Cholesterol acyltransferase (ACAT)

In cell tests, CP-113818 suppresses the formation of Aβ [1].
CP-113,818 is a fatty acid anilide derivative designed to mimic the acyl-CoA substrate of ACAT and a potent ACAT inhibitor in vitro and in cell culture [1].

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Previous cell-based studies indicate that ACAT inhibition reduces cholesteryl-ester production, which results in decreased Aβ generation (Puglielli et al., 2001). In those studies, two different ACAT inhibitors, CP-113818 and Dup128, lowered cholesteryl-ester production by up to 45% (Puglielli et al., 2001) [1].

CP-113818 (0-7.1 mg/kg/day) dramatically lowers amyloid pathology in Alzheimer's disease-modeling mice [1].
Here, we assessed the efficacy of CP-113818 in reducing AD-like pathology in the brains of transgenic mice expressing human APP(751) containing the London (V717I) and Swedish (K670M/N671L) mutations. Two months of treatment with CP-113,818 reduced the accumulation of amyloid plaques by 88%-99% and membrane/insoluble Abeta levels by 83%-96%, while also decreasing brain cholesteryl-esters by 86%. Additionally, soluble Abeta(42) was reduced by 34% in brain homogenates. Spatial learning was slightly improved and correlated with decreased Abeta levels. In nontransgenic littermates, CP-113,818 also reduced ectodomain shedding of endogenous APP in the brain. Our results suggest that ACAT inhibition may be effective in the prevention and treatment of AD by inhibiting generation of the Abeta peptide.[1]

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In order to determine the in vivo efficacy of CP-113818 in reducing mouse brain cholesteryl-ester levels, we initially treated nontransgenic animals with the ACAT inhibitor. Most ACAT inhibitors are poorly absorbed when administered orally and are quickly turned over in the blood or tissues (Marzetta et al., 1994). To minimize daily fluctuations in serum CP-113,818 levels in mice, we delivered the inhibitor via implantable slow-release biopolymer pellets. Pellets containing CP-113,818 were inserted surgically under dorsal skin to allow for continuous and controlled release of the active compound over an established period of time with effectively zero-order kinetics (Innovative Research of America). Eighteen nontransgenic mice at 3 months of age received 21 day-release biopolymer pellets containing 0, 0.2, 1.6, 3.2, 4.8, and 7.1 mg/kg/day of CP-113,818 (n = 3 per dose; Figure 1A). The highest dose that was used in these studies was far below the tolerated dose of 150 mg/kg/day for rats, even allowing for relatively low bioavailability in these animals (Marzetta et al., 1994). CP-113,818 (7.1 mg/kg/day) reduced total cholesterol levels by 29% (p < 0.007) in the serum, in the absence of any evident effect on food consumption and body weight (Figure 1A; data not shown). Hepatic free cholesterol and cholesteryl-esters were also decreased in a dose-dependent manner by up to 37% (p < 0.03) and 93% (p < 0.0004), respectively (Figure 1A). As expected, CP-113,818 had no effect on free cholesterol levels in the brain, since free cholesterol is largely immobilized in myelin membranes and is not likely perturbed by ACAT activity. Initial levels of cholesteryl-esters, 2.2 mg/g of brain tissue, were similar to reported values of approximately 3.5 mg/g of rat hippocampal tissue (Champagne et al., 2003) and corresponded to ∼8% of total brain cholesterol. We found that free cholesterol:cholesteryl-ester ratios in mouse brains were approximately 10:1 but were only 3:1 in enriched neuronal primary cultures largely devoid of myelin (Supplemental Figure S1 at http://www.neuron.org/cgi/content/full/44/2/227/DC1). CP-113,818 (7.1 mg/kg) decreased cholesteryl-ester levels in mouse brains by 86% (p < 0.0004; Figure 1A). Thus, these data indicate that 7.1 mg/kg/day of CP-113,818 is effective in markedly reducing cholesteryl-ester levels in the brains of nontransgenic mice.[1]

