All Subjects Had Their Serum Cholesterol Drawn at Baseline and Again at the Completion of the Study
Original Article
Effect of a Monoclonal Antibody to PCSK9 on LDL Cholesterol
List of authors.Abstract
Background
Proprotein convertase subtilisin/kexin ix (PCSK9), i of the serine proteases, binds to depression-density lipoprotein (LDL) receptors, leading to their accelerated deposition and to increased LDL cholesterol levels. We report three stage 1 studies of a monoclonal antibody to PCSK9 designated as REGN727/SAR236553 (REGN727).
Methods
In healthy volunteers, we performed 2 randomized, single ascending-dose studies of REGN727 administered either intravenously (40 subjects) or subcutaneously (32 subjects), equally compared with placebo. These studies were followed past a randomized, placebo-controlled, multiple-dose trial in adults with heterozygous familial hypercholesterolemia who were receiving atorvastatin (21 subjects) and those with nonfamilial hypercholesterolemia who were receiving handling with atorvastatin (xxx subjects) (baseline LDL cholesterol, >100 mg per deciliter [ii.6 mmol per liter]) or a modified diet alone (10 subjects) (baseline LDL cholesterol, >130 mg per deciliter [three.4 mmol per liter]). REGN727 doses of 50, 100, or 150 mg were administered subcutaneously on days 1, 29, and 43. The main outcome for all studies was the occurrence of adverse events. The master secondary outcome was the effect of REGN727 on the lipid contour.
Results
Among subjects receiving REGN727, at that place were no discontinuations because of agin events. REGN727 significantly lowered LDL cholesterol levels in all the studies. In the multiple-dose study, REGN727 doses of 50, 100, and 150 mg reduced measured LDL cholesterol levels in the combined atorvastatin-treated populations to 77.5 mg per deciliter (2.00 mmol per liter), 61.3 mg per deciliter (ane.59 mmol per liter), and 53.8 mg per deciliter (1.39 mmol per liter), for a difference in the change from baseline of −39.2, −53.7, and −61.0 percentage points, respectively, every bit compared with placebo (P<0.001 for all comparisons).
Conclusions
In three phase 1 trials, a monoclonal antibody to PCSK9 significantly reduced LDL cholesterol levels in good for you volunteers and in subjects with familial or nonfamilial hypercholesterolemia. (Funded past Regeneron Pharmaceuticals and Sanofi; ClinicalTrials.gov numbers, NCT01026597, NCT01074372, and NCT01161082.)
Methods
Study Designs
We performed iii separate clinical studies of REGN727. Two were studies of unmarried doses of REGN727, administered either intravenously or subcutaneously, in healthy volunteers. The third study was a multiple-dose study of subcutaneously administered REGN727 in subjects with either familial or nonfamilial hypercholesterolemia.
These studies were sponsored by Regeneron Pharmaceuticals and Sanofi. The single-dose studies were designed by Regeneron Pharmaceuticals, and the multiple-dose written report was jointly designed past the offset author and Regeneron Pharmaceuticals. The study protocols were approved past the investigational review board at each written report center, and all subjects provided written informed consent. Data were collected at the study sites by several of the coauthors and were analyzed by representatives of Regeneron Pharmaceuticals. The first draft of the manuscript was jointly written past the kickoff writer and a representative of Regeneron Pharmaceuticals, with review and revision by the other authors. Editorial assistance was provided past an employee of the PharMed Group who was paid by Regeneron Pharmaceuticals. The showtime author and Regeneron representatives fabricated the decision to submit the manuscript for publication. The academic authors vouch for the accuracy and abyss of the data and analyses as presented and for the fidelity of this report to the trial protocols, which are bachelor with the full text of this commodity at NEJM.org.
Unmarried-Dose Studies
For the single-dose studies, xl subjects were enrolled in the group receiving intravenous REGN727 and 32 were enrolled in the group receiving subcutaneous REGN727. All subjects were good for you men and women between the ages of 18 and 65 years with a body weight ranging from 50 to 95 kg and a body-mass index (the weight in kilograms divided by the foursquare of the height in meters) ranging from 18 to thirty. All subjects had a serum LDL cholesterol level of more 100 mg per deciliter (2.59 mmol per liter). The utilise of nonstudy agents to alter lipid levels was prohibited. Detailed enrollment criteria are provided in the Supplementary Appendix, available at NEJM.org.
