levels in the brains of
Targretin-treated animals compared with the vehicle cohort (Fig. 1, J and K) (*P < 0.001), thus establishing
engagement of an RXR target in
the brain. Effects of
Targretin on amyloid
plaque burden and Aβ levels in APP/PS1 mice. Representative images of hemibrain
sections from 6-month-old APP/PS1 mice treated with vehicle (A)
or 100 mg/kg Targretin (D) that were immunostained with 3D6 antibody.
Scale bar, 500 μm. (B) and (E)
Representative confocal z-stack projection images of Iba1+
microglia (green) surrounding 3D6+ amyloid plaque deposits (red) in sections
from vehicle-treated (B) or Targretin-treated
(E)
animals. (C and F)
Overlay and orthogonal views (XY/XZ
sectional) of images shown in (B) and (E), respectively. Arrows point to the
intracellular 3D6 immunoreactivity in microglia of both sample groups. These
results suggest that microglial phagocytosis of Aβ is not enhanced in
Targretin-treated animals. Scale bar, 25 μm. Sections were counterstained with
4′,6-diamidino-2-phenylindole (DAPI) (blue). (G and H)
Histograms show the amyloid plaque
area fraction and the total number of plaques in cortex (CX) or hippocampus
(HP), respectively. No significance was observed in the plaque area fraction (P = 0.122 in cortex; P = 0.234 in hippocampus)
or total plaque numbers (P = 0.174 in cortex; P = 0.183 in
hippocampus) between the treatment groups. Total
areas examined in vehicle- versus Targretin-treated groups are comparable
(194338.4 ± 5592.096 μm2 for vehicle
group versus 192936 ± 5131.078 μm2 for Targretin group, in
cortex with P = 0.437; 87837.57 ± 3391.043
μm2 for vehicle
group versus 89502.69 ± 5258.606 μm2 for Targretin group, in hippocampus with P = 0.412).
Six sections representing every sixth or twelfth
coronal serial section spanning the proximal to distal end of hippocampus,
including the cortical regions, were examined to quantify amyloid area fraction
and plaque numbers using Image J Foci dMacro and Analyze particles plug-in by
applying uniform image threshold. (I)
Histograms show fold changes in soluble (s) Aβ40 (*P = 0.279; Cohen’s d: 0.605), formic acid-extractable
insoluble (ins) Aβ40 (#P = 0.359; Cohen’s d: 0.509), soluble Aβ42 ($P = 0.247; Cohen’s d: 0.64), and insoluble Aβ42 (%P = 0.676; Cohen’s d: 0.22) after normalization against
total protein content. (J) Representative
Western blots of
detergent-soluble total protein lysates (60 μg loaded in each lane) from
hemibrains of vehicle- or Targretin-treated animals probed with antibodies to
ABCA1 or β-III tubulin. (K) Quantification of ABCA1 band intensity
normalized against β-III tubulin levels (*P < 0.001). Data in (G), (H), (I), and (K) represent mean ±
SEM; N = 7 mice per group). We then treated cohorts
of 3- to
4-month-old male 5XFAD mice [line 6799 (8)] that exhibit
robust amyloid
deposition by 2 to 2.5 months of age with Targretin or vehicle, as above. The
brains of 11 male mice per treatment group were analyzed; fig. 2, A and B, shows representative
3D6-labeled sections from individual
animals treated with vehicle or Targretin, respectively. Amyloid plaque area (Fig. 2C) and plaque
numbers (Fig. 2D) in the cortex and hippocampus were quantified and revealed no
significant differences between vehicle or Targretin groups in these measures.
