Proprotein Convertase Subtilisin/Kexin Type 9 Inhibitors Induced Neurocognitive Disorder: A Pharmacovigilance Study by Analyzing the Data from the U.S. FDA Adverse Event Reporting System

Yang Hu Peking Union Medical College Hospital Eastern Branch: Peking Union Medical College Hospital Ziyu Bai UCL: University College London Tianxin Huang Tianjin University of Traditional Chinese Medicine Xinyue Zhang UCL: University College London Qianqian Fan Peking Union Medical College Hospital Western Branch Bin Zhao (  zhaobin@pumch.cn ) Peking Union Medical College Hospital https://orcid.org/0000-0001-8555-523X Zhuang Tian Peking Union Medical College Hospital Eastern Branch: Peking Union Medical College Hospital


Introduction
The Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) is a protein that can affect normal cholesterol homeostasis regulation. It reduces plasma low-density lipoprotein receptor (LDL-R) levels by binding to and subsequently degrading them [1,2]. This will lead to reduced metabolism of low-density lipoprotein cholesterol (LDL-C) thus inducing hypercholesterolemia [3], then developing to atherosclerosis. PCSK9 inhibitor can interfere with this process, resulting in higher LDLR expression as well as lower plasma LDL levels. Evolocumab (Repatha) and Alirocumab (Praluent) are two PCSK9 inhibitors approved by the U.S.
FDA as a cholesterol-lowering agent in 2015. Later, both of them were proved for their effective performance in the management of atherosclerotic cardiovascular diseases (ASCVD) patients.
With the lipid management guidelines updating, lipid-lowering therapy has become extremely vital for ASCVD patient treatment [4]. However, previous reports showed that some neurocognitive impairment cases were possibly associated with rst-class medication, the statin. Given the effect of PCSK9 on cholesterol levels and the importance of cholesterol regulation, this is no doubt raised the concern about the PCSK9 inhibitors-related neuro-dysfunction [5,6]. Neurocognitive disorder would gradually develop into dementia. It would become 'Alzheimer' in some severe cases. Most of the time, it is commonly diagnosed among senior citizens over 65 years old, which caused great burden for patient family and healthcare staff. Previous research surmised that that 115.4 million people would suffer from this disease by 2050 [7] , [8]. To lower the occurrence of this horrible adverse event induced by a lipid-lowering medicine, FDA had urgently issued an instruction about the surveillance for PCSK9 inhibitors in 2014, but without any speci c measures to assess this kind of side-effect.
To minimize the risk of medicinal accidents in the clinic, pharmacovigilance is mainly charged to identify the potential hazards or risks associated with medicine and other pharmaceutic products. It relies on a mature system is to monitor the safety of post-marketing medicines [9,10], which including collection, detection, assessment, surveillance, and prevention of adverse events or adverse drug interactions. Due to the convenience of accessing comprehensive information, the spontaneous reporting system database is one of the main resources for pharmacovigilance studies. For example, the basic information of the patient, the usage of the drug, and detailed information about the adverse events [11]. Since some rare adverse events were hardly captured in the clinic trials, surveillance program for post-marketing medicine is particularly important for neotype drugs or long-term medicinal therapy. This is because some unexpected adverse events only occur after a large number of people receive the medicine, or the drug is being used for a longer period. Furthermore, the long-term monitoring activities are bene cial to delivering an objective appraisal of therapeutical effectiveness and long-term safety.
In recent years, some research was conducted to explore the correlation between PCSK9 inhibitors and neurocognitive disorder [12,13]. However, there is a limited number of real-world based studies related to this subject, and the existing argument is that whether the occurrence of the neurocognitive disorder is the result of the usage of PCSK9 inhibitors. In addition, a tenable mechanism of PCSK9 inhibitors inducing neurocognitive dysfunction is still unknown. Here, we conducted this pharmacovigilance study with data retrieved from the spontaneous reports of the FDA Adverse Event Reporting System (FAERS) to supplement existing insu cient PCSK9 inhibitor usage information and provide evidence-based data for its usage and potential speci c adverse effect in clinical practice.

