Novel therapeutic agents for lowering low density lipoprotein cholesterol

Pharmacology & Therapeutics 135 (2012) 31–43

Contents lists available at SciVerse ScienceDirect

Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/pharmthera

Associate editor: Y.S. Chatzizisis

Novel therapeutic agents for lowering low density lipoprotein cholesterol Tisha R. Joy ⁎ Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada, N6A 5K8

a r t i c l e

i n f o

Keywords: Anti-sense oligonucleotides Apolipoprotein B PCSK9 inhibitor Thyromimetics MTP inhibitor

a b s t r a c t Elevated low density lipoprotein cholesterol (LDL-C) levels have been associated with an increased risk for cardiovascular disease (CVD). Despite a 25–30% reduction in CVD risk with LDL-C reducing strategies, there is still a significant residual risk. Moreover, achieving target LDL-C values in individuals at high CVD risk is sometimes limited because of tolerability and/or efficacy. Thus, novel therapeutic agents are currently being developed to lower LDL-C levels further. This review will highlight some of these therapeutic agents including anti-sense oligonucleotides focused on apolipoprotein B, proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, microsomal triglyceride transfer protein inhibitors, and thyromimetics. For each therapeutic class, an overview of the mechanism of action, pharmacokinetic data, and efficacy/safety evidence will be provided. © 2012 Elsevier Inc. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . 2. Anti-sense oligonucleotides directed at apolipoprotein B 3. Proprotein convertase subtilisin/kexin type 9 inhibitors . 4. Microsomal triglyceride transfer protein inhibitors . . . 5. Thyromimetics . . . . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . 7. Conflicts of interest/disclosures . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Lowering low-density lipoprotein cholesterol (LDL-C) levels reduces the risk of cardiovascular disease (CVD) by approximately 25–30%. Currently, LDL-C reduction is primarily achieved through the use of statins although there are several other marketed medications that can lower LDL-C levels, albeit to a lesser degree. Despite these strategies for LDL-C reduction, there still remains a significant Abbreviations: Apo, apolipoprotein; ASO, antisense oligonucleotide; CHD, coronary heart disease; CVD, cardiovascular disease; CYP, cytochrome P450; ED50, effective dose; FH, familial hypercholesterolemia; HDL-C, high-density lipoprotein cholesterol; IC50, half maximal inhibitory concentration; IHTG, intrahepatic triglyceride; IV, intravenous; LDL-C, low-density lipoprotein cholesterol; LDLR, low-density lipoprotein receptor; Lp(a), lipoprotein (a); mRNA, messenger ribonucleic acid; MTP, microsomal triglyceride transfer protein; PON, paraoxonase; RCT, randomized placebo-controlled trial; SC, subcutaneous; siRNA, small interfering ribonucleic acid; t1/2, half-life; TG, triglyceride; TR, thyroid hormone receptor; VLDL, very low density lipoprotein. ⁎ Department of Medicine, University of Western Ontario, B5-107, 268 Grosvenor Street, St. Joseph's Hospital, London, Ontario, Canada, N6A 4V2. Tel.: +1 519 646 6296; fax: +1 519 646 6372. E-mail address: [email protected]. 0163-7258/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2012.03.005

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31 31 31 33 32 37 32 39 33 41 33 41 41

residual cardiovascular risk. Moreover, achieving target LDL-C values in individuals with high cardiovascular risk is sometimes limited because of tolerability and/or efficacy. Indeed, up to 40% of high-risk and 80% of very-high-risk individuals do not achieve their respective LDL-C goals (Yan et al., 2006). Thus, alternative physiologic strategies to further lower LDL-C levels effectively and safely are being actively sought. This paper will discuss several novel LDL-C lowering pharmacologic agents under consideration, including anti-sense oligonucleotides (ASOs) to apolipoprotein B (apo B), proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, microsomal triglyceride transfer protein (MTP) inhibitors, and thyromimetics. For each category, an overview of the mechanism of action, pharmacokinetic data, and efficacy/safety evidence will be provided. 2. Anti-sense oligonucleotides directed at apolipoprotein B 2.1. Mechanism of action Antisense oligoneucleotides (ASOs) are short, deoxyribonucleotide strands (8 to 50 nucleotides in length) that bind using Watson–Crick

32

T.R. Joy / Pharmacology & Therapeutics 135 (2012) 31–43

hybridization to a target messenger ribonucleic acid (mRNA) to cause inhibition of gene expression (Fig. 1). This inhibition of gene expression can occur through several mechanisms, including activation of enzymes (RNAse H or Argonaute 2) which in turn cause targeted degradation of the mRNA of interest; translational arrest through interference with ribosomal activity; or deterrence of maturation of the mRNA itself (Bennett & Swayze, 2010). The design of ASOs must ensure that these molecules have a high specificity for their target gene and are relatively resistant to degradation by endogenous nucleases. This is achieved through varying chemical modifications of the ASO, such as incorporation of a phosphodiester- or phosphorothioate-modified backbone, alteration of the ring structure, and creation of a central gap region of typically 10 modified deoxynucleotides flanked on the 5′ and 3′ ends (termed wings). The gap region activates RNAse H while the wings prevent degradation of the ASO (Ito, 2007). ISIS 301012 or mipomersen is a second-generation 20 nucleotide ASO inhibiting APOB gene expression via RNAse H activation (Ito, 2007). APOB encodes both apo B-48, required for chylomicron assembly, and apo B-100, the major lipoprotein present in LDL, very-low density lipoprotein (VLDL), and intermediate-density lipoprotein (IDL) particles. Since there is a single copy of apo B-100 in each of these atherogenic particles, apo B-100 levels are indicative of the number of atherogenic particles. Apo B-100 serves as the ligand for the LDL receptor (LDLR) and thus plays an important role in the clearance of LDL particles from the bloodstream (Hussain et al., 1999). Importantly, mutations in APOB have been associated with a clinical phenotype of significantly elevated LDL-C levels, physical stigmata (xanthelasmas, xanthomas, corneal arcus), and premature cardiovascular disease (Marsh et al., 2002). Mipomersen reduces the levels of apo B-100 and not apo B-48. Thus, using ASOs like mipomersen to directly reduce the production of apo B-100 could have a significant impact on LDL-C levels and atherosclerotic outcomes. 2.2. Pharmacokinetic and pharmacodynamic data Mipomersen is ≥85% protein-bound (Yu et al., 2007). The initial distribution half-life (t1/2) is rapid at 1.26 ± 0.16 h (Yu et al., 2007). The mean time to maximum plasma concentration is 3.4 to 4.0 h, and a loading dose is most likely not needed (Akdim et al., 2011). Urinary excretion is a minor contributor to initial plasma clearance

ASO

apo B formed Normal

No apo B formed

mRNA RNA

DNA

Fig. 1. Mechanism of action of anti-sense oligonucleotides (ASOs).Typically, once DNA is transcribed to messenger RNA (mRNA), translation of mRNA leads to formation of apolipoprotein B. ASOs bind to the mRNA and thereby inhibit apo B formation and this decreased apo B production, in turn, decreases formation of apo B containing lipoproteins, including LDL.

