Stephen Holleran

Hormonal Regulation of Lipoprotein(a) Levels: Effects of Estrogen Replacement Therapy on Lipoprotein(a) and Acute Phase Reactants in Postmenopausal Women

Estrogen lowers lipoprotein(a) [Lp(a)] levels, but the mechanisms involved have not been clarified. To address the relationship between estrogenic effects on Lp(a) and serum lipids, and on other plasma proteins of hepatic origin, 15 healthy postmenopausal women participated in a randomized, double-blinded, placebo-controlled, crossover study with 4 weeks of oral conjugated estrogens (0.625 mg/d) and placebo, separated by a 6-week period. Lp(a) levels decreased during estrogen treatment in 14 of the 15 subjects (mean decrease, 23%; P < .001). In response to estrogen, apolipoprotein A-I (apoA-I), HDL cholesterol, and triglyceride levels increased by 12% (P = .001), 11% (P < .001), and 10% (P = .02), respectively. Apolipoprotein B (apoB) and LDL cholesterol levels decreased by 7% (P = .01) and 12% (P = .03), respectively, ApoB, LDL cholesterol, and Lp(a) levels fell within 1 week of treatment, whereas apoA-I and HDL cholesterol levels rose more slowly. Levels of acid alpha 1-glycoprotein (AAG) and haptoglobin (HPT), two hepatically derived acute phase proteins, also decreased during estrogen treatment by 18% (P < .001) and 25% (P = .002), respectively. Although the changes in AAG and HPT in response to estrogen were highly correlated (r = .67, P = .009), we were unable to detect a correlation between change in either acute phase protein and change in Lp(a) (r = -.14 and -.24, P = .64 and .41). The lack of correlation between the changes in two acute phase reactants and Lp(a) suggests different underlying mechanisms for the effects of estrogen on these liver-derived proteins.

Effects of PCSK9 Inhibition With Alirocumab on Lipoprotein Metabolism in Healthy Humans

Background: Alirocumab, a monoclonal antibody to proprotein convertase subtilisin/kexin type 9 (PCSK9), lowers plasma low-density lipoprotein (LDL) cholesterol and apolipoprotein B100 (apoB). Although studies in mice and cells have identified increased hepatic LDL receptors as the basis for LDL lowering by PCSK9 inhibitors, there have been no human studies characterizing the effects of PCSK9 inhibitors on lipoprotein metabolism. In particular, it is not known whether inhibition of PCSK9 has any effects on very low-density lipoprotein or intermediate-density lipoprotein (IDL) metabolism. Inhibition of PCSK9 also results in reductions of plasma lipoprotein (a) levels. The regulation of plasma Lp(a) levels, including the role of LDL receptors in the clearance of Lp(a), is poorly defined, and no mechanistic studies of the Lp(a) lowering by alirocumab in humans have been published to date. Methods: Eighteen (10 F, 8 mol/L) participants completed a placebo-controlled, 2-period study. They received 2 doses of placebo, 2 weeks apart, followed by 5 doses of 150 mg of alirocumab, 2 weeks apart. At the end of each period, fractional clearance rates (FCRs) and production rates (PRs) of apoB and apo(a) were determined. In 10 participants, postprandial triglycerides and apoB48 levels were measured. Results: Alirocumab reduced ultracentrifugally isolated LDL-C by 55.1%, LDL-apoB by 56.3%, and plasma Lp(a) by 18.7%. The fall in LDL-apoB was caused by an 80.4% increase in LDL-apoB FCR and a 23.9% reduction in LDL-apoB PR. The latter was due to a 46.1% increase in IDL-apoB FCR coupled with a 27.2% decrease in conversion of IDL to LDL. The FCR of apo(a) tended to increase (24.6%) without any change in apo(a) PR. Alirocumab had no effects on FCRs or PRs of very low-density lipoproteins-apoB and very low-density lipoproteins triglycerides or on postprandial plasma triglycerides or apoB48 concentrations. Conclusions: Alirocumab decreased LDL-C and LDL-apoB by increasing IDL- and LDL-apoB FCRs and decreasing LDL-apoB PR. These results are consistent with increases in LDL receptors available to clear IDL and LDL from blood during PCSK9 inhibition. The increase in apo(a) FCR during alirocumab treatment suggests that increased LDL receptors may also play a role in the reduction of plasma Lp(a). Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01959971.