Data Sources.
A retrospective pharmacovigilance study was performed by retrieving spontaneous reports related to neurocognitive disorder induced by PCSK-9 inhibitors during the rst quarter of 2004 to the second quarter of 2020 in the FAERS database which is a highly reliable program supported by the FDA for the surveillance of post-marketing drugs safety. FAERS is the largest voluntary reporting database, which records substantial data including demographic information (DEMO), patient outcomes (OUTC), drug information (DRUG), adverse event (REAC), therapy start dates and end dates for reported drugs (THER), and indications for use (INDI). Healthcare professionals and consumers were the main reporting group, who can spontaneously submit adverse events resulting from medication error reports, drug interaction, and product quality complaints.
A total of 14,570,082 reports from the FAERS database and excluded duplicated records according to the FDA's recommendations by selecting the latest FDA_DT when the CASEID and FDA_DT were the same.
We ultimately included 12,198,404 reports for signal detection.

Adverse Event and Drug Identi cation.
Adverse events investigation was based on the summary of both previous research and literature review.
The FAERS database was used to collect voluntarily submitted PCSK9 inhibitor treatments in association with neurocognitive disorder reports by inputting the following preferred terms from the Medical Dictionary for Regulatory Activities Terminology (MedDRA, version 23.1): cognitive disorder[code:10057668]. The MICROMEDEX (Index Nominum) was used as a dictionary of PCSK9 inhibitors (Table 1). Reporting Odds Ratio (ROR) is one of the frequentist algorithms for data mining, which has been commonly operated in pharmacovigilance studies with an important performance [14,15]. The algorithm of ROR is de ned as below[16]: Positive signal criteria: 95%CI>1, N≥2 In the rst equation, a is the total number of reports of the target medicine associated with the neurocognitive disorder. b is the number of reports representing the other lipid-lowering medicine inducing neurocognitive disorder, except PCSK9 inhibitor. c is the number of reports containing other adverse events caused by PCSK9 inhibitor but without the neurocognitive disorder. d is the number of reports containing neither PCSK9 inhibitor nor the neurocognitive disorder. The second equation is the 95% con dence interval (95% CI), e is the natural constant, and ln is the natural logarithm.
Our study applied the above equations to calculate both ROR and 95% CI. The value of ROR was utilized to assess the correlation between two PCSK9 inhibitors and neurocognitive disorder, respectively. It can be de ned that the positive signal is identi ed, if the lower limit of the two sides of the 95% CI is over 1, with the total reports number (N) should be more than 2.

Statistical Analysis.
The clinical features of patients with PCSK9 inhibitors associated with the neurocognitive disorder were summarized by descriptive analysis. Since the data were not normally distributed, the onset time of cognitive dysfunction induced by different PCSK9 inhibitor therapies was compared by nonparametric tests (the Mann-Whitney test for dichotomous variables and the Kruskal-Wallis test for >2 subgroups of respondents). Pearson's chi-square test or Fisher's exact test was utilized to compare the disability and mortality rate of the neurocognitive disorder in patients receiving different PCSK9 inhibitors. Statistical signi cance was declared at P < 0:05 with 95% con dence intervals. Both data mining and statistical analysis were estimated by SAS version 9.4 (SAS Institute Inc., Cary, NC, USA).

Result
Within the study period, 81,272 adverse reports were related to PCSK9 inhibitor treatments, and 26,843 related to neurocognitive disorders. Through data mining technology, 152 reports correspond with our inclusion criteria. In Table 2, it demonstrates the speci c demographic and clinical characteristics of these patients as well as some important information of these reports. In terms of the gender composition, the report number of neurocognitive disorders in both males and females were quite similar, with the percentage of 47.37% and 48.03%, respectively. The range of age was from 0 to 87 years old, the proportion of patients with the age at 45 to 64 presented the highest percentage, except the unknown age patients. In terms of the reporters, the healthcare professionals were the main group who submitted the reports. Over ninety percent of reports were reported from North America. The Other Serious (Important Medical Event) took up 87.01% of the whole outcome events. No death event was reported. n is the number of the reports.
In total, there were 152 neurocognitive disorder reports related to PCSK9 inhibitor with the ROR value at 0.85. 106 reports were associated with Evolocumab, while 46 were in correlation with Alirocumab usage. According to the criteria of disproportional signal detection, the positive signal was exclusively identi ed in the patients who received Alirocumab (Table 3). n is the number of neurocognitive disorder reports. ROR is the reporting odds ratio, the value in the parentheses represents the two sides of 95% con dence interval.
The onset time of the adverse event under each medication therapy PCSK9 inhibitor was also evaluated. It was de ned as the time interval between the date patients initiated injecting PCSK9 inhibitors and the day that neurocognitive disorder onset date. The potential database errors and reporting errors, such as the start date earlier than the onset date were excluded. As a result, 16 valid data indicated that the neurocognitive disorder had occurred at the scale of 0 to 618 days, with the median at 1.5 days.