In the single-dose study involving intravenous administration, an initial group of 8 subjects was randomly assigned in a 3:ane ratio to receive either REGN727 at the lowest tested dose (0.3 mg per kilogram of body weight in six subjects) or placebo (in 2 subjects). Later safe assessment, the dose of REGN727 was increased sequentially (to 1.0, 3.0, 6.0, and 12.0 mg per kilogram) in four further iii:1 randomized comparisons with placebo, with eight subjects in each group (Fig. S1A in the Supplementary Appendix). The single-dose study of subcutaneous administration had a like pattern but included 4 sequential-dose groups receiving 50, 100, 150, and 250 mg (Fig. S1B in the Supplementary Appendix). These studies were conducted at ii contract enquiry organizations, Quintiles in Overland Park, Kansas (for intravenous and subcutaneous administration), and Comprehensive Stage One in Miramar, Florida (for subcutaneous administration).
All subjects in the single-dose studies were required to remain at the research facility for observation for 3 days after receiving a study drug. Blood was drawn for evaluation of serum lipid levels at baseline and at days 1, 2, 4, 8, 11, 15, 22, 29, 43, 64, 85 (intravenous administration only), and 106. Prophylactic assessments that were performed on the aforementioned days included an evaluation of vital signs, a physical examination, blood tests, and electrocardiography. Details of the laboratory measurements are provided in the Supplementary Appendix.
Multiple-Dose Study
The multiple-dose study included 3 dissever cohorts of subjects. The first cohort consisted of 21 subjects with heterozygous familial hypercholesterolemia (Fig. S1C in the Supplementary Appendix), and the second cohort consisted of xxx subjects with nonfamilial hypercholesterolemia (Fig. S1D in the Supplementary Appendix). All subjects in these two cohorts were receiving atorvastatin therapy and had an LDL cholesterol level of more than 100 mg per deciliter. The third cohort consisted of 10 subjects with nonfamilial hypercholesterolemia who were being treated with a modified diet just and who had an LDL cholesterol level of more 130 mg per deciliter (3.36 mmol per liter) (Fig. S1E in the Supplementary Appendix). All subjects in the multiple-dose study were between the ages of 18 and 65 years, had a body-mass index of 18 to 35, and did not have diabetes or a known atherosclerotic vascular disease. Additional enrollment criteria are provided in the Supplementary Appendix.
Subjects in the multiple-dose study were randomly assigned to receive subcutaneous REGN727 (50, 100, or 150 mg) or placebo administered on days 1, 29, and 43. All three regimens were designed to provide a 4-week prophylactic observation period betwixt the starting time and second doses of REGN727 and to assess the pharmacodynamic effects of REGN727 at fourth dimension points ii weeks after assistants (on written report days 15, 43, and 57) and 4 weeks after administration (on study days 29 and 71). The multiple-dose trial was carried out at the Metabolic and Atherosclerosis Research Heart in Cincinnati and the New Orleans Center for Clinical Research in Knoxville, Tennessee.
All subjects in the multiple-dose report were required to remain at the research facility for ascertainment for 2 days after receiving the kickoff dose of a study drug and for 2 hours after receiving the 2nd and third doses. Serum lipid levels were evaluated at screening and two days before the administration of a study drug (for subjects receiving atorvastatin only), 1 twenty-four hours before administration, and on days ane, two, three, 8, 15, 29, 43, 57, 71, 85, 99, 120, and 148 subsequently administration. Safe assessments that were performed on the same days included an evaluation of vital signs, a physical test, blood tests, and electrocardiography. Details of the laboratory measurements are provided in the Supplementary Appendix.
Statistical Assay
The primary end bespeak for all three studies was the incidence and severity of treatment-emergent adverse events. The safety population for this purpose included all subjects in each study who had received at to the lowest degree one dose of a study drug. Secondary end points included the relative and absolute change in measured and calculated serum LDL cholesterol, total cholesterol, and other serum lipid and lipoprotein levels from baseline to each visit.