Sandwich ELISA assays revealed that in contrast to a small effect size for
insoluble Aβ40 and insoluble
Aβ42, we observed a
very large effect size for soluble Aβ40 and a medium effect size for soluble Aβ42 (Fig. 2E). The significant reduction in levels of soluble Aβ40 (*P = 0.008; Cohen’s d: 1.25) in brains of
Targretin-treated mice might be a reflection of the reported rapid reduction of
interstitial fluid (ISF) Aβ by this compound (2). Finally,
we established target
engagement by demonstrating a ~2.46-fold elevation in ABCA1 levels in the
brains of Targretin-treated animals compared with the vehicle cohort (Fig. 2, F and G) (*P = 0.004). Effects of
Targretin on amyloid
plaque burden and Aβ levels in 5X FAD and APPPS1-21 mice. (A to G)
Representative images of hemibrain sections from 3- to 4-month old 5XFAD mice
treated with vehicle (A) or 100 mg/kg Targretin (B) for 7 days that were
immunostained with 3D6 antibody. Scale bar, 500 μm. (C and D) Histograms
show the amyloid plaque area fraction and the
total number of plaques in cortex (CX) or hippocampus (HP), respectively. Total
area examined in vehicle- versus Targretin-treated groups are comparable
(117841.3 ± 3303.082 μm2 for vehicle
group versus 107086.3 ± 6802.094 μm2 for Targretin group, in cortex with P = 0.17; 67039.59
± 4030.787 μm2 for vehicle
group versus 70535.3 ± 2843.477 μm2 for Targretin group, in hippocampus with P = 0.486).
(E) Histogram shows fold changes in soluble (s) Aβ40 (*P = 0.008; Cohen’s d: 1.25), insoluble (ins) Aβ40 (#P = 0.292; Cohen’s d: 0.46), soluble Aβ42 ($P = 0.102; Cohen’s d: 0.73), and insoluble Aβ42 (%P = 0.363; Cohen’s d: 0.396) levels after normalization
against total protein content. (F) Representative Western blots of
detergent-soluble total protein lysates (60 μg per lane) from hemibrains of
vehicle- or Targretin-treated 5XFAD animals using antibodies to ABCA1 or β-III
tubulin. (G) Quantification of ABCA1 band intensity normalized against β-III
tubulin levels (*P = 0.004). Data in (C), (D), (E) and (G) represent mean ± SEM; N = 11 mice per group. (H to M) Amyloid plaque burden is not
reduced in 9-month-old APPPS1-21 mice after 26 days of 100 mg/kg per day
Targretin treatment. Representative images of hemibrain sections from
9-month-old APPPS1-21 mice treated with vehicle (H2O)
(H) or 100 mg/kg Targretin (I) for 26 days that were immunostained with
Aβ-specific CN3 antibody. Scale bar, 500 μm. (J) Histogram shows the percentage
of amyloid plaque load assessed in neocortex on random sets of every 12th
systematically sampled 40-μm-thick sections, analyzed by area fraction
technique estimated with the aid of Stereologer software (mean ± SEM; N = 4 mice per group). (K) Histogram shows fold changes
in soluble
(s) Aβ40 (*P = 0.269; Cohen’s d: 0.32), insoluble (ins) Aβ40 (#P = 0.49; Cohen’s d: 0.013), soluble Aβ42 ($P = 0.182; Cohen’s d: 0.325), and insoluble Aβ42 (%P = 0.495; Cohen’s d: 0.008) levels in vehicle- versus
Targretin-treated APPPS1-21 mice, respectively. (L) Representative Western
blots of detergent-soluble total protein lysates (60 μg per lane) from
hemibrains of vehicle- or Targretin-treated APPPS1-21 mice using antibodies to
ABCA1 or β-III tubulin. (M) Quantification of ABCA1 band intensity normalized
against β-III tubulin levels (*P = 0.04). Data in (K) and (M) represent mean ± SEM; N = 3 mice per group). Concerned by the fact that
the
vehicle used for the aforementioned studies differed from that used by Cramer et al. (2), we orally
treated cohorts of 9-month-old
male APPPS1-21 mice (4) with 100 mg/kg Targretin suspended
in H2O or with H2O as the vehicle.
This strain was also used by Cramer and colleagues (2)
and exhibits robust Aβ deposition
by 8 months of age. Sections were stained with a polyclonal antibody to Aβ [CN3
(9)] and Fig. 2, H and I, are representative Aβ-labeled sections from individual animals
treated with H2O or Targretin, respectively.
