Concomitant histone deacetylase and phosphodiesterase 5 inhibition synergistically prevents the disruption in synaptic plasticity and it reverses cognitive impairment in a mouse model of Alzheimer’s disease

Background Given the implication of histone acetylation in memory processes, histone deacetylase inhibitors (HDACIs) have been postulated as potential modulators of cognitive impairment in Alzheimer’s disease (AD). However, dose-dependent side effects have been described in patients with the currently available broad-spectrum HDACIs, explaining why their therapeutic potential has not been realized for chronic diseases. Here, by simultaneously targeting two independent enzyme activities, histone deacetylase (HDAC) and phosphodiesterase-5 (PDE5), we propose a novel mode of inhibitory action that might increase the therapeutic specificity of HDACIs. Results The combination of vorinostat, a pan-HDACI, and tadalafil, a PDE5 inhibitor, rescued the long-term potentiation impaired in slices from APP/PS1 mice. When administered in vivo, the combination of these drugs alleviated the cognitive deficits in AD mice, as well as the amyloid and tau pathology, and it reversed the reduced dendritic spine density on hippocampal neurons. Significantly, the combination of vorinostat and tadalafil was more effective than each drug alone, both against the symptoms and in terms of disease modification, and importantly, these effects persisted after a 4-week washout period. Conclusions The results highlight the pharmacological potential of a combination of molecules that inhibit HDAC and PDE5 as a therapeutic approach for AD treatment. Electronic supplementary material The online version of this article (doi:10.1186/s13148-015-0142-9) contains supplementary material, which is available to authorized users.


Pharmacokinetic study of vorinostat (SAHA), tadalafil and concomitant vorinostat and tadalafil administration in plasma samples
Vorinostat and tadalafil were measured in plasma samples using a Xevo-TQ MS triple quadropole mass spectrometer with an electrospray ionization (ESI) source and an Acquity UPLC (Waters, Manchester, UK).
Vorinostat solutions were prepared by dissolving the solid in DMSO and this solution was made up to a final volume by addition of 0.9% NaCl (1/9, v:v:v, DMSO/saline). A vorinostat dosage of 12.5 mg/Kg was administered as a single intraperitoneal injection.
Tadalafil solution was administered as Cialis and prepared in the same way, with a dosage of 1 mg/Kg administered orally. For the concomitant administration, vorinostat and tadalafil were administered in the same way (IP and orally respectively). Blood was collected at predetermined times over 24 h post injection (0.17, 0.25, 0.5, 1, 2, 4, 8 and 24 h) into tubes containing citrate and plasma was obtained via centrifugation (4˚ C, 13200 rpm, 5 min) and stored at -80˚ C until analysis.
Chromatographic separation was performed by gradient elution at 0.45 mL/min using a XSelect CSH C18 column (50 x 2.1 mm, 2.5 um particle size; Waters). The mobile phase consisted of A: water with 0.1% formic acid, B: methanol with 0.08% formic acid.
The autosampler temperature was set at 10˚ C and column temperature at 40˚ C. For detection and quantification, the electrospray ionization operated in the positive mode, and the collision gas used was ultra-pure argon at a flow rate of 0.15 mL min −1 . The analytes and internal standards were detected using multiple reaction monitoring (MRM).
Quantification was achieved by external calibration using matrix-matched standards.
Concentrations were calculated using a weighted least-squares linear regression (W = 1/x). Calibration standards were prepared by adding the appropriate volume of diluted solutions of the compound (made in a mixture of methanol and water, 50:50, v:v) to aliquots of 25 L of blank plasma. 2% formic acid in acetonitrile was added to precipitate the proteins. This protein precipitation solvent contained 10 nM of vorinostat-d5 and tadalafil-d3, the internal standards. The mixture was then agitated for 5 min and centrifuged at 13200 rpm for 10 min at 4˚ C. The resulting supernatants were transferred to a Ostro plate (Waters, Manchester, UK), designed to remove phospholipids. The resulting eluents were evaporated at 37˚ C under a stream of nitrogen. Residues were dissolved in 50 L of a mixture of A and B phases (50:50, v:v).
A 1.5 L aliquot of the resulting solution was injected onto the LC-MS/MS system for analysis. Frozen plasma samples were thawed at room temperature, vortexed thoroughly and subjected to the above described extraction procedure.
The pharmacokinetic parameters were obtained by fitting the blood concentration-time  Table 1).