of the drug with b4% being excreted in urine. However, distribution to tissue is the primary mechanism of plasma clearance as the volume of distribution of mipomersen is quite large at 48.3 ± 14.7 L/kg with the tissues with highest concentration of mipomersen being liver and kidney in rodents and non-human primates (Yu et al., 2007). Mipomersen was not found to be present in the brain (Yu et al., 2007). The terminal elimination tissue t1/2 ranges from 23 (±1) day for the 50 mg dose to 30 (±11) days in the 200 mg dose (Kastelein et al., 2006). Whole-body clearance involves slow metabolism by endo- and exonucleases, followed by urinary excretion, and to a lesser extent, fecal excretion (Yu et al., 2007). 2.3. Efficacy Preclinical data for a murine ASO against apo B named ISIS 147764 has demonstrated significant dose-dependent reductions in hepatic apo B mRNA and LDL-C of 60–90% in addition to decreases in aortic atherosclerosis of 46–89% (Mullick et al., 2011). Meanwhile, use of mipomersen in transgenic mice overexpressing human apo B-100 or those overexpressing human apo B-100 and human apo (a) (to generate lipoprotein (a) [Lp(a)] particles) resulted in ~75% reduction in LDL-C and ~60% reduction in Lp(a) without a significant change in apo(a) levels at 4 weeks (Merki et al., 2008). The reduction in apo B and Lp(a) levels was sustained post-discontinuation of mipomersen, taking ~10 weeks to return to baseline values (Merki et al., 2008). Since apo B is needed for the movement of VLDL out of the liver into peripheral tissues, inhibition of apo B by ASOs could theoretically be associated with accumulation of VLDL and thus, hepatic steatosis. However, murine models testing ISIS 147764 have not demonstrated significant hepatic steatosis (Crooke et al., 2005; Mullick et al., 2011). Mipomersen effects a dose-dependent decrease in apo B, VLDL, and LDL-C levels (Kastelein et al., 2006). In those with mild LDL-C elevations receiving the 200 mg/week subcutaneous (SC) dose of mipomersen, the maximal reductions in apo B, VLDL, and LDL-C were 46%, 53%, and 45%, respectively (Akdim et al., 2011). Given the long t1/2 of mipomersen, these reductions remained significantly below baseline for up to 90 days after the last dose of mipomersen (Kastelein et al., 2006; Akdim et al., 2011). Phase II randomized placebo-controlled trials (RCTs) have been conducted in 74 individuals with hypercholesterolemia receiving stable statin therapy (Akdim et al., 2010a) and in 44 patients with heterozygous familial hypercholesterolemia (FH) (Table 1) (Akdim et al., 2010b). Significant dose-dependent decreases in apo B and LDL-C of approximately 20–50% were achieved with mipomersen at doses of 200 mg/week to 400 mg/week but more importantly, these effects demonstrated minimal attenuation at 90 days after discontinuation of the mipomersen (Akdim et al., 2010a). Moreover, 63% and 88% of those receiving the 200 mg/week and 300 mg/week mipomersen dose, respectively, were able to achieve LDL-C b 100 mg/dL. These results thereby demonstrate that mipomersen is effective in significantly lowering LDL-C levels, even when the LDL receptor is defective (Akdim et al., 2010b). This latter trial in patients with FH also demonstrated that although Lp(a) levels were not significantly decreased at 6 weeks, those receiving 300 mg/week had a 29% reduction in Lp(a) at 13 weeks follow-up (Akdim et al., 2010b). Similar results were obtained in a Phase III RCT of mipomersen in those with homozygous FH (Table 1) (Raal et al., 2010). In this 26-week trial, 34 individuals received mipomersen 200 mg/week while 17 received placebo. Compared with the placebo arm, those receiving mipomersen had significant decreases in apo B, LDL-C, and Lp(a) of 24%, 21%, and 23%, respectively. Interestingly, this was the first trial to demonstrate a significant increase of 15% in HDL-C levels in those receiving mipomersen. No significant changes in highly sensitive C-reactive protein (hsCRP) were demonstrated. A recent press release by Isis Pharmaceutical, Inc. identified positive results from 2 Phase III RCTs which have yet to be published. The first

T.R. Joy / Pharmacology & Therapeutics 135 (2012) 31–43

involves 58 individuals with severe heterozygous FH on maximally tolerated lipid-lowering medications and the second involves 158 individuals with LDL-C ≥ 100 mg/dL and at high risk of coronary heart disease (CHD) on maximally tolerated statin therapy. Both trials examined the effects of mipomersen at 200 mg/week for 26 weeks. In patients with severe heterozygous FH, mipomersen effected a net decrease in apo B and Lp(a) of 47% and 32%, respectively (“Isis Pharmaceuticals, Inc. Press Release 04/05/2011: Data from Two Mipomersen Phase 3 Trials Presented at ACC”, 2011). Similarly, in high CHD risk patients, mipomersen effected a 37% decrease in LDL-C compared with the 5% decrease in those receiving placebo. Furthermore, approximately 50% of the patients in the mipomersen arm achieved a LDL-C b 70 mg/dL (“Isis Pharmaceuticals, Inc. Press Release 04/05/2011: Data from Two Mipomersen Phase 3 Trials Presented at ACC”, 2011). These results all emphasize the significant efficacy of mipomersen in terms of LDL-C, apo B, and Lp(a) reductions. However, despite being short in duration, these trials have been hampered by adverse effects. 2.4. Safety Thus far, only 1 study has been published demonstrating no significant drug–drug interaction between mipomersen and ezetimibe or simvastatin (Yu et al., 2009). In vitro analysis of cryo-preserved human hepatocytes revealed that mipomersen did not have any evidence of inhibition of the major cytochrome P450 enzymes, namely CYP1A2, 2C9, 2C19, 2D6 and 3A4, suggesting that the likelihood of drug–drug interactions is low (Yu et al., 2009). Adverse effects however have been frequent. Injection site reactions are the most common adverse event, occurring in 80–100% of patients (Kastelein et al., 2006; Akdim et al., 2010a, 2010b; Raal et al., 2010; Akdim et al., 2011; “Isis Pharmaceuticals, Inc. Press Release 04/05/2011: Data from Two Mipomersen Phase 3 Trials Presented at ACC”, 2011). Although an injection site reaction including erythema would be considered a mild adverse event, the discontinuation rates for those assigned to mipomersen were as high as 40% with up to 2/3 of discontinuations due to adverse events (“Isis Pharmaceuticals, Inc. Press Release 04/05/2011: Data from Two Mipomersen Phase 3 Trials Presented at ACC”, 2011). The other common adverse events were flu-like illness, occurring in up to 30–50% of patients (Akdim et al., 2010a; Raal et al., 2010; Akdim et al., 2011; “Isis Pharmaceuticals, Inc. Press Release 04/05/2011: Data from Two Mipomersen Phase 3 Trials Presented at ACC”, 2011), and elevated liver enzymes >3 times the upper limit of normal (3 × ULN), occurring in up to 15% of patients (Akdim et al., 2010b; Raal et al., 2010; “Isis Pharmaceuticals, Inc. Press Release 04/05/2011: Data from Two Mipomersen Phase 3 Trials Presented at ACC”, 2011) although in one study 50% developed transaminitis (Akdim et al., 2010a). This was not associated with changes in parameters of liver function such as bilirubin or albumin. A trend toward an increase in intrahepatic TG content (IHTG) with short-term (13 weeks) mipomersen use has been noted (Visser et al., 2010), which is in contrast to prior animal data (Crooke et al., 2005; Mullick et al., 2011). It is important to signify that the study by Visser et al. examining IHTG was short-term (13 weeks) and was done in patients with heterozygous FH who were not diabetic nor obese and were all receiving statin therapy (Visser et al., 2010). Furthermore, those with high baseline IHTG values (>5%) were excluded. Thus, the potential for a greater risk of transaminitis exists if mipomersen is used in patients at higher baseline risk for steatosis such as those with diabetes, metabolic syndrome, and/or obesity. Similarly, since statins may have beneficial effects in hepatic steatosis, use of mipomersen in monotherapy may have a higher incidence of transaminitis and hepatic steatosis than when used in combination with statins. Thus, the results from Visser et al. may underestimate the true nature of hepatic steatosis with mipomersen (Visser et al., 2010). Importantly, although mipomersen