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To characterize the effect of CP-113818 treatment on AD-like pathology, we used hAPP mice that express human APP751 containing the London (V717I) and Swedish (K670M/N671L) mutations under the regulatory control of the murine Thy-1 promoter (mThy1-hAPP751) (Rockenstein et al., 2001). These animals develop detectable plaques in the neocortex and hippocampus at the ages of 4 and 6 months, respectively, and show memory deficits starting at 6 months of age. Given our prior observations that CP-113,818 reduced Aβ generation and not clearance, we used relatively young hAPP mice to minimize initial Aβ deposition. hAPP mice (n = 12) at 4.5 months of age were administered 60 day-release biopolymer pellets containing 13 mg of CP-113,818 (7.2 mg/kg/day), while age-matched animals were implanted with placebo pellets (n = 12). Recent reports have shown that female mice have higher levels of Aβ and amyloid pathology than age-matched males in a variety of AD transgenic mouse models Callahan et al. 2001, Lee et al. 2002, Wang et al. 2003 and that lovastatin may function in a gender-specific manner (Park et al., 2003). Therefore, we included similar numbers of male and female animals in both placebo and CP-113,818 groups to detect possible gender-specific effects of the inhibitor in our mouse model. Following 4 days of Morris water maze (MWM) tests, the animals were sacrificed at day 57 to ensure continued presence of the inhibitor. At 7.2 mg/kg/day of CP-113,818, there was no histologic evidence of toxicity found in adrenal cortical cells, a potential class-related effect of ACAT inhibitors (Figure 1B). Serum total cholesterol and liver free cholesterol/cholesteryl-esters were reduced to levels similar to those previously observed in nontransgenic animals (Figures 1A and 1C). It is noteworthy that liver cholesteryl-ester levels decreased by 87% (p < 0.0001) in hAPP mice treated for 2 months with CP-113,818 (Figure 1C). Taken together, these experiments demonstrated that 2 months of CP-113,818 treatment effectively reduced cholesteryl-ester levels of hAPP mice in the absence of adrenal toxicity. The tissue requirements for assessment of Aβ burden in the brains of these animals precluded direct examination of their brain cholesteryl-ester levels.[1]

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We next used a sandwich enzyme-linked immunosorbent assay (ELISA) to measure Aβ1-40 and Aβ1-42 levels in 70% formic acid extracts from brain homogenates (“insoluble” Aβ) and in the initial Tris-buffered saline (TBS) fraction of the same homogenates (soluble Aβ). Similar to plaque load, Aβ levels varied widely, as expected in transgenic mice specifically at early stages of pathology Johnson-Wood et al. 1997, Kawarabayashi et al. 2001. Treatment with CP-113818 decreased Aβ1-40 and Aβ1-42 in the formic acid fraction to almost undetectable concentrations. An overall 92% (p < 0.024) decrease in “insoluble” Aβ1-40 as well as 83% (p < 0.032) in “insoluble” Aβ1-42 was detected in all animals after treatment with CP-113,818, with no significant difference in the Aβ1-42/Aβ1-40 ratio (Figure 3A). Since brain extracts were obtained from entire hemispheres including cortices and hippocampi, the difference between male and female Aβ levels at this age was accentuated by the slowly developing amyloid pathology in male hippocampi (see Figure 2B). When analyzed according to gender, only female “insoluble” Aβ was sufficiently elevated to obtain a highly significant decrease by CP-113,818 treatment, with Aβ1-40 down by 96% (p < 0.014) and Aβ1-42 down by 90% (p < 0.014; Figure 3A). Importantly, soluble Aβ levels were also decreased in the initial TBS fraction, with Aβ1-42 showing a significant 34% reduction (p < 0.0014). This decrease was statistically significant in both genders. Soluble Aβ1−40 levels were close to the limit of detection, but a trend toward decrease was observed (20%) in all animals, significant only in males (Figure 3B). Thus, three different methods of detection, thioflavin S and Aβ stainings and Aβ ELISAs, independently showed that CP-113,818 treatment is highly effective in reducing amyloid pathology in hAPP mice. The decrease in soluble Aβ suggests that CP-113,818 may either reduce Aβ generation or accelerate its catabolism or clearance.[1]