Results for all subjects in the placebo groups were pooled within each of the single-dose studies. In the multiple-dose study, results for all subjects in the placebo groups were pooled for each report cohort (subjects with heterozygous familial hypercholesterolemia or nonfamilial hypercholesterolemia receiving statin therapy and subjects with nonfamilial hypercholesterolemia receiving a modified diet just). Missing values were imputed with the final ascertainment carried frontward (in v subjects in the single-dose report of intravenous assistants, six subjects in the single-dose written report of subcutaneous administration, and no subjects in the multiple-dose study). In the multiple-dose study, the results on report day 57, the point at which the effects of administration at a biweekly interval could exist observed, were chosen to compare the lipid and lipoprotein furnishings.
In the unmarried-dose studies, the enrollment of six subjects per dose group was estimated to provide a power of at least 80% to detect a mean (±SD) departure of thirty±15% between REGN727 and placebo in the mean percent change in LDL cholesterol from baseline, with a two-sided test at a significance level of 0.05. In the multiple-dose study, comparing the pooled grouping of 6 placebo-treated subjects with familial hypercholesterolemia with the five who received REGN727 in each dose group was likewise estimated to provide a power of at least lxxx% to discover a treatment difference of 30±15% on the basis of like assumptions.
Furnishings of treatments on efficacy variables were assessed with the use of analysis-of-covariance models, with the report group as the fixed upshot and the relevant baseline value equally a covariate. Least-squares means of differences between the treatment group and the placebo grouping, 95% confidence intervals, and P values for comparing betwixt the handling grouping and the placebo group co-ordinate to visit were obtained inside the framework of assay of covariance. For values for triglycerides and lipoprotein(a), a rank-based analysis of covariance was used. Categorical variables were analyzed with the use of Fisher'south verbal test. A P value of 0.05 was considered to indicate statistical significance. No adjustments were made for multiple comparisons.
Results
Study Populations
Table i.
Table ane. Baseline Characteristics of Healthy Volunteers and Subjects with Familial Hypercholesterolemia (FH) or Non-FH. Enrollment and outcomes for subjects in all three studies are summarized in Figure S1 in the Supplementary Appendix. For the unmarried-dose studies, 40 and 32 subjects were enrolled in the intravenous and subcutaneous studies, respectively. For the multiple-dose report, 21 subjects with heterozygous familial hypercholesterolemia and 41 subjects with nonfamilial hypercholesterolemia were enrolled. One subject with nonfamilial hypercholesterolemia who was randomly assigned to receive REGN727 did not receive the drug owing to poor venous admission for the required pharmacokinetic sampling. Baseline characteristics of the subjects in the iii studies are provided in Tabular array i.
Rubber
Two subjects in the unmarried-dose studies had serious adverse events: a 33-year-old man receiving intravenous placebo, who had intestinal pain and rectal bleeding on study twenty-four hour period 83, and a xix-twelvemonth-one-time man with a history of appendectomy receiving l mg of subcutaneous REGN727, who had a small-bowel obstruction that was diagnosed on study mean solar day 75.
In the unmarried-dose intravenous study, a 54-year-old man who received 12 mg per kilogram of REGN727 had a slightly elevated full bilirubin level at screening and throughout nigh of the trial (highest value, 2.v mg per deciliter [43 μmol per liter] on day 15). Too, a 26-year-onetime man who received 0.3 mg of REGN727 per kilogram had a transient meridian of serum creatine kinase to more than ten times the upper limit of the normal range (highest value, 5382 U per liter on solar day 22, with a return to less than 3 times the upper limit of the normal range on study day 43) in temporal proximity to strenuous physical practice; at that place was no associated muscle hurting or serum creatinine elevation. No bailiwick in the unmarried-dose trials discontinued participation early because of an adverse event.
In the multiple-dose report, no subject had a serious adverse consequence, and all subjects completed all visits. No subject field receiving REGN727 in any of the three studies had an elevation of aspartate aminotransferase or alanine aminotransferase to more than than 3 times the upper limit of the normal range or an increase in creatinine to more than than i.seven mg per deciliter (150 μmol per liter). Also in this study, amidst subjects who received REGN727, 5 of 39 subjects (13%) who were besides receiving atorvastatin had creatine kinase levels that were more 3 times the upper limit of the normal range, but none had levels that were more than 10 times the upper limit of the normal range; of the 8 subjects who were non receiving atorvastatin, none had such increased levels. Among subjects who received placebo, creatine kinase levels of more than iii times the upper limit of the normal range occurred in 0 of 12 subjects who were receiving atorvastatin and in 2 of 2 subjects who were non receiving atorvastatin. There were a few injection-site reactions, which were balmy.
