Technical Comments

Comment on “ApoE-Directed Therapeutics Rapidly Clear β-Amyloid and Reverse Deficits in AD Mouse Models”

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Science  24 May 2013:
Vol. 340, Issue 6135, pp. 924
DOI: 10.1126/science.1233937


Cramer et al. (Reports, 23 March 2012, p. 1503; published online 9 February 2012) tested bexarotene as a potential β-amyloid–lowering drug for Alzheimer’s disease (AD). We were not able to reproduce the described effects in several animal models. Drug formulation appears very critical. Our data call for extreme caution when considering this compound for use in AD patients.

ApoE4 is the most important genetic risk factor for Alzheimer’s disease (AD) (1), and we therefore read with great interest the paper by Cramer et al. (2) demonstrating spectacular results of bexarotene on β-amyloid (Aβ) accumulation in the brain of mouse models for AD. Bexarotene is a retinoid X receptor (RXR) agonist and approved by the U.S. Food and Drug Administration (FDA) for treatment of cutaneous T cell lymphoma.

We decided to replicate the data by Cramer et al. (2) in mice and dogs before considering trials in humans. A single 100 mg per kg of weight (mg/kg) oral dose of bexarotene (Ontario Chemical, Inc., Canada) administered to wild-type male Swiss CD1 mice did not affect endogenous levels of soluble Aβx-37, Aβx-38, Aβx-40, and Aβx-42 in brain at different time points (Fig. 1A), despite the drug’s reaching high concentrations in brain and plasma (Fig. 1B). JNJ42601572, a known γ-secretase modulator, affected Aβ levels as expected (Fig. 1A). Similarly, 25 and 100 mg/kg oral bexarotene in beagle dogs did not affect Aβ levels in cerebrospinal fluid (CSF) (Fig. 1C). The drug reached high concentrations in plasma (Fig. 1D).

Fig. 1 Soluble brain and CSF Aβ levels are not significantly reduced by bexarotene in wild-type CD1 mice and beagle dogs.

(A) Soluble Aβx-37, Aβx-38, Aβx-40, and Aβx-42 levels (enzyme-linked immunosorbent assay) in whole-brain homogenate of wild-type CD1 mice treated with one dose of oral bexarotene (100 mg/kg; 20% Captisol; N = 6 mice) for 7, 24, 49, and 72 hours, and vehicle (N = 6 mice). Samples of two CD1 mice treated with the gamma-secretase modulator (GSM) JNJ42601572 for 4 hours (30 mg/kg; N = 2) were included as positive controls. Mean ± SEM calculated as percentage compared to vehicle control. (B) Bexarotene levels were measured in plasma and brain at different time points after administration. BQL, below quantification limit. (C) CSF Aβx-37, Aβx-38, Aβx-40, and Aβx-42 levels were sequentially measured at 4, 8, 24, 49, and 72 hours in beagle dogs (N = 4 dogs per group) treated with vehicle or a single dose of 25 or 100 mg/kg bexarotene in 20% 2-Hydroxypropyl-b-cyclodextrin (HP-b-CD) + Tween. Results shown are mean ± SEM calculated as percentages compared with baseline CSF levels before treatment. (D) Maximal bexarotene concentration, half life (T1/2), maximum concentration (Cmax), and area under the curve (AUC) levels measured in the plasma of treated beagle dogs.

We tested chronic oral bexarotene treatment (100 mg/kg per day for 19 days) in 10-month-old male hAPP/PS1 mice (3) and euthanized the mice 24 hours after the last dose. Plaque burden and plaque number in the right hemispheres (Fig. 2, A to D) and soluble Aβ1-40 in the left hemispheres was not altered (Fig. 2E). ABCA1 levels were significantly up-regulated (Fig. 2F), demonstrating target engagement. Changes in APP, APP-CTF fragments, or apoE levels were not observed. Thus, previously observed acute and chronic effects of bexarotene on brain Aβ levels (2) were not reproduced.

Fig. 2 Aβ levels and deposition are not reduced by chronic bexarotene treatment of hAPP/PS1 mice.