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To analyze the effect of CP-113818 treatment on cognitive function of hAPP mice, we performed MWM spatial learning and memory tests on the animals. Since hAPP mice develop cognitive deficits starting at 6 months of age, the 6.5-month-old animals in our study were only expected to show slight deterioration due to Aβ accumulation, when compared to nontransgenic littermates. Consistently, length of swimming path and time latency required for finding the submerged platform revealed only a slight disturbance in spatial learning and memory in transgenic animals, compared to their littermates. Clear impairment of cognitive function due to Aβ accumulation was only observed in female transgenic mice. In these animals, CP-113,818 treatment reversed the observed impairment in spatial learning (p < 0.014; Figure 4). Compared to placebo, CP-113,818 treatment resulted in a significant improvement of learning between day 1 and day 3 (p < 0.016). CP-113,818 treatment did not affect spatial performance of nontransgenic female mice, consistent with lack of Aβ deposition in these animals. Only on day 4, CP-113,818-treated male nontransgenic animals performed better than the placebo-treated cohort regarding swimming path (p < 0.04; Supplemental Figure S3 at http://www.neuron.org/cgi/content/full/44/2/227/DC1) but not escape latency; however, the improvement between day 1 and day 3 or 4 was identical for both groups. All hAPP mice taken together showed a trend toward acquiring the task quickly and reaching a plateau of performance on day 3 when treated with CP-113,818 (p < 0.07; Figure 4). This could have been due to a ceiling effect owing to maximum swimming speed. Compared to placebo, the improvement between day 1 and day 3 due to CP-113,818 treatment was significant (p < 0.033). As expected, CP-113,818 did not induce significant changes in synaptic density as assessed by synaptophysin immunoreactivity, consistent with lack of differences between nontransgenic versus transgenic animals given their young age (Supplemental Figure S4). Although testing of more mice at an older age will be needed to further assess the effect of decreased amyloid pathology on spatial learning and memory, improved performance in a MWM test correlated well with the highly significant decrease of overall plaque load and Aβ in CP-113,818-treated female animals.[1]

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Behavioral performance assessing spatial navigation was evaluated for each animal three times per day for 4 days, between days 54 and 57 of treatment with CP-113818. In each Morris water maze (MWM) trial, swimming path (length in meters required to reach the platform) and escape latency (seconds to reach the platform) were measured. The top two panels show data collected from all animals in our study, regardless of gender. In addition to hAPP transgenic mice, we also used equal numbers of nontransgenic littermates untreated and treated with CP-113,818 in this study. The results show only a slight impairment in MWM trials in transgenic mice when compared to nontransgenics, thus leaving little room for improvement due to reduced amyloid accumulation by CP-113,818. The lower panels show exclusively results obtained with female mice. Correlating with 2-fold increased amyloid load in the female relative to the male cohort, only female transgenic animals exhibited a significant decline in performance when compared to nontransgenic littermates. Treatment with CP-113,818 improved this performance in the female cohort (p < 0.014 on day 3), so that treated hAPP transgenic animals performed similarly to nontransgenics in both the swimming path and escape latency tests. Data represent mean ± SEM; n = 3 trials per animal per day; statistical differences were calculated with the Mann-Whitney U test.[1]

