Therapeutic efficacy and safety of PCSK9-monoclonal antibodies on familial hypercholesterolemia and statin-intolerant patients: A meta-analysis of 15 randomized controlled trials

Proprotein convertase subtilisin/kexin9 monoclonal antibodies (PCSK9-mAb) have been studied intensively to identify their effect in lowering levels of low density lipoprotein cholesterol (LDL-C). However, the applicable target of PCSK9-mAbs remains inconclusive so far. Therefore, this first meta-analysis was carried out to clarify the therapeutic efficacy and safety of PCSK9-mAbs on the potential patients: familial hypercholesterolemia and statin-intolerant patients. All randomized controlled trials that met the search terms were retrieved in multiple databases. Efficacy outcomes included parameter changes from baseline in LDL-C and other lipid levels. Therapeutic safety were evaluated by rates of common adverse events. A total of 15 studies encompassing 4,288 patients with at least 8 weeks duration were selected. Overall, the therapeutic efficacy was achieved with significant reduction in LDL-C, TC, TG, Lp(a), Apo-B versus placebo. The decline in familial hypercholesterolemia patients (−53.28%, 95% CI: −59.88 to −46.68%) was even more obvious than that in statin-intolerant patients (−34.95%, 95% CI: −41.46 to −28.45%). No obvious safety difference was found out in the rates of common and serious adverse events. To conclude, PCSK9-mAb contributes to the decreased level of LDL-C and other lipids in familial hypercholesterolemia and statin-intolerant patients with satisfactory safety and tolerability.

In the last 4 years, early clinical trials have proven that PCSK9-mAb can lower the plasma LDL-C level in FH and statin-intolerant patients. The other lipids and lipoproteins; high density lipoprotein cholesterol (HDL-C), total cholesterol (TC), triglycerides (TG), lipoprotein(a) (Lp(a)), apolipoprotein-B (Apo-B) and apolipoprotein-A1 (Apo-A1) can also benefit. Currently, there is no report to comprehensively pinpoint the applicable targets of PCSK9-mAbs-FH and statin-intolerant patients with sufficient clinical outcomes. To compare the efficacy and safety of PCSK9-mAb on FH and statin-intolerant patients, a total of 15 articles were assessed in this meta-analysis.

Results
Study selection and characteristic. A total of 178 studies were searched in our systematic literature, with 43 duplicate publications. 106 studies were unable to meet the inclusion criteria excluded; 14 studies were further ruled out, where 6 studies were articles, 2 studies had no control groups, 1 study was not RCT, 1 was open label trial and 3 were not meta-analysis with quantitative synthesis. (Figure 1) As a result, 15 studies encompassing a total of 4,288 patients were selected [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] . Among them, 7 trials used evolocumab (AMG 145) and 8 studies with alirocumab (SAR236553/REGN727) treatment. Baseline characteristics were detailed giving the substantially similar basic values between PCSK9-mAbs and controls. Mean age of the subjects ranged from 31 to 65 years old. All trials were published between 2012 and 2015, followed up for 8 to 78 weeks, with a low risk of bias (Table 1 and Figure 2).

Discussion
As far as we know, this is the first meta-analysis using sufficient clinical outcomes comprehensively to define the applicable targets of PCSK9-mAbs-FH and statin-intolerant patients. In the present analysis, a total of 15 studies encompassing 4,288 patients with FH and statin-intolerence were included. The main point addressed is whether PCSK9-mAb treatment have the ability to improve the lipid levels of FH and statin-intolerant patients with satisfactory safety and tolerability.
At present, additional on-going large-sized multicenter randomized studies were not included in our study, such as the Phase III FOURIER, GLAGOV and GAUSS-3 trial 41-43 , which are intended to determine if PCSK9-mAbs can help to not only reduce LDL-C level obviously, but also improve cardiovascular related events such as myocardial infarction(MI), and the rates of morbidity and mortality. Nevertheless, the concept of LDL-C reduction, as a surrogate for prevention of long-term cardiovascular outcomes in high-risk patients with acute coronary syndromes, was provided by the recently published IMPROVE-IT study with the strongest clinical trial evidence. The more reduction of LDL-C, the rate of major vascular events decreases 44 . (Figure 7) Given the consistent effects on LDL-C reduction, PCSK9-mAbs appears to be a promising approach to cut down the risk for cardiovascular events in the patients selected.
In a broader sense, LDL particles, non-HDL-C, the Apo-B/Apo-A1 ratio, and Lp(a) may be the better replacement markers for cardiovascular mortality in the foreseeable future, compared with LDL-C levels [45][46][47][48] . Overall, results of the study are suggestive of therapeutic efficacy of PCSK9-mAbs in curtailing the major risk factors (including LDL-C, TC, TG, Lp(a) and Apo-B) of CVD, and increasing HDL-C and Apo-A1 by detecting both  evelocumab and alirocumab. It has been previously suggested that Apo-B/Apo-A1 ratio is a better risk indicator for CVD and MI than the level of lipids 49 . In our study, Apo-B decreased while Apo-A1 elevated in the PCSK9-mAb treatment group. The decrease in Apo-B/Apo-A1 ratio obviously indicates that PCSK9-mAb therapy greatly lowers the primary risk factors of heart disease. We also found that the reduction of LDL-C in FH patients (−53.28%, 95% CI: −59.88 to −46.68%) was greater than that in statin-intolerant patients (−34.95%, 95% CI: −41.46 to −28.45%). In terms of safety, PCSK9-mAbs may lead to many events, none of which is life-threatening; the serious events do not increase compared with the control group; more large RCTs are needed to further confirm the safety.
Recently, there have been four meta-analyses comparing the effects of PCSK9-mAbs over placebo controls, with or without considering statin therapy [50][51][52][53] . In these studies, AMG145/Evolocumab, REGN727/SAR236553/ Alirocumab were primarily included. However, novel clinical studies are being done to evaluate the target of LDL-C, such as RN316/bococizumab, RG7652, LY 3015014, ALN-PCSSC, which has been genetically validated to lower the cardiovascular risk.
The mechanism of improving the survival of patients treated with PCSK9mAb remains obscure and its role in the observed survival benefit, for its encouraging result, should be interpreted with caution. The efficacy of these agents in reducing lipid levels (particularly LDL-C) and rates of MI due to plaque stabilization is well-evaluated. Moreover, the lower frequency of PCSK9-mAbs intake may lead to slighter hepatotoxicity.  Table 2. Pre-specified Safety End Points. No statistical differences between PCSK9-mAbs and control group except any and paraesthesia events, which were positive in the PCSK9-mAbs group. PCSK9-mAbs = PCSK9-monoclonal antibodies; CHF = congestive heart failure; Any = any of the common adverse events, ALT = alanine aminotransferase; AST = aspartate aminotransferase; ULN = upper limit of normal; CK = creatinine kinase ; hsCRP = hypersensitive C reactive protein. However, subcutaneous injection of PCSK9-mAb makes it an alternative for drug adherence. What's more, studies reveal that LDL-R can act as the entry point for hepatitis C virus, which may increase the risk of infection 54 .