Anacetrapib lowers LDL by increasing ApoB clearance in mildly hypercholesterolemic subjects

BACKGROUND Individuals treated with the cholesteryl ester transfer protein (CETP) inhibitor anacetrapib exhibit a reduction in both LDL cholesterol and apolipoprotein B (ApoB) in response to monotherapy or combination therapy with a statin. It is not clear how anacetrapib exerts these effects; therefore, the goal of this study was to determine the kinetic mechanism responsible for the reduction in LDL and ApoB in response to anacetrapib. METHODS We performed a trial of the effects of anacetrapib on ApoB kinetics. Mildly hypercholesterolemic subjects were randomized to background treatment of either placebo (n = 10) or 20 mg atorvastatin (ATV) (n = 29) for 4 weeks. All subjects then added 100 mg anacetrapib to background treatment for 8 weeks. Following each study period, subjects underwent a metabolic study to determine the LDL-ApoB-100 and proprotein convertase subtilisin/kexin type 9 (PCSK9) production rate (PR) and fractional catabolic rate (FCR). RESULTS Anacetrapib markedly reduced the LDL-ApoB-100 pool size (PS) in both the placebo and ATV groups. These changes in PS resulted from substantial increases in LDL-ApoB-100 FCRs in both groups. Anacetrapib had no effect on LDL-ApoB-100 PRs in either treatment group. Moreover, there were no changes in the PCSK9 PS, FCR, or PR in either group. Anacetrapib treatment was associated with considerable increases in the LDL triglyceride/cholesterol ratio and LDL size by NMR. CONCLUSION These data indicate that anacetrapib, given alone or in combination with a statin, reduces LDL-ApoB-100 levels by increasing the rate of ApoB-100 fractional clearance. TRIAL REGISTRATION ClinicalTrials.gov NCT00990808. FUNDING Merck & Co. Inc., Kenilworth, New Jersey, USA. Additional support for instrumentation was obtained from the National Center for Advancing Translational Sciences (UL1TR000003 and UL1TR000040).

Lack of effect of lovastatin therapy on the parameters of whole-body cholesterol metabolism.

UNLABELLED The effects of lovastatin therapy on the parameters of body cholesterol metabolism were explored in nine hypercholesterolemic patients. Long-term cholesterol turnover studies were performed before therapy, and were repeated after 15 mo of lovastatin therapy (40 mg/d) while continuing on therapy. The major question addressed was whether a reduction in plasma cholesterol level with lovastatin would be associated with a reduction in the whole-body production rate of cholesterol or with the sizes of exchangeable body cholesterol pools as determined by the three-pool model of cholesterol turnover. The mean plasma cholesterol level decreased 19.4% (from 294 to 237 mg/dl), and low-density lipoprotein cholesterol decreased 23.8% (from 210 to 159 mg/dl) with lovastatin therapy. Changes in high-density lipoprotein cholesterol level were not significant. The cholesterol production rate did not change significantly with therapy (1.09 +/- 0.10 [mean +/- S.D.] vs. 1.17 +/- 0.09 g/d). By comparison, colestipol and niacin treatment in three other subjects more than doubled the cholesterol production rate (1.14 +/- 0.28 vs. 2.42 +/- 0.34 g/d). Thus, hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibition by lovastatin at the therapeutic dose used here did not change the steady-state rate of whole-body cholesterol synthesis. Despite the changes in plasma cholesterol levels, no significant changes were seen in the values of M1, of M3 or of Mtot, the sizes of the pools of rapidly, of slowly, and of total body exchangeable cholesterol. CONCLUSION lovastatin therapy to lower plasma cholesterol does not lead to corresponding reductions in body cholesterol pools or to a reduction in the rate of whole-body cholesterol synthesis. In the new steady state that exists during long-term lovastatin therapy, along with increased expression of the genes for HMG-CoA reductase and the LDL receptor, the body compensates for the effects of the drug so that cholesterol production rate and tissue pool sizes are not changed from pretreatment values.

