Systemic clearance and brain distribution of carbazole-based cyanine compounds as Alzheimer’s disease drug candidates

SLM and SLOH, two analogues of carbazole-based cyanine compounds, have been shown to inhibit β-amyloid peptide aggregation in vitro and in Alzheimer’s disease model mice, which could be potentially developed into drugs for disease treatment. To pave the way for further pharmacokinetics-pharmacodynamics study, we set to investigate these compounds’ systemic clearance pathways and their brain exposure. We found that they generally exhibited relatively low plasma clearance which comprised of hepatic clearance and biliary clearance. Phase I oxidative metabolites for SLM and for SLOH upon microsomes incubation were identified, and the metabolism by CYP3A4 were found to be the major (>70%) hepatic clearance pathway, while the efflux by P-gp and BCRP located in the canalicular membrane of hepatocytes led to high biliary clearance. The permeation of SLM and SLOH through the brain endothelium was affected by the efflux transporters (P-gp and BCRP) and influx transporter (OATP2B1). The unbound interstitial fluid to plasma ratio (K puu,brain) was 8.10 for SLOH and 11.0 for SLM, which favored brain entry and were several folds higher than that in wild-type mice. Taken together, these carbazole compounds displayed low plasma clearance and high brain permeability, which entitle further development.

Systemic clearance and brain distribution of carbazole-based cyanine compounds as Alzheimer's disease drug candidates Wei Zhou, Xiaohui Hu, and Kin Yip Tam * Faculty of Health Sciences, University of Macau, Macau SAR, PR China. Table S1 Mass spectral characteristics of the in vitro metabolites of SLOH.

Table S2
Mass spectral characteristics of the in vitro metabolites of SLM.

Table S4
Blood cell partitioning, non-specific plasma protein binding, microsomes binding, and brain distribution using brain slices of positive controls (n=3, Mean ± SD).

Table S5
The Papp and efflux ratio values of positive controls in Caco-2 cell transport (n=3, Mean ± SD).

Table S6
Liver microsomes stability and hepatocyte stability in mice and human of positive controls (n=3, Mean ± SD).         represent pharmacokinetics profiles; A2 and B2 represent linearity analysis between AUC and dosages (n=5, Mean ± SD).

Fig. S11
The pharmacokinetics and brain exposure in WT and transgenic AD model mice. A: SLOH, B: SLM. A1 and B1 represent pharmacokinetics profiles; A2 and B2 represent brain exposure in ISF.

Fig. S12
The efflux ratio values of SLOH and SLM in the presence or absence of specific OATP2B1 inhibitor (Erlotinib) in Caco-2 cell transport. Statistical significance in parameters obtained from multiple groups was estimated using One-way ANOVA followed by Dunnett's test. (vs control group), & p＜0.05 and && p ＜0.01 (n=3, Mean ± SD).

Fig. S13
The Vu,brain values of SLOH and SLM in the presence or absence of P-gp and BCRP inhibitor in brain slice experiment. Statistical significance in parameters obtained from multiple groups was estimated using One-way ANOVA followed by Dunnett's test. (vs control group), * p＜0.05 and ** p＜0.01 (n=3, Mean ± SD).

Blood-to-plasma ratio
Aliquots of fresh whole blood and reference plasma (CCP) are spiked with SLOH and SLM (1 μM) and incubated for an hour at 37 O C. At the end of the incubation period, plasma (CP) is separated from whole blood by centrifugation and all plasma samples are analyzed after protein precipitation using 2 folds acetonitrile (ACN) containing berberine chloride as internal standard (IS) by LC-MS/MS (Analytical method seen below). The blood cell partitioning was calculated as follows.
(40 nM). The samples were centrifuged at approximately 1,900 g for 10 mins before LC-MS/MS analysis.

LC-HR MS (LTQ-Orbitrap XL hybrid MS) conditions for metabolites identification of SLOH and SLM
LC-MS experiments were carried out on a Thermo Electron LTQ-Orbitrap XL hybrid MS (ThermoFinnigan, Bremen, Germany) equipped with an ESI interface. An Accela HPLC system (ThermoElectron) was equipped with an autosampler, a vacuum degasser unit and a quaternary pump.
The MS employing positive ionization was calibrated using calibration standards mixture allowing for mass accuracies less than 5 ppm in external calibration mode. The ionization voltage was 4.2 KV and the capillary temperature was set at 300 o C. Nitrogen was used as both the sheath gas (40 units) and auxiliary gas (10 units). The resolving power was 15000 for full-scan and 7500 for the MS 2 scans.
A Thermo Xcaliber 2.1 workstation was used for the data acquisition and processing. For the computer-based MDF approach, representative structures with predicted mass defect windows were set as filtering templates for screening homologous compounds. Thermo Scientific TM MetWorks TM and Mass Frontier software automated structural elucidation.

Bio-sample preparations and LC-MS/MS (TQD) analysis for quantitation of SLOH and SLM
The LC-MS/MS condition was reported previously [4]. Briefly, under positive mode of Waters Xevo TQD, SLM (m/z 437＞347 as quantifier ion and m/z 437＞168 as qualifier ion), SLOH (m/z 467＞333 as quantifier ion and m/z 467＞154 as qualifier ion) and berberine (as internal standard, m/z 336＞320 as quantifier ion and m/z 336＞278 as qualifier ion) were simultaneously monitored. Plasma, urine and bile samples preparation method was completed as previously described [4]. For brain sample, brain was weighted and PBS buffer (pH=7.4) was added based on 10-fold volume of each brain weight. Brain samples were cut into smaller pieces before homogenization. Then 20 µL of homogenate were vortex 8 mixed with nitrogen-dried IS for 1 min. 40 µL of ACN was then added to precipitate the protein. After vortexed for 1.5 mins, the samples were centrifuged at 9,659 g for 5 mins. The supernatant was transferred to an autosampler vial and an aliquot of 1 µL was injected onto the LC-MS/MS system for analysis.    Unbound brain distribution (Vu, brain mL/g brain) Verapamil 36.0 ± 9.70 -Gabapentin 2.20 ± 1.00 -