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Our previous cell-based studies have shown that ACAT inhibition reduces Aβ generation and alters APP processing by all three secretases (Puglielli et al., 2001). To determine whether CP-113818 treatment modified brain APP processing in hAPP mice, we initially resolved formic acid-extracted brain proteins by SDS-PAGE to detect APP and its proteolytic derivatives. Western blot analysis showed that formic acid-extracted full-length APP and APP C-terminal fragments (CTFs) C83 and C99 were unchanged by CP-113,818. However, levels of the secreted cleavage products of both α- and β-secretases (sAPP) were reduced by treatment with the ACAT inhibitor, albeit not nearly as dramatically as Aβ (Supplemental Figure S5 at http://www.neuron.org/cgi/content/full/44/2/227/DC1). Inhibition of sAPP production in the absence of a change in CTFs was somewhat surprising and possibly an artifact caused by formic acid extraction. Because tissue from treated hAPP mice had been extracted with formic acid or fixed for histological studies, we looked at levels of APP-CTFs in total brain lysates from the nontransgenic littermates used in the MWM tests. Using nontransgenic animals for these experiments also afforded an assessment of the effects of CP-113,818 on endogenous APP processing. CP-113,818 reduced liver cholesterol/cholesteryl-esters and serum cholesterol of nontransgenic littermates similarly to the transgenic animals shown in Figure 1C (data not shown). Endogenous mouse APP-CTF levels in brain lysates from CP-113818-treated animals decreased by 44% when compared to placebo-treated mice (n = 8; p < 0.005; values normalized to APP holoprotein levels; Figures 5A and 5B). We could not resolve C99 and C83 from these samples due to smearing in the low molecular weight range because of high protein load (Figure 5A). However, we were able to specifically detect secreted APPα (sAPPα), the N-terminal product of α-secretase cleavage, in the soluble TBS fraction of the initial homogenate. sAPPα showed a trend toward decreasing (3.4%; p < 0.14 = not significant) in CP-113818-treated animals, while total sAPP, which also includes the β-secretase product sAPPβ, was significantly reduced by 19.1% (p < 0.038; Figures 5A and 5B). A 36% decrease (p < 0.013) in the ratio between total sAPP and sAPPα indicates that inhibition of α-secretase in ACAT inhibitor-treated mice is not as pronounced as that of other ectodomain-shedding proteases (e.g., BACE). We next asked whether γ-secretase component levels and γ activity were affected by CP-113,818 in the nontransgenic brain lysates. We did not detect significant changes in levels of PS1 N- and C-terminal fragments, nicastrin, and pen-2 (Figure 5C; 8 of 16 samples shown, as in all other figures). Nicastrin maturation was also unaffected by CP-113,818 treatment. To detect potential changes in γ- and α-secretase-like activities for substrates other than APP, we tested two γ-secretase substrates, Notch and N-cadherin De Strooper et al. 1999, Marambaud et al. 2003. Both proteins are also known to undergo α-secretase-like cleavages, mediated by TACE for Notch and a metalloprotease for N-cadherin Brou et al. 2000, Marambaud et al. 2003. Notch intracellular domain (NICD) represents the final γ-secretase cleavage product, and its levels are indicative of changes in both α-secretase-like and γ-secretase activities for Notch. NICD levels were statistically unchanged by CP-113,818 treatment, when the protein was immunoprecipitated from nontransgenic mouse brain lysates (Figure 5C). Similarly, levels of N-cadherin-CTF1, the product of metalloprotease cleavage and a substrate for γ-secretase, were unaffected by the ACAT inhibitor (Figure 5C). These data indicate that processing of at least two γ-secretase substrates was not altered by CP-113818 and that α-secretase-like cleavages of these substrates were also unaffected. BACE protein levels were also unchanged in brain lysates of CP-113,818-treated animals (Figure 5D). One mechanism that would explain our data is that ACAT inhibition may result in an interaction between APP and an unknown protein, blocking access of the three secretases to APP instead of direct inhibition of each secretase. Expression of ApoE, an essential protein in both brain lipid metabolism and Aβ deposition/clearance, was also unchanged by ACAT inhibition (Figure 5D). In an effort to exclude potential direct effects of CP-113,818 on β- and γ-secretase cleavages of APP in the absence of changes in cholesterol metabolism, we performed in vitro β- and γ-secretase assays employing purified recombinant BACE1 and membrane fractions isolated from Chinese hamster ovary (CHO) cells harboring endogenous γ-secretase, respectively. Both BACE1 and γ-secretase activities were unaffected by increasing amounts of CP-113,818 in the reaction mixture (Figures 5E and 5F). Finally, we tested whether CP-113818 directly modulates in vitro aggregation of Aβ40 promoted by zinc or Aβ42 Bush et al. 1994, Jarrett et al. 1993. CP-113,818 failed to directly inhibit aggregation of Aβ40 in vitro, when promoted by zinc (p < 0.56) or Aβ42 (p < 0.96) (Figure 5G). In the absence of zinc or Aβ42, Aβ40 alone did not significantly aggregate over 4 days (p < 0.671). Together, these data show that APP-CTFs, total sAPP, and, to a lesser extent, sAPPα levels are decreased in the brains of CP-113818-treated nontransgenic mice in the absence of any apparent change in the levels or processing of all control proteins tested. Although these data, together with decreased soluble Aβ in hAPP mice, argue strongly that the ACAT inhibitor reduces Aβ generation, they do not exclude additional beneficial effects of altered cholesterol metabolism on Aβ aggregation, catabolism, and/or clearance [1].