Analysis of amyloid plaque load in four male mice per group failed to reveal a
significant difference between H2O or
Targretin-treated animals (Fig. 2J),
and Meso Scale Discovery (MSD) analysis using the human 6E10
Aβ triplex assay revealed a medium effect size for soluble Aβ species and a
small effect size for insoluble Aβ species (Fig. 2K). Finally,
we demonstrated an ~1.5-fold elevation in ABCA1
levels in the brains of Targretin-treated animals compared with the vehicle
cohort (Fig. 2, L and M (*P = 0.04), thus establishing engagement of an RXR target in the
brain. In summary, although our
studies have
not examined the rapid effects of bexarotene on ISF Aβ levels or behavior, we
have failed to support earlier findings by Cramer et al. (2) that Targretin
is efficacious in
reducing plaque burden in transgenic mouse models of cerebral Aβ deposition. Received
for publication 22
January 2013. Accepted
for publication 26
April 2013. , Mutant genes in familial
Alzheimer’s disease and transgenic models. Annu. Rev. Neurosci. 21, 479 (1998). ., ApoE-directed therapeutics
rapidly clear β-amyloid and reverse deficits in AD mouse models. Science 335, 1503 (2012). 1.
., Aβ42-driven cerebral
amyloidosis in transgenic mice reveals early and robust pathology. EMBO Rep. 7, 940 (2006). , Gender differences in the
amount and deposition of amyloidbeta in APPswe and PS1 double transgenic mice. Neurobiol. Dis. 14, 318 (2003). ., Multiple effects of aspartate
mutant presenilin 1 on the processing and trafficking of amyloid precursor
protein. J. Biol. Chem. 276, 43343 (2001). 2.
, Combining effect size estimates
in meta-analysis with repeated measures and independent-groups designs. Psychol. Methods 7, 105 (2002). 3.
., Intraneuronal beta-amyloid
aggregates, neurodegeneration, and neuron loss in transgenic mice with five
familial Alzheimer’s disease mutations: Potential factors in amyloid plaque
formation. J. Neurosci. 26, 10129 (2006). 4.
., Peripherally applied
Aβ-containing inoculates induce cerebral β-amyloidosis.Science 330, 980 (2010). 2.
Acknowledgments: This work was supported by the Cure Alzheimer’s
Fund (S.S.S.,
R.V., D.M.H., and R.E.T.). The authors thank V. Bindokas for expert assistance
with confocal microscopy at the Digital Integrated Microscopy Facility of the
University of Chicago, N. H. Varvel (University of Tubingen) for assistance in
the analysis of the APPPS1-21 mice, X. Zhang for technical assistance, and Elan
Pharmaceuticals, Inc., for providing Aβ-specific 3D6 antibody. D.M.H. was the
head of the Neuroscience Therapeutic area scientific advisory group for Pfizer
from September 2011 to September 2012. S.S.S is a paid consultant of Eisai
Research Labs Inc. but is not a shareholder in any company that is a maker or
owner of a FDA-regulated drug or device. TECHNICAL COMMENTSResponse to Comments on
“ApoE-Directed Therapeutics Rapidly Clear β-Amyloid and Reverse Deficits in AD
Mouse Models” Science 24 May 2013: 924. TECHNICAL COMMENTSComment on “ApoE-Directed
Therapeutics Rapidly Clear β-Amyloid and Reverse Deficits in AD Mouse Models” Science 24 May 2013: 924. TECHNICAL COMMENTSComment on “ApoE-Directed
Therapeutics Rapidly Clear β-Amyloid and Reverse Deficits in AD Mouse Models” Science 24 May 2013: 924. TECHNICAL COMMENTSComment on “ApoE-Directed
Therapeutics Rapidly Clear β-Amyloid and Reverse Deficits in AD Mouse Models” Science 24 May 2013: 924. REPORTApoE-Directed Therapeutics Rapidly Clear β-Amyloid and Reverse
Deficits in AD Mouse Models Science 23 March 2012: 1503-1506.Published online 9 February 2012 Response to Comments on "ApoE-Directed Therapeutics Rapidly
Clear {beta}-Amyloid and Reverse Deficits in AD Mouse Models"Science 24 May 2013: 924.
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