33

is currently in Phase III trials (Table 2), the approval and use of this agent in more generalizable, at-risk populations will likely be limited due to concerns of these side effects. 3. Proprotein convertase subtilisin/kexin type 9 inhibitors 3.1. Mechanism of action PCSK9, a member of the proteinase K subfamily of subtilases, is a protease that is involved in regulating the level of circulating LDL-C through effects on LDLR. In particular, PCSK9 accelerates the degradation of hepatic LDLR (Maxwell & Breslow, 2004; Park et al., 2004). Thus, sustained elevation of PCSK9 levels through gain of function variants would be associated with reductions in LDLR and increases in LDL-C. In fact, these gain of function variants have been described with the phenotype of FH (Abifadel et al., 2003). Since inactivation of PCSK9 or reduction in PCSK9 levels would theoretically lead to reductions in LDL-C levels, investigators have examined several different strategies for inhibiting PCSK9, including through ASOs, antibodies (polyclonal or monoclonal), small peptides that hinder the interaction between PCSK9 and LDLR, and small interfering RNA (siRNA) (Chan et al., 2009; Duff et al., 2009; Frank-Kamenetsky et al., 2008; Graham et al., 2007; Gupta et al., 2010; Liang et al., 2012; Lindholm et al., 2012; Ni et al., 2010, 2011; Palmer-Smith & Basak, 2010). The mechanism of ASOs has been described above, and in this case, the ASOs are directed against mRNA of PCSK9. The antibodies derived for PCSK9 inhibition thus far work through disruption of the interaction and formation of the PCSK9/LDLR complex or through inhibition of PCSK9 internalization (Chan et al., 2009; Duff et al., 2009; Ni et al., 2010, 2011). Meanwhile, siRNAs, although mechanistically different from ASOs, ultimately lead to the cleavage or translational repression of mRNA. PCSK9 inhibition via these various methods is only in phase I/II trials currently and thus, there is limited human data available. 3.2. Pharmacokinetic and pharmacodynamic data When examined in nonhuman primates, the onset and duration of LDL-C reduction seems to vary with the method of PCSK9 inhibition employed. For antibody-mediated PCSK9 inhibition, LDL-C reduction occurred as early as 8 h post injection with the decrease maintained for at least 10 days but could be sustained for over 2 weeks depending on the monoclonal antibody used (Chan et al., 2009; Ni et al., 2011). siRNA-based PCSK9 inhibition had a slower onset, with a significant reduction in LDL-C levels beginning 3 days after the dose but LDL-C levels returned to baseline by 2 to 3 weeks (Frank-Kamenetsky et al., 2008). The decrease in LDL-C seemed most sustained with ASO-based therapy, with reductions in total cholesterol levels occurring continuously over the first 3 weeks after injection and returning to baseline levels by day 56 (Lindholm et al., 2012). The t1/2 of the LDL-C lowering effect of this latter ASO-based PCSK9 inhibitor, SPC5001, was determined to be 24 days (Lindholm et al., 2012). In humans, 2 trials with ASO-based PCSK9 inhibitors, SPC 5001 and BMS-844421 have been prematurely terminated without explanation of cause (“Multiple Ascending Dose Study of SPC5001 in Treatment of Healthy Subjects & Subjects With FH”, 2012; “Safety Study of BMS-844421 for Treatment of Hypercholesterolemia”, 2011). Meanwhile, preliminary phase I/II data are available for the 2 monoclonal antibodies in development (AMG-145 and REGN727/ SAR236553) as well as the siRNA-based PCSK9 inhibitor, ALN-PCS (“Alnylam Pharmaceuticals. Alnylam Reports Positive Preliminary Clinical Results for ALN-PCS, an RNAi Therapeutic Targeting PCSK9 for the Treatment of Severe Hypercholesterolemia”, 2012; Dias et al., 2011; “Regeneron Pharmaceuticals Inc.: Sanofi and Regeneron

34

Table 1 Published Phase II and Phase III randomized placebo-controlled clinical trials of select novel LDL-lowering therapies. Population

Visser et al., 2010

21 patients with heFH

Raal et al., 2010

51 patients with hoFH

Akdim et al., 2010a

74 patients with hypercholesterolemia on statin therapy with fasting LDL-C between 100 and 200 mg/dL

Akdim et al., 2010b

44 patients with heFH

Akdim et al., 2011

50 patients with hyperlipidemia

Design

Duration of study

HDL-C change

LDL-C change

CVD outcome

Adverse effects

Single SC dose of 50–400 mg mipomersen followed by 4-week multiple-dosing regimen with the same assigned dose SC injection of 200 mg mipomersen or placebo weekly Mipomersen 200 mg SC vs placebo weekly (randomization 2:1) RCT of 6 cohorts randomized 4:1 (active: placebo) 5 dose-escalation cohorts receiving mipomersen 30, 100, 200, 300, or 400 mg SC for 7 doses over 5 weeks 1 cohort receiving mipomersen 200 mg SC 3 times in the first week and then weekly for 12 weeks. 4 cohorts: Mipomersen (50, 100, 200, or 300 mg) SC vs. placebo (4:1 randomization to active: placebo)

4 weeks

No significant change

Dose-dependent reduction in LDL-C, with −35% change in LDL-C for the 200 mg dose

None assessed

Most common AE was injection site reaction, affecting ~70% of the subjects

13 weeks

No significant change

−22%

None assessed

26 weeks

No significant change

−21.3% Also 23% decrease in Lp(a)

None assessed

No significant change

Doses of 100–400 mg/week associated with 21–52% decrease in LDL-C

None assessed

Trend towards increased intrahepatic TG content in those receiving mipomersen Main AEs were injection site reactions and >3× ULN increase in transaminases levels Major AEs were injection site reactions and >3× ULN increase in transaminases levels

5 weeks

8 SC doses over 6 weeks but 300 mg cohort continued for additional 7 weeks

5 cohorts randomized 4:1 (active: placebo)

2 cohorts had 2-week loading dose period (200 mg SC dose 2 times per week) followed by 100 or 200 mg SC every other week for 11 weeks

−36% at 200 mg dose for 13 weeks

13 weeks

No significant change

No significant change

11 weeks for the 50 mg/ week and 100 mg/week cohorts (who had the loading period)

None assessed At 6 weeks, 200 and 300 mg doses associated with reductions of 21% and 34%, respectively. At 13 weeks, 300 mg dose associated with 37% decrease in LDL-C. Also, reductions in Lp(a) of 29% at 13 weeks. None assessed Dose-dependent reductions in LDL-C ranging from 7 to 71% in the 50 to 400 mg dose groups. Significant 32–49% and 46–53% reductions in Lp(a) and TG, respectively, in the 200–400 mg cohorts

Major AEs were injection site reactions and >3× ULN increase in transaminases levels

Major AEs were injection site reactions and >3x ULN increase in transaminases levels

T.R. Joy / Pharmacology & Therapeutics 135 (2012) 31–43

1. Antisense oligonucleotides of APOB Kastelein et 36 individuals with al., 2006 mild dyslipidemia

3 cohorts were dosed at 200, 13 weeks for the 200, 300, 300, or 400 mg SC weekly for and 400 mg per week 13 weeks without the loading cohorts period 2. MTP inhibitors Samaha et al., 2008

3. Thyromimetics Goldman et al., 2009 and a subsequent report by Ladenson PW et al. (Ladenson et al., 2010b)

12 weeks Randomized to ezetimibe alone 10 mg daily; AEGR-733 titrated to a dose of 10 mg PO daily; or combination therapy

Ezetimibe alone +6%; Ezetimibe alone −20%; AEGR-733 alone −6%; AEGR-733 alone −30%; combination therapy −46% combination therapy −9%