The proportions of subjects who had at to the lowest degree one handling-emergent adverse issue were similar among subjects who received intravenous REGN727 and those who received placebo. Equally compared with subjects who received placebo, a college proportion of subjects who received subcutaneous REGN727 had an adverse event in the unmarried-dose and the multiple-dose studies. Headache was the virtually common agin event. Boosted rubber data (treatment-emergent adverse events, clinical chemical analyses, and hematologic measurements) are provided in Tables S1 through S7 in the Supplementary Appendix.
Lipid and Lipoprotein Response
Table 2.
Table 2. Baseline and Lowest Values for Depression-Density Lipoprotein (LDL) Cholesterol in Single-Dose Studies, According to Route of Administration. Figure one. 
Figure 1. Mean Percent Change from Baseline in LDL Cholesterol Values amid Healthy Volunteers in Unmarried-Dose Studies. Among subjects receiving increasing single doses of REGN727, values are shown for intravenous administration (Panel A) and subcutaneous administration (Panel B). LDL cholesterol values were calculated with the utilise of the Friedewald formula. The I bars point standard errors.
Single administration of intravenous or subcutaneous REGN727 in healthy volunteers resulted in a least-squares mean deviation in the change from baseline in LDL cholesterol of upwards to 65 per centum points, as compared with placebo (Table two and Figure 1). The degree and duration of LDL cholesterol lowering were dose-dependent, with higher doses producing prolonged reductions that were sustained up to day 64 (Figure 1).
Table 3.
Table three. Baseline and Day 57 Values for LDL Cholesterol among Subjects with Familial Hypercholesterolemia (FH) or Non-FH in the Multiple-Dose Written report, According to Atorvastatin Use. Effigy two. 
Figure 2. Mean Pct Modify from Baseline in LDL Cholesterol Values among Subjects with Familial Hypercholesterolemia (FH) or Non-FH. Among subjects receiving multiple doses of REGN727, values are shown for subcutaneous doses of 50 mg (Panel A), 100 mg (Panel B), and 150 mg (Panel C) in subjects who were besides receiving atorvastatin and for a subcutaneous dose of 150 mg in subjects who were not receiving atorvastatin (Panel D). All LDL cholesterol values, which were calculated with the use of the Friedewald formula, were bachelor for all subjects at all visits in this report. The I bars indicate standard errors.
In the multiple-dose report, REGN727 doses of 50, 100, and 150 mg reduced measured LDL cholesterol in the combined atorvastatin-treated populations to 77.five mg per deciliter (2.00 mmol per liter), 61.3 mg per deciliter (1.59 mmol per liter), and 53.8 mg per deciliter (i.39 mmol per liter), for a difference in the alter from baseline of −39.2, −53.7, and −61.0 percentage points, respectively, as compared with placebo (P<0.001 for all comparisons) (Table S8 in the Supplementary Appendix). The degree and duration of LDL cholesterol reduction in the unlike dose groups corresponded to the reduction of gratis PCSK9 in plasma (Fig. S2 in the Supplementary Appendix). The mean difference in the change from baseline in LDL cholesterol, as compared with placebo, exceeded −46 percentage points 2 weeks later get-go administration of 150 mg in all three cohorts studied (Tabular array 3 and Figure ii). All but one patient (who had heterozygous familial hypercholesterolemia and was receiving atorvastatin) who received 150 mg in the multiple-dose study had a difference in the change from baseline in LDL cholesterol of at least −40 percentage points, equally compared with placebo (Fig. S3C in the Supplementary Appendix).
The LDL cholesterol response was similar in all subjects, regardless of whether they had familial or nonfamilial hypercholesterolemia or whether they were treated with atorvastatin or with a modified nutrition alone. Corresponding changes were observed in levels of total and non–high-density lipoprotein (HDL) cholesterol and apolipoprotein B, and reductions were besides observed in lipoprotein(a) (Tables S8 through S10 and Fig. S4 through S8 in the Supplementary Appendix). In addition, every bit compared with placebo, increases were seen in both HDL cholesterol (upwardly to 18 pct points) and apolipoprotein A1 (up to 13 percentage points) in subjects taking atorvastatin.