(A) Representative pictures of cortical sections of control (vehicle) and bexarotene-treated (100 mg/kg per day; 20% Captisol) 10-month-old hAPP/PS1 transgenic mice (N = 9 per group) stained with horseradish peroxidase (HRP)–labeled antibody 6E10 to Aβ and tyramide-fluorescein (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Mice were treated once a day for 19 days. (B) Higher magnification of a typical Aβ deposit in bexarotene-treated mice. (C) Plaque number counted in hippocampus by visual inspection blinded toward genotype. (D) 6E10-immunoreactive area quantified in four pictures per area of three sections per mouse, using Image J software. (E) Soluble Aβ40 levels measured with an Aβ40-specific alphaLISA in hippocampal and cortical brain extracts. (F) ABCA1 levels measured in cortical brain extracts by immunoblot. Band intensities given as fold intensity toward control. Each dot represents one mouse. ****P < 0.0001; unpaired Student’s t test with Welch’s correction. Social recognition memory was recovered in bexarotene-treated hAPP/PS1 as indicated by raw exploration data (G) and ratio data (H). Bexarotene-treated hAPP/PS1 displayed less exploration than wild-type control and control hAPP/PS1 mice [(G) and (I)] and spent significantly more time in total grooming (J) and maximum grooming sequence (K). In the passive avoidance (L), bexarotene-treated hAPP/PS1 mice showed significantly longer step-through latency during retention testing than control hAPP/PS1 mice. Body weight was monitored during the entire experiment (M and N). Wild-type control and control hAPP/PS1 mice showed no significant changes in weight during the experiment, whereas bexarotene-treated hAPP/PS1 mice displayed a sharp drop in body weight after 4 days of treatment (M). At the end of the experiment, bexarotene-treated hAPP/PS1 mice lost ~10% of their initial weight (N). Data are expressed as means ± SEMs. ###P < 0.001, significant within group exploration difference between S1 (familiar mouse) and S2 (novel mouse), Two-way repeated-measures analysis of variance (ANOVA) with Tukey test; *P < 0.05, **P < 0.01, ***P < 0.001, significant difference between groups, one-way-ANOVA with Tukey test.

We also tested cognitive status in chronically treated hAPP/PS1 mice. Although social recognition memory seemed improved after 14 days of treatment (Fig. 2, G and H), exploratory tendency was generally reduced in treated versus untreated hAPP/PS1 and control wild-type mice (Fig. 2I). Reanalyzing the recordings revealed a significant increase of grooming behavior in bexarotene-treated hAPP/PS1 mice (Fig. 2, J and K). Skin irritation and itching appear to be common reactions in patients taking Targretin (4). In addition, bexarotene-treated mice showed significant weight loss, increased irritability during handling and oral gavage, and difficulty breathing, which indicate severe adverse effects of the treatment (Fig. 2, M and N). Those might interfere with the execution of behavioral tests. The retention test of the passive avoidance task showed longer step-through latency in the bexarotene-treated hAPP/PS1 mice (Fig. 2L), but, again, adverse effects of the treatment confound interpretation. Thus, although we cannot exclude an effect of bexarotene on memory, the adverse effects of the drug make a definitive conclusion impossible.

Bexarotene is insoluble in water, and we therefore used Captisol (Cydex Pharmaceuticals), a widely used formulation for hydrophobic drugs, and 2-Hydroxypropyl-b-cyclodextrin (HP-b-CD/Tween), another β-cyclodextrine, for drug administration in mice and dogs, respectively. Cramer et al. wrote that they solubilized bexarotene in water (2), but we learned afterward that they actually used Targretin capsules. These capsules contain additional ingredients (5). Because Cramer et al. (2) used a different formulation (dimethyl sulfoxide and intraperitoneal injections) for their tissue bioavailability studies, we are not sure how to relate the different experiments published in (2) with regard to formulation and administration routes. The FDA filing mentions increased bexarotene uptake when the drug is taken together with high-fat food (5); thus, differences in fat content of mouse chow might further confound these studies. Our data demonstrate that we had good brain penetration of the drug and ABCA1 target engagement, but no effects on Aβ. We want to alert the field to this important issue. Given the toxicity of bexarotene, our study clearly strongly cautions against testing this drug in AD patients at this time.

References and Notes

  1. Acknowledgments: We would like to thank H. Borghys and D. Dhuyvetter for help with the dog studies. This work was supported by the Fund for Scientific Research Flanders, the KU Leuven, a Methusalem grant from the KU Leuven and the Flemish government, and the Foundation for Alzheimer Research (SAO/FRMA) to B.D.S. B.D.S. is the Arthur Bax and Anna Vanluffelen chair for Alzheimer’s disease. A.C.L. was financed by FP7-Alphaman. Bart De Strooper is a consultant for Janssen Pharmaceutica, Envivo Pharma, and Remynd NV.

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