86 patients with stable CHF NYHA II–IV with LVEF ≤ 40%

DITPA (90 to 360 mg/day) PO vs. placebo

24 weeks with 4 week off study drug period

No significant change

−30%

Epritorome (25, 50, or 100 μg) PO daily vs. placebo

12 weeks

Approximately −6%

None assessed Dose-dependent decrease in LDL-C ranging from 22 to 32% Also dose-dependent decreases in Lp(a) and TG of 27–43% and 16–33%, respectively

Ladenson et al. article describes the 48 patients (21 DITPA and 27 placebo) who completed the entire 28 weeks 189 patients on statin therapy with an LDL-C≥ 116 mg/dL)

None assessed

Major AE was transaminase elevation occurring in 18 of 56 patients receiving AEGR-733 (vs. 0 receiving placebo)

Adverse trend seen in the primary composite CHF outcome with a worsening in patient global assessment in those receiving DITPA (Goldman et al., 2009)

Suppression of TSH with decreased body weight (6%) accompanied by significant increase in heart rate and in serum markers of bone turnover – Although common in both groups, a higher percentage of patients noted fatigue and diarrhea in the DITPA arm with 25 of the original 57 randomized to DITPA dropping out due to adverse events. No significant changes in body weight, heart, rate, BP, cardiac rhythm or ECG findings. No significant changes in serum markers of bone turnover. Mild reversible increases in aminotransferase levels Main AEs seemed to be mild and consist of gastrointestinal symptoms or nasopharyngitis Dose-dependent decreases in total and free thyroxine levels but these remained within the normal range Dose-dependent increases in sex-hormone binding globulin but no events related to sexual dysfunction were noted

Abbreviations: AE, adverse event; BP, blood pressure; CHF, congestive heart failure; ECG, electrocardiogram; HDL-C, high-density lipoprotein cholesterol; heFH, heterozygous familial hypercholesterolemia; hoFH, homozygous familial hypercholesterolemia; IV, intravenous LDL-C, low-density lipoprotein cholesterol; Lp(a), lipoprotein (a); LVEF, left ventricular ejection fraction; NYHA, New York Heart Association; PO, per os; RCT, randomized controlled trial; SC, subcutaneous; TG, triglycerides; and ULN, upper limit of normal.

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Ladenson et al., 2010a

84 patients with hypercholesterolemia

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T.R. Joy / Pharmacology & Therapeutics 135 (2012) 31–43

Table 2 Current stage of development of LDL-C lowering agents tested in humans. Class

Mechanism of action

Agent name

Route of administration

Stage of development

1. Inhibition of apo B production 2. PCSK9 inhibition

ASO a. ASO

Mipomersen BMS-844421 SPC 5001 AMG-145 REGN727/SAR236553 ALN-PCS Lomitapide Implitapide JTT-130 SLx-4090 DITPA Eprotirome

SC SC

Phase III Phase I prematurely terminated

SC

Phase II

IV PO PO PO

Phase I Phase II On hold Phase II

PO PO

Discontinued Phase II

b. Monoclonal antibody

3. MTP inhibitors

c. siRNA a. Non-specific b. Intestine-specific

4. Thyromimetic

TRβ1-selective

Abbreviations: apo B, apolipoprotein B; ASO, antisense oligonucleotide; IV, intravenous; MTP, microsomal triglyceride transfer protein; PCSK9, proprotein convertase subtilisin kexin type 9; PO, per os; SC, subcutaneous; and siRNA, small interfering ribonucleic acid.

Report Positive Preliminary Phase 2 Program Results for Anti-PCSK9 Antibody in Hypercholesterolemia”, 2011; Simon, 2012). In a total of 7 cohorts (n = 8 per cohort), AMG-145 was administered as a single SC dose in 5 cohorts and as a single intravenous (IV) dose in 2 cohorts (Dias et al., 2011). Time to nadir and duration of effect were all dose-dependent. In 20 healthy volunteers with LDL-C>116 mg/dL, ALN-PCS was administered as a single IV dose of 0.015, 0.045, 0.090, 0.150, and 0.250 mg/kg with pretreatment with steroids, histamine blocker, and acetaminophen (“Alnylam Pharmaceuticals. Alnylam Reports Positive Preliminary Clinical Results for ALN-PCS, an RNAi Therapeutic Targeting PCSK9 for the Treatment of Severe Hypercholesterolemia”, 2012; Simon, 2012). There was rapid, dose-dependent silencing of PCSK9 protein levels in plasma with the mean nadir reduction being 60% at day 4 in the high dose group of 0.25 mg/kg IV. The greatest reduction in LDL-C levels also occurred at approximately day 4. The results seemed to be durable enough to support once monthly administration. 3.3. Efficacy Dose-dependent reductions in LDL-C levels have been demonstrated with PCSK9 inhibition regardless of method. Reductions in LDL-C levels ranged between 20 and 80% with the average reduction being 30–50% (Chan et al., 2009; Frank-Kamenetsky et al., 2008; Graham et al., 2007; Lindholm et al., 2012; Ni et al., 2011). Peer-reviewed publication of human data is currently awaited. However, presentation of results on company websites and in abstract form has revealed significant reductions in LDL-C levels with PCSK9 inhibition. Dose-dependent reductions have been demonstrated for AMG-145 and REGN727/SAR236553 (Dias et al., 2011; “Regeneron Pharmaceuticals Inc.: Sanofi and Regeneron Report Positive Preliminary Phase 2 Program Results for Anti-PCSK9 Antibody in Hypercholesterolemia”, 2011). In patients with heterozygous FH on lipid lowering therapy (statins with or without ezetimibe), REGN727/ SAR236553 effected reductions in LDL-C ranging from approximately 30% to over 65% depending on dose (“Regeneron Pharmaceuticals Inc.: Sanofi and Regeneron Report Positive Preliminary Phase 2 Program Results for Anti-PCSK9 Antibody in Hypercholesterolemia”, 2011). In another Phase II trial with REGN727/SAR236553, patients with hypercholesterolemia treated with low-dose atorvastatin (10 mg daily) were randomized to 3 arms: continuation of atorvastatin 10 mg daily with addition of REGN727/SAR236553 or titration to atorvastatin 80 mg daily with addition of placebo or of REGN727/SAR236553 (“Regeneron Pharmaceuticals Inc.: Sanofi and Regeneron Report Positive Preliminary Phase 2 Program Results for Anti-PCSK9 Antibody in Hypercholesterolemia”, 2011). A greater than 65% reduction in LDLC levels was achieved with the addition of REGN727/SAR236553 in contrast to a drop in LDL-C levels of 17% with titration alone to 80 mg daily (“Regeneron Pharmaceuticals Inc.: Sanofi and Regeneron Report