Pre-specified Safety End Points
Finally, it still remains unknown whether the reduced LDL-C by PCSK9-mAbs can improve the long-term clinical outcomes 55 . Several limitations should be taken into consideration. First, a few studies have only been reported in abstracts or presented at meetings, leading to added detection bias. Besides, significant heterogeneities were observed in most of the reported outcomes, but we failed to reveal the heterogeneities by dividing subgroups or sensitivities methods. Some large multicenter RCTs are still under investigation. Therefore, caution should be taken in interpreting the results of the meta-analysis when combining the heterogeneous data sets. Despite these limitations, our meta-analysis covers the most applicable targets of PCSK9-mAbs-FH and statin-intolerant patients, and their clinical outcomes are sufficient enough to compensate our clinical guidelines. Hopefully, more RCTs could be carried out to provide more evidences about its long-term therapeutic efficacy, safety, and clinical outcomes.

Methods
Study searching. In order to identify appropriate RCTs, a comprehensive literature search was performed throughout PubMed, EMBASE and The Cochrane Library databases, with the following terms and key words: evolocumab, AMG 145, alirocumab, SAR236553, REGN727x, PCSK9 monoclonal antibodies, FH, statin intolerant and randomized controlled trial from it's inception in 2012 to May 26, 2016. All the selected studies, restricted to English, were initially screened for relevance at the abstract level. Study selection. The inclusion criteria are as follows: (1) RCTs; (2) population: FH, statin intolerant patients; (3) safety and efficacy outcomes of PCSK9-mAbs; (4) mean differences (MDs) with 95% corresponding confidence interval (95% CI) and so on. Studies that were not randomized, special-population-targeted or designed to test PCSK9-mAbs with limited number of trials (such as bococizumab, RG7652, LY3015014 and ALN-PCSSC) were excluded.
Outcomes. The primary efficacy endpoints included parameter changes following PCSK9-mAbs treatment from baseline: (1) LDL-C reduction; (2) other lipid profile changes. Therapeutic safety was evaluated by rates of the common adverse events, serious events and laboratory adverse events respectively. Data collection. Two investigators (LJQ and YG) performed eligibility assessment with a standardized data extraction individually and another reviewer (YMZ) checked the data. Basic information was extracted as follows: study/author, year, design, diagnosis, control, drug regimen, duration, patient number, mean age (y) at baseline. As a precedence, we extracted the corresponding mean differences, 95% CI from baseline for each lipid items, such as LDL-C, HDL-C, TC, TG, Apo-B and Apo-A1 levels, before and after PCSK9-mAbs treatment as the primary outcomes. Safety endpoints covering the common adverse events, serious events and laboratory adverse events were compared between the treatment and control groups. In addition, the common adverse events included injection site reaction (e.g. generalized pruritis, hypersensitive reaction, erythema, rash, swelling, discoloration, or pain), nasopharyngitis, upper respiratory tract infections, influenza, cough, nausea, myalgia, myositis, headache, diarrhea, fatigue, abnormal pain, rectal bleeding, dehydration, arthralgia, and back pain. The serious events, fatal and life threatening, require hospital admission or prolonged stay in the hospital for the possibility of persistent or significant disability. The laboratory adverse events evaluated hepatotoxicity with ALT or AST levels ≥3 × ULN. Quality assessment. Two reviewers (LJQ and YG) assessed the risk of bias with the Cochrane Collaboration's tool (Review Manager version 5.3) in the included trials. For efficacy outcomes, comparisons of LDC-C reduction were performed separately on FH and statin-intolerant patients. The changes of each lipid items stratified by forms and dosages of PCSK9-mAbs were carried out versus placebo or ezetimibe treatment. We assessed the publication bias, including the risk of selection bias, performance bias, detection bias, attrition bias, reporting bias and other bias, by using the Risk-of-bias graph and summary table.
Statistical analysis. All analyses were conducted with Review Manager version 5.3. A fixed-effect model was selected if there was no unexplained statistical heterogeneity, otherwise, a random-effect model was used in the meta-analysis. Cochrane Q test to measure the heterogeneity among the included trials and I 2 statistics to assess the magnitude of heterogeneity were performed separately. χ 2 statistics was to assess the magnitude of heterogeneity and P value < 0.05 was considered to be statistically significant.