Effects of a 1,3-diacylglycerol oil-enriched diet on postprandial lipemia in people with insulin resistance.

Postprandial hypertriglyceridemia is common in individuals with insulin resistance, and diets enriched in 1,3-diacylglycerol (DAG) may reduce postprandial plasma triglycerides (PPTGs). We enrolled 25 insulin-resistant, nondiabetic individuals in a double-blind, randomized crossover trial to test the acute and chronic effects of a DAG-enriched diet on PPTG. Participants received either DAG or triacylglycerol (TAG) oil, in food products, for 5 weeks. Fasting lipids, and two separate postprandial tests, one with DAG oil and one with TAG oil, were performed at the end of each 5 week diet period. We found no acute or chronic effects of DAG oil on PPTG. Thus, neither the DAG oil PPTG (h/mg/dl) on a chronic TAG diet [area under the curve (AUC) = 503 ± 439] nor the TAG oil PPTG on a chronic DAG diet (AUC = 517 ± 638) was different from the TAG oil PPTG on a chronic TAG diet (AUC = 565 ± 362). Five weeks of a DAG-enriched diet had no acute or chronic effects on PPTG in insulin-resistant individuals. We suggest further studies to evaluate the effects of DAG on individuals with low and high TG levels.

Cholesteryl Ester Transfer Protein Inhibition With Anacetrapib Decreases Fractional Clearance Rates of High-Density Lipoprotein Apolipoprotein A-I and Plasma Cholesteryl Ester Transfer Protein

Objective—Anacetrapib (ANA), an inhibitor of cholesteryl ester transfer protein (CETP) activity, increases plasma concentrations of high-density lipoprotein cholesterol (HDL-C), apolipoprotein A-I (apoA)-I, apoA-II, and CETP. The mechanisms responsible for these treatment-related increases in apolipoproteins and plasma CETP are unknown. We performed a randomized, placebo (PBO)-controlled, double-blind, fixed-sequence study to examine the effects of ANA on the metabolism of HDL apoA-I and apoA-II and plasma CETP. Approach and Results—Twenty-nine participants received atorvastatin (ATV) 20 mg/d plus PBO for 4 weeks, followed by ATV plus ANA 100 mg/d for 8 weeks (ATV-ANA). Ten participants received double PBO for 4 weeks followed by PBO plus ANA for 8 weeks (PBO-ANA). At the end of each treatment, we examined the kinetics of HDL apoA-I, HDL apoA-II, and plasma CETP after D3-leucine administration as well as 2D gel analysis of HDL subspecies. In the combined ATV-ANA and PBO-ANA groups, ANA treatment increased plasma HDL-C (63.0%; P<0.001) and apoA-I levels (29.5%; P<0.001). These increases were associated with reductions in HDL apoA-I fractional clearance rate (18.2%; P=0.002) without changes in production rate. Although the apoA-II levels increased by 12.6% (P<0.001), we could not discern significant changes in either apoA-II fractional clearance rate or production rate. CETP levels increased 102% (P<0.001) on ANA because of a significant reduction in the fractional clearance rate of CETP (57.6%, P<0.001) with no change in CETP production rate. Conclusions—ANA treatment increases HDL apoA-I and CETP levels by decreasing the fractional clearance rate of each protein.