Discussion
In 3 early-phase randomized trials, nosotros evaluated the effects of REGN727, a monoclonal antibody that blocks the interaction of PCSK9 with LDL receptors. In all three trials, REGN727 significantly reduced LDL cholesterol levels, every bit compared with placebo. This consequence was significant both in good for you volunteers and in subjects with familial or nonfamilial forms of hypercholesterolemia. The effect was also significant in subjects who were concomitantly taking atorvastatin.
Our results confirm a role for PCSK9 in the regulation of LDL cholesterol levels. In improver, the demonstration of a expert correlation between a reduction in gratis PCSK9 levels and a reduction in LDL cholesterol levels after the administration of REGN727 in humans supports previous reports from studies involving rodents and nonhuman primates that PCSK9 in the circulation, not intracellular PCSK9, is primarily responsible for regulating hepatic LDL receptors.10,eleven
In our studies, the furnishings of REGN727 and atorvastatin in lowering LDL cholesterol appeared to be condiment, not synergistic, since hateful percent reductions were like when REGN727 was administered lonely or in subjects already receiving atorvastatin. Although REGN727 and atorvastatin both lower LDL cholesterol by increasing hepatic LDL-receptor activity, atorvastatin does and then primarily past enhancing the production of receptors, whereas REGN727 decreases the degradation of receptors. REGN727 induced a maximum lowering of LDL cholesterol within ii weeks, whereas statins typically have longer.12 The different modes of activeness of these agents may account for the more than rapid effect seen with REGN727.
Statins increase the expression of sterol regulatory element–bounden protein two, a transcription factor that in turn enhances the expression of both the low-density lipoprotein receptor factor (LDLR) and PCSK9. This apparent counter-regulation may limit the effectiveness of statins by PCSK9-mediated devastation of LDL receptors.xiii Subjects with no detectable circulating PCSK9 have LDL cholesterol levels of approximately 15 mg per deciliter (0.39 mmol per liter), a level that is non achieved by even the most efficacious statins.14,15 Thus, the charge per unit of devastation of LDL receptors appears to be an important determinant of LDL cholesterol levels. Statin-stimulated product of PCSK9, while not apparently altering the maximum LDL-lowering effect, might affect the elapsing of action of therapeutic antibodies because higher rates of PCSK9 product may result in greater clearance of costless antibody. Thus, the elapsing of action of REGN727 may be longer in subjects who are treated with nutrition modification alone than in those treated with atorvastatin (Figure 2C and 2d).
As might be predictable with an amanuensis that significantly reduces LDL cholesterol, REGN727 also significantly reduced apolipoprotein B levels. In subjects receiving both REGN727 and atorvastatin, there was a significant increment in HDL cholesterol levels. A reduction in lipoprotein(a) was also seen, although this consequence was not pregnant at all doses. Other agents that up-regulate LDL receptors, such as statins and bile acid sequestrants, likewise raise HDL cholesterol levels, perchance by reducing the transfer of cholesterol from HDL to LDL particles. However, none of these drugs reduce levels of lipoprotein(a), which is non idea to be cleared through LDL receptors.16 The caption for a possible effect on lipoprotein(a) is thus uncertain, and additional work is required to ostend and explicate this observation.
In our trials, nosotros saw no clear evidence of drug-related agin events. 5 subjects in the multiple-dose trial who were receiving REGN727 with concomitant atorvastatin had cursory elevations in creatine kinase to more than iii times the upper limit of the normal range. Given the pocket-size number of subjects and the short duration of exposure, our power to evaluate the safe contour of REGN727 in these trials was express. Boosted studies will be required to make a clearer assessment of this agent for potential adverse effects.
In summary, we evaluated the effects of REGN727, a fully human monoclonal antibody that blocks the interaction of PCSK9 with LDL receptors. In three small early-phase trials, REGN727 significantly reduced LDL cholesterol levels. This issue was meaning both in good for you volunteers and in subjects with familial or nonfamilial forms of hypercholesterolemia. The effect was also significant in subjects who were concomitantly taking atorvastatin.
Supplementary Material
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