Discussion
In this study, disproportionality analysis revealed that Alirocumab may correlate with the neurocognitive disorder due to the presenting positive signal, while the total PCSK9 inhibitors and Evolocumab presented a negative signal with a weak correlation. From the report system, we retrieved 77 neurocognitive disorder outcome events which should pay more attention to the clinic, including primary and long-term hospitalization (7.79%), disability (3.90%) and life-threatening (1.30%). In addition, the origin of reports was summarized to accumulate more adverse event information of PCSK9 inhibitors in the real world.
Disproportionality signal can help to establish a new potentially causal association between PCSK9 inhibitors and neurocognitive disorder [17]. Alirocumab was highly associated with the neurocognitive disorder, but Evolocumab was not related. This result is consistent with the nding obtained in two previous RCTs. found that there was an increased occurrence of neurocognitive disorder after the usage of PCSK9 inhibitors [5,19]. Although their result further supports the idea that there is a signal toward PCSK9 inhibitors associated with the neurocognitive disorder, they did not identify which PCSK9 inhibitor existed the potential relation with neurocognitive impairment. Our study is distinguishing from the anterior RCTs and meta-analysis study because we had comprehensively applied the pharmacovigilance study method to detect the drug-event signal. Thus, a larger amount of data can be obtained to reveal the hidden relationship between the adverse event and PCSK9 inhibitors. For example, the meta-analysis conducted by Lipinski et al. enrolled 17 RCTs, 13,083 patients participated, whereas, this study included 12,198,404 reports, which is much more than these four mentioned studies. Consequently, the su cient real-world datasets can transform into valuable evidence to improve the insight of the medication effect.
However, there would be a concern that the signal of the Alirocumab report was a false positive.
Alirocumab may not have any association with the neurocognitive disorder as Karatasakis A. et al. studied it [13]. Their nding was based on a meta-analysis, which involved 21 RCTs including 42,668 patients who participated in analyzing neurocognitive adverse events. Some potential biases may lead to an alpha error in our study. For example, PCSK9 inhibitors were exclusively prescribed for very-high risk ASCVD patients for managing their LDL-C level, which caused the channelling bias. In addition, the notoriety bias was existed, because some stimulating reports were raised after the FDA had issued the surveillance. With the existence of the above bias, evaluating the false positive signal by a single study is impossible. But it can motivate the healthcare professionals to pay more attention to monitoring the neurocognitive dysfunction among the patients who received Alirocumab. The follow-up data and cognitive evaluation report are vital sources for case analysis then assessing the false positive signal.
Moreover, the negative disproportion signal should not be interpreted as a safety endorsement [16,17].
Although the signal of Evolocumab is negative, it cannot infer that neurocognitive disorder is unassociated with Evolocumab. Since disproportion analysis is only used for adverse signal detection and prioritization, it cannot identify the risk and mechanism of medicine. Therefore, establishing the causality of neurocognitive disorder and PCSK9 inhibitors should require more comprehensive evidence.
Recent research proposed the mechanism that the neurocognitive adverse event may pose by sustained low LDL-C levels. While the treatment effect of PCSK9 inhibitor can promote the above process, thus neurocognitive dysfunction becomes the potential adverse event [12,20]. Based on this mechanism and the disproportional analysis, it is necessary to keep vigilant for the usage of PCSK9 inhibitors in the clinic and record the effect. The collected data would further prove the causality of PCSK9 inhibitor and neurocognitive disorder.
From the traits of the report, neurocognitive adverse events occurred after a period of using PCSK9 inhibitors. We found that neurocognitive related events occurred among 5 patients within one year. This result matched those observed from Koren et al [21]. They reviewed the OSLER-1 trial including 12,251 patients, and 8 patients presented neurocognitive disorder in the rst year after using Evolocumab. However, data related to the onset time of Alirocumab have remained absent because many RCTs did not demonstrate the detailed information of adverse event onset time. Although the sample size of onset time was limited in this study, we still calculated the median for analysis, however, less valuable information was obtained. By analyzing the age of the patient provides, we nd that the neurocognitive disorder was concentrated in the age of 45 to 64 patients. Normally, dementia would occur among the patient who is over 65 years old, except the Frontotemporal dementia because it can induce onset earlier [7,22]. In addition, around 60% of reports were submitted by the healthcare professionals, the source of data was reliable. These reports may relate with the PCSK9 inhibition, but it lacks the follow-up data or the subsequent report. For example, whether the patients recovered after withdrawing the PCSK9 inhibitors. Therefore, it still requires more evidence.