Determination of brain to plasma concentration ratios of vorinostat and tadalafil after administration of vorinostat, tadalafil or the combination
Vorinostat was injected (12.5 mg/Kg, i.p.) to mice and 0.25 h after injection plasma and brain samples were collected. A second study was conducted: vorinostat was administered in the same way (12.5 mg/Kg, i.p) and tadalafil was administered orally as Cialis with a dosage of 1 mg/Kg; 0.33 h after injection plasma and brain samples were collected. Samples were frozen at 80˚ C until further process for analysis.
Chromatographic separation was performed by gradient elution at 0.4 mL/min using an Acquity UPLC BEH C18 column (100 x 2.1 mm, 1.7 µm particle size; Waters). The mobile phase consisted of A: water with 0.1% formic acid, B: methanol. The autosampler temperature was set at 10˚ C and column temperature at 45˚ C. Compound detection in plasma and brain samples was carried out as described previously for plasma samples for PK. Quantification and sample preparation in the case of brain samples was similar to that applied to plasma samples. Brain samples were thawed unassisted at room temperature and homogenized using a Branson 250 ultrasonic sonifier (Branson, Danbury, Connecticut, USA). When homogenized, 75 mg were weighted and extracted as described previously, with the difference that residues were dissolved in 100 L of a mixture of methanol and water with 0.1% formic acid (75:25, v:v).
BBB permeability was reported as logBB, where BB is the ratio of the brain to serum concentration. When only vorinostat was administered, concentration values led to a logBB of -1.17 which indicates that about 6.7% of the compound in the bloodstream crosses the BBB to reach the brain. According to previous studies performed in our laboratory, when only tadalafil was administered, concentration values led to a logBB of -0.89 (Garcia-Barroso et al. Neuropharmacology, 2013, 64, 114-123). In the case of concomitant vorinostat and tadalafil administration, concentration values obtained determined 20 minutes after administration led to a logBB value equal to -1.3 for vorinostat and -1.0 for tadalafil, which indicate that about 5.5% of vorinostat (SAHA) and 11.0% of tadalafil in plasma cross the BBB; thus, corresponding brain concentrations are 345.5 and 30 nmol/Kg respectively. (Supplementary Table 1).

Immunoblotting
In all cases, the membranes were blocked and incubated overnight with the following

Affymetrix microarray hybridization and data analysis
The hippocampi were dissected and RNA was extracted with TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. As a last step of the extraction procedure, the RNA was purified with the RNeasy Mini-kit (Qiagen, Hilden, Germany).
Before cDNA synthesis, RNA integrity from each sample was confirmed on Agilent Additional Table 1. Pharmacokinetic parameters and blood-brain barrier permeability for vorinostat and tadalafil. Pharmacokinetic parameters were estimated by fitting the experimental data to a non-compartmental model using Winnonlin software for pharmacokinetic analysis for vorinostat (12.5 mg/Kg, i.p. administration), tadalafil (1 mg/Kg, oral administration) and concomitant vorinostat and tadalafil administration (12.5 mg/Kg, i.p. administration; and 1 mg/Kg, oral administration, respectively). The parameters are: Area Under the Curve (AUC); half life of the product (T ½); clearance (Cl); volume of distribution (Vz); time to reach maximum plasma concentration (T max ); blood-brain barrier permeability (LogBB); brain to plasma concentration ratio (BP ratio).