Positive Preliminary Phase 2 Program Results for Anti-PCSK9 Antibody in Hypercholesterolemia”, 2011). For ALN-PCS, the reduction in LDL-C in a phase I trial was up to 50%, with a significant mean reduction of 39% at the highest dose of 0.25 mg/kg occurring as early as 4 days after the single injection (“Alnylam Pharmaceuticals. Alnylam Reports Positive Preliminary Clinical Results for ALN-PCS, an RNAi Therapeutic Targeting PCSK9 for the Treatment of Severe Hypercholesterolemia”, 2012; Simon, 2012). These reductions continued for at least 14 days. No effects on HDL-C were observed for ALN-PCS and AMG-145 (Dias et al., 2011; Simon, 2012). 3.4. Safety Safety data is very limited. PCSK9 inhibition has not revealed any detectable adverse effects on liver transaminase levels nor glucose levels (Gupta et al., 2010; Lindholm et al., 2012). HDL-C levels have been decreased in some studies of antibody-mediated PCSK9 inhibition, but the effect was not consistent across the different methods (Chan et al., 2009; Frank-Kamenetsky et al., 2008; Lindholm et al., 2012; Ni et al., 2011). Although a potential concern raised with PCSK9 inhibition has been increased cholesterol accumulation within the liver, this was not evident in nonhuman primates (Lindholm et al., 2012). In fact, cholesterol and triglyceride content has been demonstrated to be lower in animals treated with PCSK9 inhibitors, suggesting possible implications of benefit in patients with non-alcoholic fatty liver disease (Gupta et al., 2010; Lindholm et al., 2012). In humans, thus far, antibody-mediated and siRNA-mediated PCSK9 inhibition appears well-tolerated. Preliminary results from AMG-145 and REGN727/SAR236553 have not identified a significant safety signal (Dias et al., 2011; “Regeneron Pharmaceuticals Inc.: Sanofi and Regeneron Report Positive Preliminary Phase 2 Program Results for Anti-PCSK9 Antibody in Hypercholesterolemia”, 2011). No elevations in CK levels occurred. The most significant reactions were injection site reactions (“Regeneron Pharmaceuticals Inc.: Sanofi and Regeneron Report Positive Preliminary Phase 2 Program Results for Anti-PCSK9 Antibody in Hypercholesterolemia”, 2011). Only one patient in the atorvastatin 80 mg with REGN727/ SAR236553 arm developed further transaminase elevation to 3 × ULN but this individual already had a baseline elevation (“Regeneron Pharmaceuticals Inc.: Sanofi and Regeneron Report Positive Preliminary Phase 2 Program Results for Anti-PCSK9 Antibody in Hypercholesterolemia”, 2011). One patient in the REGN727/SAR236553 study discontinued the study due to a hypersensitivity reaction and one patient was noted to have a severe reaction (dehydration) but this was deemed not related to the study (“Regeneron Pharmaceuticals Inc.: Sanofi and Regeneron Report Positive Preliminary Phase 2 Program Results for Anti-PCSK9 Antibody in Hypercholesterolemia”, 2011). Preliminary results with

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ALN-PCS have also revealed no serious adverse events with no liver transminase elevations (“Alnylam Pharmaceuticals. Alnylam Reports Positive Preliminary Clinical Results for ALN-PCS, an RNAi Therapeutic Targeting PCSK9 for the Treatment of Severe Hypercholesterolemia”, 2012; Simon, 2012). Five individuals, including 2 who received placebo, developed a mild rash. PCSK9 inhibitors seem to be a very promising modality for further LDL-C reduction but are still very early in their development (Table 2). The premature termination of the ASO-based therapies for PCSK9 inhibition raises concerns whether there had been a safety signal identified. Meanwhile, siRNA-based and antibody-mediated therapies seem to be doing well in human trials thus far. siRNAbased therapies require a focus on ensuring specificity of target (i.e. avoiding off-target effects) and maximizing efficient delivery. Ultimately, these multiple methods of PCSK9 inhibition will need to be investigated thoroughly to determine which method reigns supreme in terms of both efficacy and safety. 4. Microsomal triglyceride transfer protein inhibitors 4.1. Mechanism of action Microsomal triglyceride transfer protein (MTP) transfers triglyceride (TG) to nascent apo B, aiding the formation of TG-rich lipoproteins, namely chylomicrons and VLDL in enterocytes and hepatocytes, respectively (Wetterau et al., 1997). Thus, MTP inhibition would lead to decreases in chylomicrons and VLDL (Fig. 2). Additionally, since LDL is formed from VLDL, MTP inhibitors would additionally decrease plasma LDL-C levels. This has been indeed evident in the rare disorder abetalipoproteinemia (ABL), in which MTP gene mutations produce non-functional MTP, resulting in a significant decrease in apo B-containing lipoproteins, including LDL (Rader & Brewer, 1993; Wetterau et al., 1992). Consequently, individuals with abetalipoproteinemia are relatively spared from atherosclerosis, but the lack of MTP is also associated with intestinal fat malabsorption, leading to steatorrhea and fat-soluble vitamin deficiency. Namely, the deficiency in vitamins A, D, E, and K in abetalipoproteinemia can be manifest as night blindness, rickets or osteomalacia, neuropathy and ataxia, and coagulopathy, respectively. Furthermore, the lack of functional MTP in patients with abetalipoproteinemia is associated with hepatic steatosis. Given the known consequences of absent MTP in abetalipoproteinemia, careful evaluation of the merits of MTP inhibition is warranted. There are several MTP inhibitors being investigated, including lomitapide (AEGR-733, previously known as BMS-201038), implitapide (formerly AEGR-427 or Bayer BAY-13-9952), CP-34086, and the

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intestine-specific MTP inhibitors — dirlotapide, JTT-130, and SLx-4090 (Table 2). The intestine-specific inhibitors, except dirlotapide, are early in human clinical trials. Meanwhile, of the non-specific inhibitors, lomitapide is furthest along, currently being tested in Phase III clinical trials. Unfortunately, the development of both CP-34086 and implitapide is currently stalled. Despite beneficial effects in terms of reductions in LDL-C and TG in both rodents and humans with administration of CP-346086, further development of CP-346086 seems to be at a halt currently, possibly due to its increases in hepatic TG levels (Chandler et al., 2003). Similar to CP-346086, implitapide effected VLDL and LDL-C reductions of ~70% without significant changes in HDL (Shiomi & Ito, 2001). Additionally, implitapide decreased aortic atherosclerotic lesion area by 83% in apo E(−/−) mice possibly linked through its effects on postprandial hypertriglyceridemia (Ueshima et al., 2005). However, implitapide use results in markedly increased fecal fat excretion (Ueshima et al., 2005) and reductions in plasma vitamin E levels in correlation with LDL levels (Shiomi & Ito, 2001). In 2007, the Food and Drug Administration instituted a partial clinical hold on MTP inhibitor clinical trials and requested further preclinical data to assess the long-term risk for pulmonary phospholipidosis (“Aegerion Pharmaceuticals Inc. Quarterly Report”, 2011). The partial clinical hold was lifted for lomitapide but remains in effect for implitapide until such preclinical data is submitted (“Aegerion Pharmaceuticals Inc. Quarterly Report”, 2011). Thus, the discussion on non-specific MTP inhibitors will be restricted to lomitapide. 4.2. Lomitapide 4.2.1. Pharmacokinetic and pharmacodynamic data In Zucker fatty rats, TG secretion rate was significantly reduced by 47% by a single dose of lomitapide at a dose of 1 mg/kg (Dhote et al., 2011). A recent Phase II clinical trial in patients with homozygous FH revealed that lomitapide decreases apo B production by ~70% at a dose of 1 mg/kg (Cuchel et al., 2007). 4.2.2. Efficacy In hamsters, lomitapide has been shown to have dose-dependent decreases in VLDL and LDL cholesterol combined of 19% at 1 mg/kg, 66% at 3 mg/kg and 89% at 6 mg/kg (Wetterau et al., 1998). TG reductions were also seen in a dose-dependent manner, ranging from 8% at 1 mg/kg to 49% reduction at 6 mg/kg. Unfortunately, HDL-C reductions were also evident, as little as 5% at 1 mg/kg to most marked at 6 mg/kg where HDL-C levels were reduced by 92% (Wetterau et al., 1998). These effects were evident after just 7 days of treatment. Further evaluation of lomitapide at a dose of 10 mg/kg for 14 days in

MTP

MTP

apoB48

apoB100 MTP Inhibitor

Chylomicron

Enterocyte

MTP

VLDL

Inhibitor

Hepatocyte

Fig. 2. Mechanism of action of microsomal triglyceride transfer protein (MTP) inhibitors.MTP transfers triglyceride to apo B48 or apo B100, aiding the formation of TG-rich lipoproteins, namely chylomicrons and VLDL, in enterocytes and hepatocytes, respectively. MTP inhibitors thereby prevent the formation of chylomicrons and VLDL. Inhibition of VLDL formation, in turn, leads to inhibition of LDL formation.