Since PCSK9 inhibitor is pricy for patients, the potential adverse event possibly leads to the increasing of therapy cost, then patient life-quality would decrease. A systematic review concludes that although adverse events associated with PCSK9 inhibitors were rare in most outcome studies and RCTs, the severe events were still existing [23]. Another research based on the real-world experience [24] inferred that although the adverse effect did not interfere with the usage of PCSK9 inhibitors since it was rare, Gurgoze MT. et al. pointed out that balancing the long-term bene ts and potential adverse events is a critical process. The rare adverse events should not be neglected, but the real-world data of PCSK9 inhibitors was limited. Thus, it is vital to collect more evidence of adverse events associated with PCSK9 inhibitor [24][25][26]. Here, we therefore advocate performing more drug pharmacovigilance programs for PCSK9 inhibitors, consequently, the awareness of those serious adverse effects among patients and clinicians will be enhanced, which is also bene cial for the patient to remain safe and reduce medical costs.
As real-world study is developed for post-approved drug surveillance, it performs differently from the RCTs in re ecting the real clinical treatment, especially for drug safety assessment [27,28]. Since the realworld data is based on clinical observation, some rare but important clinical features can preserve as supplementary evidence to the pharmacovigilance study. Besides, real-world evidence also helps to provide a practical and persuasive clinical suggestions for healthcare professionals [27].
Although the recognition of the potential of big data analytics is rising, its application in pharmacovigilance research is still emerging. FAERS database might be a useful tool for pharmaceutical companies to collect related e cacy and safety information because the preserved data size and worldwide coverage of this database are conducive to provide robust evidence for the conduction of spontaneous reporting data analysis. Moreover, the substantial volume of data contributes to real-world study, in terms of saving the research cost and time compared with the RCTs [29,30].
Data mining of the FAERS database can obtain the previously unknown, but clinically important drug and adverse events associations [31]. Since the valuable information comes from the raw data, the application of data mining technology is vital for the pharmacovigilance study. Establishing the association between the drug and adverse event is a complex process, the connection requires deep investigation, such as the inducement mechanism. A disproportional signal is a symbol to guild the researchers to nd out the new objective. With the comprehensive and multifaceted study, drug safety can be reached to a higher level.
There were several limitations in this study. First, the subjectivity is the main factor in drug-related adverse event research [32]. As the FDA did not require the reporters should be an expert or professionals in pharmacovigilance activities, the quality of the adverse reports is particularly different. There might contain underreported or submitted with false and incomplete information. Second, the worldwide coverage of the FAERS may become a two-edged sword. It provides a substantial sample size, but the different reporting culture from different countries is another major source of heterogeneity, which affects the quality of the reports as well. Third, the causal relationship between medication and the adverse event may not consistent, because the report lacks relative evidence. Furthermore, the spontaneous reporting system cannot quantitatively measure the signals of neurocognitive disorder base on the total number of adverse reactions. It also cannot calculate the exact incidence of potential adverse events, the main reason is the spontaneous reporting system has insu cient exposure data, which corresponds to the denominator of incidence. Despite the above limitation, the FAERS database is still a reliable source of real-world data for the disproportional signal study. In the future pharmacovigilance study, the analysis for causality of PCSK9 inhibitor and neurocognitive disorder should together with clinical follow-up and cognitive function evaluation.

Conclusion
In conclusion, we conducted the disproportional analysis between PCSK9 inhibitors and neurocognitive disorder in a number of real-world reports based on the FAERS database. Although disproportional signal was detected, both Alirocumab and Evolocumab require further study to reassess the safety. Due to the usage of PCSK9 inhibitors is expected to rise in the clinic for the next few years, pharmacovigilance study is extremely necessary and vital for monitoring neurocognitive disorders. As the number of long-term follow-up reports increases, the collected real-world data will signi cantly increase the evidence quality.
Furthermore, it will assist to facilitate the administration of PCSK9 inhibitors in the clinic.

Declarations
Funding