Complex effects of inhibiting hepatic apolipoprotein B100 synthesis in humans

Targeting apoB synthesis with mipomersen (KYNAMRO) can be effective for lowering plasma levels of apoB lipoproteins without reducing the secretion of VLDL. Making sense of antisense Mipomersen is an FDA-approved antisense oligonucleotide that lowers low density lipoprotein (LDL) in patients with high cholesterol by targeting apolipoprotein B (apoB) synthesis. Although safe, how mipomersen works exactly in humans is unclear. Reyes-Soffer and colleagues found in healthy volunteers that the drug reduced levels of LDL and its precursor, very low density lipoproteins (VLDL), by increasing clearance of both of these vessel-clogging lipoproteins rather than reducing their secretion from the liver. The direct clearance of VLDL led to reduced production of LDL. Studies in mice and cell lines demonstrated how the liver compensates for reduced apoB synthesis to potentially avoid hepatic steatosis. Mipomersen is a 20mer antisense oligonucleotide (ASO) that inhibits apolipoprotein B (apoB) synthesis; its low-density lipoprotein (LDL)–lowering effects should therefore result from reduced secretion of very-low-density lipoprotein (VLDL). We enrolled 17 healthy volunteers who received placebo injections weekly for 3 weeks followed by mipomersen weekly for 7 to 9 weeks. Stable isotopes were used after each treatment to determine fractional catabolic rates and production rates of apoB in VLDL, IDL (intermediate-density lipoprotein), and LDL, and of triglycerides in VLDL. Mipomersen significantly reduced apoB in VLDL, IDL, and LDL, which was associated with increases in fractional catabolic rates of VLDL and LDL apoB and reductions in production rates of IDL and LDL apoB. Unexpectedly, the production rates of VLDL apoB and VLDL triglycerides were unaffected. Small interfering RNA–mediated knockdown of apoB expression in human liver cells demonstrated preservation of apoB secretion across a range of apoB synthesis. Titrated ASO knockdown of apoB mRNA in chow-fed mice preserved both apoB and triglyceride secretion. In contrast, titrated ASO knockdown of apoB mRNA in high-fat–fed mice resulted in stepwise reductions in both apoB and triglyceride secretion. Mipomersen lowered all apoB lipoproteins without reducing the production rate of either VLDL apoB or triglyceride. Our human data are consistent with long-standing models of posttranscriptional and posttranslational regulation of apoB secretion and are supported by in vitro and in vivo experiments. Targeting apoB synthesis may lower levels of apoB lipoproteins without necessarily reducing VLDL secretion, thereby lowering the risk of steatosis associated with this therapeutic strategy.

Registry-linked electronic influenza vaccine provider reminders: a cluster-crossover trial.

OBJECTIVE: To determine the impact of a vaccination reminder in an electronic health record supplemented with data from an immunization information system (IIS). METHODS: A noninterruptive influenza vaccination reminder, based on a real-time query of hospital and city IIS, was used at 4 urban, academically affiliated clinics serving a low-income population. Using a randomized cluster-crossover design, each study site had “on” and “off” period during the fall and winter of 2011–2012. Influenza vaccination during a clinic visit was assessed for 6-month to 17-year-old patients. To assess sustainability, the reminder was active at all sites during the 2012–2013 season. RESULTS: In the 2011–2012 season, 8481 unique non-up-to-date children had visits. Slightly more non–up-to-date children seen when the reminder was ‘on’ were vaccinated than when ‘off’ (76.2% vs 73.8%; P = .027). Effects were seen in the winter (67.9% vs 62.2%; P = .005), not fall (76.8% vs 76.5%). The reminder also increased documentation of the reason for vaccine non-administration (68.1% vs 41.5%; P < .0001). During the 2011–2012 season, the reminder displayed for 8630 unique visits, and clinicians interacted with it in 83.1% of cases where patients required vaccination. During the 2012–2013 season, it displayed for 22 248 unique visits; clinicians interacted with it in 84.8% of cases. CONCLUSIONS: An IIS-linked influenza vaccination reminder increased vaccination later in the winter when fewer vaccine doses are usually given. Although the reminder did not require clinicians to interact with it, they frequently did; utilization did not wane over time.