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Watanabe-heritable hyperlipidemia (WHHL) rabbits, an animal model of homozygous FH, revealed normalization of the plasma levels of atherogenic apo B-containing lipoproteins (Wetterau et al., 1998). More recently, lomitapide has been examined in an animal model of metabolic syndrome — Zucker fatty rats. In this model, 14-day therapy with lomitapide reduced TG levels by 87% and LDL by 29% at a dose of 1 mg/kg (Dhote et al., 2011). Treatment with lomitapide had other favorable effects including significant reductions in insulin resistance and lipid peroxidation in the liver and aorta as well as increases in superoxide dismutase activity. Moreover, compared with control rats, those receiving lomitapide decreased their food intake and demonstrated attenuated weight gain (Dhote et al., 2011). As obesity, hyperlipidemia, insulin resistance, and generation of reactive oxygen species are important to the development of atherosclerosis, these favorable changes via the MTP inhibitor lomitapide would certainly imply favorable effects on atherosclerosis. To date, there have only been 2 human clinical trials involving lomitapide. Given the significant LDL improvements in the WHHL rabbits, lomitapide was initially tested in a dose-escalation study in 6 patients with homozygous FH (Cuchel et al., 2007; Wetterau et al., 1998). All patients were instructed to follow a low fat diet and after cessation of all other lipid-lowering therapies for 4 weeks, received lomitapide orally for 4 weeks. Dose-dependent reductions in LDL-C and TG levels were observed; those receiving the 1 mg/kg dose effected a significant 51% and 65% decrease in LDL-C and TG levels, respectively (Cuchel et al., 2007). HDL-C and apo A-I levels did not change significantly while apo B levels decreased by 56% at the 1.0 mg/kg dose. Samaha et al. conducted a double-blind RCT of lomitapide in 84 patients with hypercholesterolemia (Table 1) (Samaha et al., 2008). All patients were instructed to follow a low-fat diet and were randomly assigned to lomitapide at escalating oral doses of 5 mg, 7.5 mg, and 10 mg daily for 4 weeks each plus placebo (lomitapide alone arm), ezetimibe 10 mg daily plus placebo (ezetimibe alone arm), or ezetimibe 10 mg daily plus lomitapide using the same dose-titration regimen (combination therapy arm). After 12 weeks, the mean percentage decreases in the LDL-C levels were 20%, 30%, and 46% in the ezetimibe alone, lomitapide alone, and combination arms, respectively. Thus, the use of lomitapide in monotherapy or in combination therapy was associated with significantly greater reductions in LDL-C levels compared with ezetimibe monotherapy. Lp(a) levels were also significantly reduced by ~16% in the lomitapide groups compared with the ezetimibe arm alone. Unlike the study by Cuchel and colleagues, no significant changes in TG levels were noted by Samaha et al. (Cuchel et al., 2007; Samaha et al., 2008). Unfortunately, lomitapide therapy was associated with decreases in HDL-C and apo A-I levels of 6–9% and 8–11%, respectively — effects similar to those seen in animals (Samaha et al., 2008; Wetterau et al., 1998). 4.2.3. Safety In vitro, lomitapide has been demonstrated to inhibit CYP3A4 (Dunbar et al., 2009a). Consequently, co-administration of lomitapide with several lipid-lowering agents has been examined for possible drug–drug interactions. In humans, although a 7-day course of lomitapide increased the area-under-the-curve concentrations of single-dose atorvastatin (metabolized by CYP3A4) or rosuvastatin (not metabolized by CYP3A4) by ~30% and simvastatin by 39%, the clinical significance of these increases is uncertain (Duffy et al., 2007, 2009a). Lomitapide also effects a 29% decrease in maximal concentration of fenofibrate, although this change is also of uncertain clinical significance (Dunbar et al., 2007). Importantly, lomitapide does not seem to have a significant effect on the pharmacokinetics of nicotinic acid (Dunbar et al., 2009b). Rodent studies have demonstrated that lomitapide has been associated with elevations in hepatic TG content although this returned to baseline 48 h after discontinuation of lomitapide (Wetterau et al.,

1998). As well, although the rodents did not exhibit steatorrhea, there was visual and biochemical evidence of fat accumulation within the enterocytes. In human trials, gastrointestinal and liver adverse effects have been noted. Despite significant reductions in LDL-C in patients with homozygous FH, these beneficial effects were hindered by adverse effects of increased stool frequency, which occurred in 5 of the 6 patients (Cuchel et al., 2007). These episodes were transient and often linked to consumption of a high fat meal. The other major adverse events in this study were serum transaminase elevation and hepatic steatosis, where hepatic fat content as measured by magnetic resonance imaging (MRI) ranged from less than 10% to 40% (normal b1%) and were consistent with changes seen in animal studies (Chandler et al., 2003; Cuchel et al., 2007; Raabe et al., 1999). Four weeks after discontinuation of therapy, transaminase and hepatic fat levels returned to baseline levels in all patients except one, who had a delayed normalization until 14 weeks (Cuchel et al., 2007). The 2 individuals who had the greatest increases in hepatic fat content had other risk factors for hepatic steatosis including marked hypertriglyceridemia and increased alcohol intake. In the trial by Samaha et al. (2008), hepatic fat was not assessed. However, transaminase elevation occurred in 32% (18/56) of patients receiving lomitapide and resulted in discontinuation in 9 of these patients. This was in contrast to the patients randomized to ezetimibe, none of whom had transaminase elevations. Transaminase levels did return to baseline in all patients at 2 weeks after discontinuation of the lomitapide. Yet, given the associated morbidity and mortality with the management of homozygous FH, the potential benefits of further LDL-C reduction may outweigh the potential harms and as such, similar to mipomersen, lomitapide continues in Phase III testing, particularly focused on patients with FH. 4.3. Intestine-specific microsomal triglyceride transfer protein inhibitors Given the adverse effects of hepatic steatosis and decreases in HDL-C with non-specific MTP inhibitors, intestine-specific MTP inhibitors such as dirlotapide, JTT-130 and SLx-4090 have been developed. Dirlotapide is currently marketed for the management of obesity in canines (Wren et al., 2007). Meanwhile, JTT-130 and SLx-4090 are being developed for human use and have limited published human data available. 4.3.1. Pharmacokinetic and pharmacodynamic data In in vitro studies, JTT-130 demonstrated potent TG and cholesteryl ester transfer with half maximal inhibitory concentration (IC50) values of 0.83 and 0.74 nM, respectively. The IC50 value for apo B secretion was 9.5 nM but importantly JTT-130 had no effect on apo A-I secretion. JTT-130 does not inhibit hepatic TG secretion even at doses of up to 1000 mg/kg in hamsters but can significantly suppress postprandial hypertriglyceridemia after olive oil loading with as low a dose as 0.3 mg/kg, thereby demonstrating the intestinal specificity of JTT-130 (Mera et al., 2011). After administration to hamsters at doses of 40 mg/kg, the parent compound JTT-130 was not detected in the plasma; instead, a large amount of its hydrolysed metabolite was found. Characterization of the intestinal effects of JTT-130 revealed that fat accumulation by JTT-130 primarily occurred in the upper small intestine, especially the jejunum (Mera et al., 2011). As in the case with JTT-130, SLx-4090 demonstrated in vitro potent apo B secretion with an IC50 of 9.6 nM but no significant effect on apo A-I secretion at the highest concentrations tested (Kim et al., 2011). SLx-4090 demonstrated a dose-dependent and rapid ability to decrease postprandial hypertriglyceridemia, occurring within 1 h of administration and with an effective dose (ED50) of approximately 7 mg/kg (Kim et al., 2011). The effect was sustained for up to 6 h. Importantly, since SLx-4090 is a nonsystemically available compound, it is rapidly metabolized via cytochrome P450 with a t1/2 of