In Vitro Fluorometric BACE1 Activity Assay [1]
A fluorometric β-secretase activity kit was used for detecting BACE1 activity in vitro. Reaction was done according to the manufacturer's instructions with slight modifications. Briefly, 1 μg of purified recombinant human BACE1 (R&D systems) was resuspended in 10 μl of 1× cell extraction buffer and incubated with EDANS/DABCYL-REEVNLDAEFKR substrate peptide in the absence or presence of indicated amount of CP-113818 for 2 hr. Released EDANS fluorescence was measured by a fluorescent plate reader. As a negative control, a specific β-secretase inhibitor, GL-189, was added to one of the samples with the indicated concentration.

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In Vitro Generation of APP Intracellular Domain [1]
Membrane preparation and in vitro generation of APP intracellular domain (AICD) were performed as described Gu et al. 2001, Kim et al. 2002. P2 membrane fractions were prepared from CHO cells overexpressing wild-type APP and resuspended in Buffer H (20 mM HEPES, 150 mM NaCl, 10% glycerol, 5 mM EDTA [pH 7.4]) with protease inhibitors. In vitro cleavage experiments were performed by incubating the membrane fractions at 0°C or 37°C for 1 hr with the indicated amounts of CP-113818. As a negative control, a specific γ-secretase inhibitor, L-685,458, was added to one of the samples.

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Turbidity Assay [1]
Aβ40 aggregation was monitored according to the method of Jarrett et al. (1993). Briefly, Aβ40 (100 μg/ml) was incubated at 37°C in TBS in a 384-well plate (60 μl/well). Well turbidity was monitored for 4 days by following absorbance at 400 nm. Aβ40 aggregation was promoted by the addition of “aged” (preincubated for 2 weeks at room temperature) Aβ42 (Aβ42/Aβ40 = 1/10) (Jarrett et al., 1993) or Zn/histine buffer (50 μM Zn2+ in 350 μM histine) (Bush et al., 1994). Absorbance of test wells was blanked on signal from samples without Aβ40. For some experiments, incubation buffers included CP-113818 (10 μM).

Animal/Disease Models: C57BL/6, hAPP (human amyloid precursor protein) transgenic mice [1]
Doses: 0, 0.2, 1.6, 3.2, 4.8 and 7.1 mg/kg/day
Route of Administration: via implantable sustained release Biopolymer particles, 21 days for non-transgenic mice or hAPP mice 60 days
Experimental Results: Serum total cholesterol levels were diminished by 29%, liver free cholesterol and cholesterol esters were also diminished by 37% and 93%, respectively, in a dose-dependent manner . Non-transgenic mice. Effectively lowered cholesterol ester levels, diminished plaque number, and diminished amyloid burden in hAPP mice in a sex-independent manner without adrenal toxicity. Levels of “insoluble” and soluble Aβ1-40 and Aβ1-42 were diminished in the brains of hAPP transgenic mice. Normal spatial learning and memory in female hAPP mice were restored in the Morris water maze test. Reduces processing of endogenous APP, but not Notch or N-cadherin, and does not directly inhibit β- and γ-secretase activity or Aβ aggregation in non-transgenic littermates.

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Animals and Drug Treatment [1]
hAPP transgenic mice overexpress human APP751 with the London (V717I) and Swedish (K670M/N671L) mutations under the regulatory control of the neuron-specific murine (m)Thy-1 promoter (mThy-1-hAPP751; heterozygous with respect to the transgene, on a C57BL/6 F3 background) (Rockenstein et al., 2001). The hAPP colony was sustained by crossing transgenic APP751 with C57BL/6. Corresponding littermates were used for control studies. All mice were housed according to standard animal care protocols, fed ad libitum with standard chow diet, and maintained in a pathogen-free environment in single ventilated cages at JSW-Research. The transgenic status of each animal was confirmed by real-time PCR of tail snips using specific primers and the appropriate hybridization probe. A modified Irvine test was regularly performed prior to the experiment to assess the neurological status of the animals; those showing disturbances were excluded from the experiments. Mice were then randomly assigned to different treatment groups and individually coded. Investigators performing behavioral testing, biochemical analyses, and histomorphological evaluations of the brains were blinded in terms of group allocation of the mice. CP-113818, a potent inhibitor of both ACAT1 and ACAT2 (Chang et al., 2000), was used. Only the test compound, but not carrier biopolymers and substances in the pellets, was released into the bloodstream. Pellets were generated to provide either 21 days or 60 days of continuous drug delivery. For implantation of pellets, animals were anesthetized with isoflurane. Then, sterile pellets containing either CP-113,818 or placebo were implanted subcutaneously along the anterolateral aspect of the left shoulder with a special precision trocar in accordance with the supplier's instructions. There was no evident need for additional hemostasis, and no signs of infection, discomfort, or distress were observed in association with the implantation and treatment.