Impacting Delayed Pediatric Influenza Vaccination: A Randomized Controlled Trial of Text Message Reminders

BACKGROUND Influenza vaccination coverage is low, especially among low-income populations. Most doses are generally administered early in the influenza season, yet sustained vaccination efforts are crucial for achieving optimal coverage. The impact of text message influenza vaccination reminders was recently demonstrated in a low-income population. Little is known about their effect on children with delayed influenza vaccination or the most effective message type. PURPOSE To determine the impact of educational plus interactive text message reminders on influenza vaccination of urban low-income children unvaccinated by late fall. DESIGN Randomized controlled trial. SETTING/PARTICIPANTS Parents of 5,462 children aged 6 months-17 years from four academically affiliated pediatric clinics who were unvaccinated by mid-November 2011. INTERVENTION Eligible parents were stratified by their childs age and pediatric clinic site and randomized using a 1:1:1 allocation to educational plus interactive text message reminders, educational-only text message reminders, or usual care. Using an immunization registry-linked text messaging system, parents of intervention children received up to seven weekly text message reminders. One of the messages sent to parents in the educational plus interactive text message arm allowed selection of more information about influenza and influenza vaccination. MAIN OUTCOME MEASURES Influenza vaccination by March 31, 2012. Data were collected and analyzed between 2012 and 2014. RESULTS Most children were publicly insured and Spanish speaking. Baseline demographics were similar between groups. More children of parents in the educational plus interactive text message arm were vaccinated (38.5%) versus those in the educational-only text message (35.3%; difference=3.3%, 95% CI=0.02%, 6.5%; relative risk ratio (RRR)=1.09, 95% CI=1.002, 1.19) and usual care (34.8%; difference=3.8%, 95% CI=0.6%, 7.0%; RRR=1.11, 95% CI=1.02-1.21) arms. CONCLUSIONS Text message reminders with embedded educational information and options for interactivity have a small positive effect on influenza vaccination of urban, low-income, minority children who remain unvaccinated by late fall.

CETP (Cholesteryl Ester Transfer Protein) Inhibition With Anacetrapib Decreases Production of Lipoprotein(a) in Mildly Hypercholesterolemic Subjects

Objective— Lp(a) [lipoprotein (a)] is composed of apoB (apolipoprotein B) and apo(a) [apolipoprotein (a)] and is an independent risk factor for cardiovascular disease and aortic stenosis. In clinical trials, anacetrapib, a CETP (cholesteryl ester transfer protein) inhibitor, causes significant reductions in plasma Lp(a) levels. We conducted an exploratory study to examine the mechanism for Lp(a) lowering by anacetrapib. Approach and Results— We enrolled 39 participants in a fixed-sequence, double-blind study of the effects of anacetrapib on the metabolism of apoB and high-density lipoproteins. Twenty-nine patients were randomized to atorvastatin 20 mg/d, plus placebo for 4 weeks, and then atorvastatin plus anacetrapib (100 mg/d) for 8 weeks. The other 10 subjects were randomized to double placebo for 4 weeks followed by placebo plus anacetrapib for 8 weeks. We examined the mechanisms of Lp(a) lowering in a subset of 12 subjects having both Lp(a) levels >20 nmol/L and more than a 15% reduction in Lp(a) by the end of anacetrapib treatment. We performed stable isotope kinetic studies using 2H3-leucine at the end of each treatment to measure apo(a) fractional catabolic rate and production rate. Median baseline Lp(a) levels were 21.5 nmol/L (interquartile range, 9.9–108.1 nmol/L) in the complete cohort (39 subjects) and 52.9 nmol/L (interquartile range, 38.4–121.3 nmol/L) in the subset selected for kinetic studies. Anacetrapib treatment lowered Lp(a) by 34.1% (P⩽0.001) and 39.6% in the complete and subset cohort, respectively. The decreases in Lp(a) levels were because of a 41% reduction in the apo(a) production rate, with no effects on apo(a) fractional catabolic rate. Conclusions— Anacetrapib reduces Lp(a) levels by decreasing its production. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00990808.