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approximately 8 min (Kim et al., 2011). After IV administration, t1/2 was noted to be 1.6 h and when orally administered at 10 mg/kg, no detectable amounts of SLx-4090 were noted up to 24 h post-dose. The oral bioavailability of SLx-4090 after single doses of 10 or 30 mg/kg was demonstrated to be essentially 0%. Similarly, the absorption of SLx-4090 after doses of 25 and 100 mg/kg is very low within the portal circulation (Kim et al., 2011). Thus, SLx-4090 is not absorbed from the gut and does not undergo exposure to the liver, thereby potentially limiting its adverse effects. Although JTT130 and SLx-4090 have been both tested in humans in phase I trials, the data have not yet been published (“Japan Tobacco Inc. Clinical development (as of May 12, 2011),” 2011; Webster, 2008). 4.3.2. Efficacy and safety Treatment of hamsters with JTT-130 at a dose of 10.6 mg/kg for 2 weeks reduced plasma TG and cholesterol levels by 28% and 38%, respectively, and unlike non-specific MTP inhibitors, effected a decrease in hepatic TG content by 43% (Mera et al., 2011). Lack of hepatotoxicity with JTT-130 has also been demonstrated in guinea pigs (Aggarwal et al., 2005). Furthermore, JTT-130 has been demonstrated to decrease food intake, increase plasma levels of gut peptides important for appetite and insulin regulation (namely, glucagon-like peptide 1 and peptide YY), increase mRNA levels of glucose transporter 4 and lipoprotein lipase, and in rats, suppress high-fat diet-induced obesity and glucose intolerance (Hata et al., 2011a, 2011b, 2011c). In apo E(−/−) mice, SLx-4090 administered for 10 weeks suppressed diet-induced weight gain, decreased visceral fat weight, decreased LDL-C by 26–70%, and increased HDL-C by 14–123% depending on the dose of SLx-4090 and the dietary fat intake (Kim et al., 2011). The increase in HDL-C is unlike other MTP inhibitors tested. No adverse events were observed with Sprague–Dawley mice administered doses as high as 1000 mg/kg (Kim et al., 2011). There were no significant effects on vitamins A, C, and E, although a 24% decrease in mean circulating levels of vitamin E was observed after 14 days of SLx-4090 administration (Kim et al., 2011). Phase 2a preliminary data of SLx-4090 has revealed significant reductions in postprandial TG levels and fasting LDL-C levels at 14 days of treatment although details are not available (Webster, 2008). There was also ‘clinically significant weight loss’ without evidence of adverse gastrointestinal or liver effects (Webster, 2008). Peer-reviewed publication of these results is eagerly awaited (Webster, 2008). Thus, compared with non-specific MTP inhibitors, intestine-specific inhibitors may be associated with less adverse effects and thereby warrant further investigation into ensuring safety and efficacy for humans. 5. Thyromimetics 5.1. Mechanism of action The association between hyperthyroidism and decreased plasma cholesterol levels has been noted since 1930 (Mason et al., 1930). Consequently, the use of thyroid hormone to reduce cholesterol levels has been examined in several studies, including the Coronary Drug Project (CDP) (Strisower et al., 1954, 1955, 1959; Hollister & Arons, 1962; “The coronary drug project. Findings leading to further modifications of its protocol with respect to dextrothyroxine. The coronary drug project research group”, 1972). Unfortunately, these studies were associated with evidence of overt hyperthyroidism and increased adverse events among patients receiving thyroid hormone. In CDP, although the increased incidence of adverse events was attributed to the contamination of the thyroid hormone analog examined, dextrothyroxine, by levothyroxine, investigation into the use of thyromimetics or thyroid hormone analogs for management of hyperlipidemia was essentially curbed once statins came onto the market (Young et al., 1984).

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Recently, there has been renewed interest in the interplay between thyroid hormones and cholesterol levels since the identification of the roles of the thyroid hormone receptors (TRs) in cardiac and lipid effects. There are two main TRs in humans — TRα (TRα1 and TRα2) and TRβ (TRβ1 and TRβ2). Although TRα1 and TRβ1 are expressed virtually in all tissues, TRα1 is primarily present in muscle and brown adipose tissue while TRβ1 is predominant in the liver. The effect of thyroid hormones on tachycardia is mediated via TRα1 while that on plasma cholesterol levels is mediated via TRβ1, thereby positing that the development of TRβ1-selective thyromimetics would be useful in lowering plasma cholesterol levels without the untoward cardiac side effects (Gloss et al., 2001; Gullberg et al., 2002; Johansson et al., 1998). Thus, several TRβ1-selective thyromimetics have been under investigation, including DITPA (3,5-diiodothyropropionic acid), sobetirome (GC-1), eprotirome (KB2115), MB07811, KB-141 and T-0681. Although some agents have been tested for use in heart failure, the discussion here will focus primarily on the use of thyromimetics for dyslipidemia. These agents have been noted to have several different effects on lipoproteins, including reductions in LDL-C, HDL-C, and TG (Fig. 3). The mechanisms of actions are varied. The primary mechanism of LDL-C lowering is postulated to occur through enhanced hepatic LDLR expression by TRβ as increased hepatic LDLR expression has been noted in several other animal models in response to different thyromimetics and neither T-0681 nor MB07811 affected plasma cholesterol levels in LDLR knockout mice (Erion et al., 2007; Tancevski et al., 2010). However, this effect of upregulation of LDLR expression is not consistent, as sobetirome and T-0681 do not induce hepatic LDLR expression in wild-type mice and mice deficient in the hepatic HDL receptor, scavenger receptor B-I (SR-BI), do not respond to treatment with T-0681 (Johansson et al., 2005; Tancevski et al., 2010). These findings have prompted Tancevski et al. to postulate that upregulation of LDLR by thyromimetics occurs primarily in the setting of reduced and/or absent SR-BI-mediated uptake of plasma cholesterol (Tancevski et al., 2010). Further mechanisms for LDL-C reduction include stimulation of bile acid synthesis (through upregulation of the rate-limiting enzyme, cholesterol 7-hydroxylase [CYP7A1]) and stimulation of biliary excretion (through increased expression of ATP-binding cassette proteins G5/G8 [ABCG5/G8]) (Erion et al., 2007; Johansson et al., 2005; Tancevski et al., 2010). These latter effects on CYP7A1 and ABCG5/G8 are evidence also of the effects of thyromimetics on enhancing reverse cholesterol transport, which is the mechanism for movement of cholesterol from the periphery to the liver for excretion. T-0681 has been shown to enhance in vivo reverse cholesterol transport in mice, concomitant with increased hepatic expression of CYP7A1, ABCG5/8, and SR-BI (Tancevski et al., 2010). However, use of T-0681 in cholesteryl ester transfer protein (CETP) transgenic mice did not result in upregulation of CYP7A1 nor ABCG5/8 while SR-BI was still upregulated. Whether this lack of effect on CYP7A1 and ABCG5/8 is evident in other animal models containing CETP naturally, such as rabbits, or in humans remains to be seen. The upregulation of SR-BI, also evident with sobetirome, may explain the decrease in HDL-C levels observed with thyromimetics (Johansson et al., 2005). Thus, thyromimetics seem to exert changes in early and late stages of reverse cholesterol transport. Finally, the decreases in TG levels are proposed to be mediated through possible repression of sterol regulatory binding protein (SREBP)-1c (Erion et al., 2007; Johansson et al., 2005). Hence, the effects of thyromimetics, although not completely elucidated, are variable and potentially beneficial in reducing atherogenic lipoproteins and enhancing reverse cholesterol transport. 5.2. Pharmacokinetic and pharmacodynamic data GC-1 binds to TRβ1 with the same affinity as T3 but binds to TRα1 with only one-tenth the affinity as T3 (Chiellini et al., 1998). The ED50 for GC-1 has been noted to be 190 nmol/kg/day (Grover et al., 2004).

