Pitavastatin

The REAL-CAD study (Randomized Evaluation of Aggressive or Moderate Lipid Lowering Therapy With Pitavastatin in Coronary Artery Disease) is a prospective, multicenter, randomized, open-label, blinded end point, physician-initiated superiority trial to determine whether high-dose (4 mg/d) compared with low-dose (1 mg/d) pitavastatin therapy could reduce cardiovascular events in Japanese patients with stable CAD. Pitavastatin is a statin with potent LDL-C–lowering effects developed by Kowa Pharmaceutical Co Ltd (Tokyo, Japan). Pitavastatin doses of 1 and 4 mg were reported to reduce LDL-C by 33.6% and 47.2%, respectively, in Japanese patients.^{20 (link)} A similar magnitude of LDL-C reduction was also reported in white and East Asian patients.^{21 (link)–23 (link)} Pitavastatin 4 mg is the maximum approved dose in Japan and has demonstrated effects comparable to atorvastatin 20 mg in terms of both LDL-C reduction and coronary plaque regression assessed by intravascular ultrasound, whereas pitavastatin 1 mg has an LDL-C–lowering effect comparable to that of atorvastatin 5 mg.^{24 (link),25 (link)}Eligible patients were men and women 20 to 80 years of age with stable CAD as defined by a history of acute coronary syndrome or coronary revascularization >3 months ago or a clinical diagnosis of CAD with angiographically documented coronary artery stenosis of at least 75% diameter narrowing according to the American Heart Association classification.^{26 (link)} We excluded those patients with LDL-C <100 mg/dL without statin therapy before enrollment because the label in the instructions for pitavastatin restricted use to patients with hypercholesterolemia. Detailed inclusion and exclusion criteria are provided in the online-only Data Supplement. Patients were enrolled on an outpatient basis through academic and general hospitals and clinics across Japan. Eligible patients who provided informed consent were enrolled and received pitavastatin 1 mg once daily orally for a run-in period of at least 1 month. Patients were evaluated for secondary eligibility, excluding those patients with LDL-C ≥120 mg/dL after the run-in period, onset of acute coronary syndrome and/or coronary revascularization within the past 3 months, poor medication adherence to pitavastatin, occurrence of primary end point events, or adverse events prohibiting study continuation during the run-in period.
Patients who met the secondary eligibility criteria were randomized in a 1-to-1 fashion to oral pitavastatin, either 4 mg/d (high-dose group) or 1 mg/d (low-dose group), with an electronic data capture system and dynamic allocation stratified by facility, age (<65 or ≥65 years), sex, diabetes mellitus, and statin use before enrollment. The assignment algorithm was determined by the study statistician. This is an open-label trial. However, the independent event committee adjudicated all the end point events while blinded to the assigned group (online-only Data Supplement).
During follow-up, the patients’ visits dictated by the protocol were at 6 and 12 months in the first year and every 12 months thereafter. Serum lipid levels such as LDL-C, total cholesterol, triglycerides, and high-density lipoprotein cholesterol, as well as other blood tests such as creatine kinase, alanine aminotransferase, aspartate aminotransferase, creatinine, and hemoglobin A_1c, were to be measured at baseline, at 6 and 12 months, and yearly thereafter, whereas high-sensitivity C-reactive protein (hsCRP) was to be measured at baseline and at 6 months.
The site investigators reported follow-up information through the web-based electronic data capturing system. Data were monitored by the data center, and the logical inconsistencies were resolved by queries. Final clinical follow-up data were collected through January to March 2016. From 2012 to 2016, site audits were performed for 3914 patients from 28 centers, and the independent data monitoring committee regularly assessed the safety aspect of study conduct.

PD-GSCs were harvested from a T75 flask and passaged into replicate T75 flasks for either pitavastatin (6μM) or vehicle (DMSO) treatment (2.0e6 cells/flask). Concomitantly, a portion of those PD-GSCs were used to inoculate laminin-treated 96 well plates for drug-dosing analysis (see IC₅₀Analysis section). On D4, PD-GSCs were harvested using Accutase (1mL/25cm²) as described previously. Cell suspensions were spun at 1000rpm (193g) for five minutes. Cell pellets were then resuspended with serum-free culture media (200,000 cells/mL) to inoculate 96 well plates (100μL/well, 20,000 cells/well) for subsequent IC₅₀ determination. PD-GSCs were incubated in serum-free culture media in 96 well plates for 48 hours to allow for cell attachment prior to replacing spent media with serum-free media with pitavastatin (or vehicle). Treated cells were incubated at 37°C for four days. Following the four-day treatment, cell viability was measured via MTT assay as described below.

PD-GSCs were incubated in serum-free culture media (described above) with pitavastatin (6μM). Stock pitvastatin calcium (Selleck Chemicals LLC) was dissolved in DMSO to obtain a stock concentration of 10mg/mL and stored in aliquots at −80°C. Stock pitavastatin calcium solution was serially diluted in serum-free culture media to 100μM and then to the final concentration of 6μM with a final DMSO concentration of 0.053% (v/v).
To monitor longitudinally PD-GSC response to pitavastatin, we performed a reverse time-course treatment by adding pitavastatin to SN520 and SN503 cultures in a staggered fashion such that the longest (4-day) treatment would have drug added first. Subsequent addition of pitavastatin would occur on following days for 3- and 2-day treatment, respectively. This reverse time course design allowed us to collect all samples simultaneously on day four following the initial addition of pitavastatin. Because pitavastatin was added to PD-GSCs on different days, flasks were inoculated at slightly different cell densities to account for cell growth that would occur in between inoculation and time of pitavastatin addition. Consequently, scRNA-seq library preparation of all samples for a particular PD-GSC population occurred simultaneously to minimize batch effects due to individual sample processing (table S16)
Prior to T25 flask (BioLite^™) inoculation for pitavastatin treatment, PD-GSCs were first expanded in a T75 flask (BioLite^™). Once the culture was confluent, the culture was harvested and split into laminin-treated T25 flasks. Upon inoculation, cells were incubated in serum-free culture media at 37°C for 24 hours to allow cells to adhere to the interior surface of the flask. Following the first 24 hours, serum-free culture media was replaced with serum-free culture media with pitavastatin (6μM) in T25 flasks predetermined to receive a 4-day treatment. Spent culture media would then be replaced with fresh culture media with pitavastatin (6μM) on subsequent days for D3 and D2 treatment conditions.
Upon the completion of the 4-day treatment, spent media was removed and cells were harvested using Accutase^™ (1mL/25cm²). To prevent any cell-free DNA/RNA from treatment-induced lysed cells contaminating single-cell samples, we first processed a portion of the cell harvest solution using the dead cell removal kit (Miltenyi Biotec 130–090-101) to remove any cell debris to avoid any free RNA from lysed cells from getting mixed in with mRNA to be extracted from live cells. Samples were processed per vendor’s specifications. The result was a cell suspension of the remaining live cells post vehicle- or pitavastatin-treatment. Cell suspension was then processed for scRNA-seq profiling per the 10X Chromium platform.