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Tissue Sampling [1]
Animals were sacrificed the day after the last MWM training, on day 57 of treatment. Blood was obtained by cardiac puncture, and total serum was used for cholesterol determination. Transcardiac perfusion with 4C PBS was performed, followed by dissection. Brains were removed, divided along the sagittal plane, and then either frozen in liquid nitrogen or immersion fixed with 4% paraformaldehyde for histologic evaluation. Liver was frozen in liquid nitrogen, and adrenal glands were fixed in 4% paraformaldehyde followed by standard paraffin embedding and sectioning. Hematoxylin and eosin-stained 6–8 μm sections were analyzed for evidence of CP-113818 toxicity.

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Brain Plaque Load and Synaptophysin Analyses [1]
For histomorphological analyses, 8 to 15 histological sections (10 μm) were prepared from one hemisphere of 12 animals (six placebo, six CP-113818; three male, three female per group). Number and surface area of amyloid plaques were evaluated in both cortex and hippocampus. 6E10 (1:5000; monoclonal) as primary antibody and Cy3 fluorescent goat anti-mouse IgG as secondary antibody were used for Aβ staining. Sections of the same brains were also used for synaptophysin staining (1:5000; monoclonal). Synaptophysin immunoreactive spots were counted by light microscopy in the hippocampal CA1 region. The estimation of immunohistochemical reactions was done by computer-assisted quantification.

[1]. The ACAT inhibitor CP-113,818 markedly reduces amyloid pathology in a mouse model of Alzheimer's disease. Neuron. 2004 Oct 14;44(2):227-38.

Our previous cell-based studies indicate that ACAT inhibition reduces cholesteryl-ester production, which results in decreased Aβ generation (Puglielli et al., 2001). In those studies, two different ACAT inhibitors, CP-113818 and Dup128, lowered cholesteryl-ester production by up to 45% (Puglielli et al., 2001). Our current study reveals a more robust decrease in mouse brain cholesteryl-ester levels, perhaps attributable to the slow-release biopolymer carrier, which ensured continuous delivery of the inhibitor. While it is difficult to predict final concentrations of CP-113818 in the brain, crossing of the blood-brain barrier is suggested by the structure of this ACAT inhibitor, a small fatty acid analog, and the marked reduction in brain cholesteryl-esters relative to serum cholesterol, by 86% and 29%, respectively. A previous study had reported 100-fold fluctuations of CP-113818 blood levels in monkeys after oral administration of the compound (Marzetta et al., 1994). Our own attempts to quantitate liver or brain CP-113,818 have failed, consistent with fast turnover or clearance of the inhibitor (Marzetta et al., 1994). Although neuronal Aβ generation could be affected by serum cholesterol, as suggested by a number of studies correlating serum cholesterol with brain Aβ levels (Puglielli et al., 2003), it is unlikely that a 29% decrease in serum cholesterol would reduce brain Aβ to the levels found in the current study. A more likely explanation is that altered intracellular cholesterol metabolism, evidenced by the 86% decrease in brain cholesteryl-esters, is responsible for the observed reduction in Aβ accumulation in CP-113,818-treated animals.

In transgenic mice, soluble Aβ42 only decreased by 34%, while “insoluble” Aβ decreased by 83%–99%. These data may be explained by the complex metabolism of these peptides in the brain, such that the net amount of soluble Aβ is the result of not only its rate of generation alone, but also its rates of deposition, degradation, and clearance (Saido, 1998). On the other hand, reduction in Aβ generation alone could largely account for the surprisingly robust effect of CP-113818 on “insoluble” Aβ and amyloid plaques in mouse brains. Small changes in Aβ generation, exemplified by a 50% increase in APP gene dosage in Down's syndrome patients, or catabolism may disproportionately accelerate Aβ pathology and age of onset of AD (Saido, 1998). Similarly, FAD mutations in the presenilin genes only increase Aβ42 generation by approximately 1.2- to 3-fold and yet cause early-onset forms of the disease that can strike from the third decade onward Borchelt et al. 1996, Citron et al. 1997, Duff et al. 1996, Holcomb et al. 1998. Although it is tempting to attribute decreased Aβ pathology in CP-113,818-treated mice to reduced Aβ production, we cannot exclude the possibility that altered brain cholesterol metabolism may also affect Aβ aggregation, catabolism, and/or clearance in addition to APP processing. Further studies will be necessary to evaluate these aspects of our findings.

Direct comparison between the efficacy of CP-113818 and statins (or statin-like compounds) is nearly impossible due to differences in animal models, methods of drug administration, and length of treatment employed in each study. Refolo et al. (2001) and Petanceska et al. (2002) employed a double transgenic mouse line, PSAPP, in which Aβ deposition begins at 12 weeks of age (Holcomb et al., 1998). The cholesterol-lowering drug BM15.766 (250 mg/kg/day) (Refolo et al., 2001) or 30 mg/kg/day atorvastatin (Petanceska et al., 2002) was administered orally starting at 8 weeks of age and ending after 5 and 8 weeks, respectively. BM15.766 reduced amyloid load by 53%, formic acid-extracted Aβ40 by 58%, and Aβ42 by 48% (Refolo et al., 2001). Atorvastatin lowered amyloid load by ∼62% (2.7-fold), with formic acid-extracted Aβ40 and Aβ42 decreasing by ∼60 (2.5-fold) and ∼50% (2-fold), respectively (Petanceska et al., 2002). In another study conducted in guinea pigs, ∼250 mg/kg/day of orally administered simvastatin reduced endogenous detergent-extracted total Aβ by ∼45% in 3 weeks of treatment (Fassbender et al., 2001). Interestingly, one report has shown that lovastatin treatment of 12-month-old Tg2576 mice (expressing FAD mutant APP) for 3 weeks did not affect amyloid load or brain Aβ levels in males while increasing Aβ pathology in female animals (Park et al., 2003). It is not clear whether this contrasting result reflects differences in genetic backgrounds or the short treatment time beginning at a late age, after development of large Aβ deposits. Female Tg2576 mice have been reported to develop amyloid pathology before males, perhaps due to higher synaptic zinc content in female brains by the age of 12 months Callahan et al. 2001, Lee et al. 2002. Although difficult to compare in efficacy to CP-113818, it is clear that statins or statin-like drugs strongly reduce Aβ pathology in different animal models when administered prior to Aβ deposition. It can be speculated that the influence of ACAT inhibition on APP metabolism is additive to effects resulting from changes in total cholesterol levels. Thus, in principle statins and ACAT inhibitors together could exhibit synergy in positively impacting AD pathology in patients affected by the disease.

CP-113818 has never been tested in clinical trials, while CI-1011 (avasimibe), a well-studied ACAT inhibitor produced by Pfizer, previously entered phase III trials for vascular disease and atherosclerosis. Avasimibe is considered safe for human use, with a good therapeutic window. Our results suggest that slow-release biopolymer administration of ACAT inhibitors may be considered as a potential strategy for the treatment and prevention of Alzheimer's disease, alone or in combination with statins. [1]

C24H42N2OS3

470.7981

470.245

C, 61.23; H, 8.99; N, 5.95; O, 3.40; S, 20.43

135025-12-6

164373

White to off-white solid powder

8.8

1

5

17

30

439

1

CCCCCCCCC(C(=O)NC1=C(C=C(N=C1SC)C)SC)SCCCCCC

XAMYAYIMCKELIP-FQEVSTJZSA-N

InChI=1S/C24H42N2OS3/c1-6-8-10-12-13-14-16-20(30-17-15-11-9-7-2)23(27)26-22-21(28-4)18-19(3)25-24(22)29-5/h18,20H,6-17H2,1-5H3,(H,26,27)/t20-/m0/s1

(2S)-2-hexylsulfanyl-N-[6-methyl-2,4-bis(methylsulfanyl)pyridin-3-yl]decanamide

135025-12-6; cp-113818; CP 113818; N-(2,4-Bis(methylthio)-6-methylpyridin-3-yl)-2-(hexylthio)decanoic acid amide; CP 113,818; CP-113,818; Decanamide, 2-(hexylthio)-N-(6-(methyl-2,4-bis(methylthio)-3-pyridinyl)-, (S)-; (2S)-2-hexylsulfanyl-N-[6-methyl-2,4-bis(methylsulfanyl)pyridin-3-yl]decanamide;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)