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Bile acids SREBP-2

7 hydroxylase

ABCG5/8

LDLR

Intestine

LDL LDL

SR-BI

LDL

CE

CE

Liver

LDL

Thyromimetics

CE

Reverse cholesterol transport

C

Macrophage

Fig. 3. Postulated mechanisms of action of thyromimetics.Thyromimetics may have a number of effects on lipid metabolism, including: a) stimulating low-density lipoprotein receptor (LDLR) expression, thereby leading to increased LDL catabolism and lowered LDL-C levels; b) activating cholesterol 7α hydroxylase activity, the rate limiting step in the conversion of cholesterol to bile acids; c) enhancing expression of ATP-binding cassette protein G5/G8 (ABCG5/G8), leading to increased biliary cholesterol secretion, which in turn, further stimulates LDLR expression via sterol regulatory binding protein 2 (SREBP-2); and d) promoting reverse cholesterol transport (RCT), which is the mechanism for removal of cholesterol (C) from the periphery (including in macrophages) to the liver for excretion as cholesteryl esters (CE). The effects on RCT include enhancing HDL formation, promoting cholesteryl ester transfer protein (CETP) activity, and increasing scavenger receptor B-I (SR-BI) activity for the uptake of CE. Though not shown on the figure, triglyceride levels are decreased likely via repression of SREBP-1c.

Meanwhile, greater pharmacokinetic data is available for MB07811 and eprotirome. MB07811 is a prodrug which selectively enters hepatocytes and then is intracellularly cleaved by CYP3A4 to generate the TRβ1 agonist MB07344 (Erion et al., 2004, 2007). For MB07811, pharmacokinetic studies in rats have revealed that it has an oral bioavailability of 39% and undergoes significant first-pass hepatic extraction of ~55%. It is rapidly cleared with a volume of distribution of 14.8 ± 4.0 L/kg and t1/2 of 1.23 ± 0.15 h (Erion et al., 2007). The main route of elimination of MB07811 and MB07344 is biliary with no evidence of enterohepatic recirculation of MB07344 (Fujitaki et al., 2008). MB07811 reduced total plasma cholesterol with an ED50 of 0.4 mg/kg compared with an ED50 of 0.05 mg/kg for KB-141 (Erion et al., 2007). Human pharmacokinetic data for eprotirome is limited. The pharmacokinetics were linear, with rapid drug absorption (t1/2 ~ 2 h). No accumulation was observed (Berkenstam et al., 2008). Thus, these compounds seem to demonstrate rapid absorption and low efficacy doses. 5.3. Efficacy In animal models, total cholesterol reductions of 60–67% have been noted with significant reductions in LDL-C and TG of up to 30–40% while the effects on HDL-C and apo A-I have ranged from neutral to reductions of up to 20–25% (Erion et al., 2007; Johansson et al., 2005; Tancevski et al., 2009, 2010). Treatment with KB 141 for as few as 7 days can result in reductions in total cholesterol by 35% (Grover et al., 2003). Addition of MB07911 to atorvastatin therapy results in an additional ~20–30% decrease in total plasma cholesterol levels in animal models (B. R. Ito et al., 2009). Some thyromimetics such as KB-141 and GC-1 have demonstrated reductions in Lp(a) of ~50% in primates (Grover et al., 2003) (Grover et al., 2004). Hepatic TG and liver weight have also been reduced by 30–40% in animals receiving MB07811 or KB141, indicating a possible application for non-alcoholic fatty liver disease (NAFLD) (Erion et al., 2007; Johansson et al., 2005; Tancevski et al., 2010) although this effect was not evident with GC-1 (Johansson et al., 2005).

In New Zealand White (NZW) rabbits fed a 2% cholesterol diet for 8 weeks, treatment with T-0681 resulted in substantial reductions in cholesterol and TG levels of 60% and >80%, respectively (Tancevski et al., 2009). More importantly, in these rabbits, T-0681 significantly decreased the development of atherosclerosis by 80% (Tancevski et al., 2009). Similarly, in apo E knockout mice, although there was a slight increase in mean atherosclerotic lesion area at 4 weeks, prolonged treatment for 8 weeks resulted in inhibition of progression of these atherosclerotic lesions (Tancevski et al., 2010). The exact mechanism for the early slight increase is uncertain although the authors suggested that this may have been mediated by the upregulation of SR-BI in macrophages leading to increased reuptake of cholesterol into macrophages at that early phase of atherosclerosis (Tancevski et al., 2010). Weight loss of 3–4% and of up to 7% have been demonstrated in primates with 7 days of treatment with GC-1 and KB-141, respectively (Grover et al., 2003, 2004). These changes in body weight were not accompanied by changes in food intake or other biochemical or clinical parameters to suggest wasting or sickness and regain of weight was quick upon discontinuation of the thyromimetic (Grover et al., 2003, 2004). After 2 weeks of treatment, diet-induced obese (DIO) mice exhibited a weight loss of as much as 12% and 3–4% for KB-141 and MB07811, respectively (Erion et al., 2007). These effects were accompanied by improvements in blood glucose levels (Erion et al., 2007). Recent trials with thyromimetics in humans are limited (Table 1). A randomized clinical trial of 86 patients examining the effect of DITPA on parameters of heart failure was disappointing (Goldman et al., 2009). Although there was a 29% and 27% reduction in LDL-C and TG, respectively, there was a high drop out rate with only 37% of the initially enrolled study patients remaining on DITPA for the entire 24 weeks (Ladenson et al., 2010b). The placebo group also had a high drop out rate. Although fatigue was common in both arms, gastrointestinal complaints were more common in the DITPA arm (Goldman et al., 2009). DITPA unfortunately also did demonstrate adverse cardiac and metabolic effects including tachycardia,

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weight loss, and increased bone turnover markers, accompanied by a decrease in serum TSH to the hyperthyroid range (Ladenson et al., 2010b). Thus, the adverse effects of DITPA have limited further investigation of this compound in humans. Eprotirome (KB2115) has been well-tolerated with no adverse events reported in the phase I clinical trial (Berkenstam et al., 2008). LDL-C level was reduced an additional 29% in those receiving eprotirome compared with those receiving placebo and the level returned to baseline within 5 days after cessation. No significant changes in HDL-C, TG, or Lp(a) levels were noted (Berkenstam et al., 2008). A larger RCT examined the efficacy and safety of addition of eprotirome for 12 weeks to individuals already receiving statin therapy (namely, simvastatin or atorvastatin) (Table 1) (Ladenson et al., 2010a). Dose-dependent reductions in LDL-C, TG, and Lp(a) were evident. A dose of 100 μg/day of epritorome was associated with significant reductions in LDL-C, TG, and Lp(a) of 32%, 33%, and 43%, respectively (Ladenson et al., 2010a). HDL-C levels were only minimally reduced with the use of epritorome. 5.4. Safety Concern regarding the use of thyromimetics has been focused on the development of adverse cardiac or metabolic effects based on TRβ1:TRα1 selectivity. Differences among the different agents in the risk for these adverse events have been demonstrated. Effects of cardiomyopathy and increased heart rate have only been demonstrated at high doses of KB-141 (Erion et al., 2007; Grover et al., 2003). Meanwhile, MB07811 did not effect changes in heart rate or heart weight at even higher equivalent doses compared with KB-141 (Erion et al., 2007). Similarly, GC-1 only demonstrated a significant increase in heart rate at the highest dose tested of 10,000 nmol/kg/day in rats (Grover et al., 2004). No significant increase in heart rate or blood pressure was evident though in primates receiving GC-1 (Grover et al., 2004). Another possible adverse effect from thyromimetics is relative hypothyroidism in some tissues secondary to effects on the hypothalamic–pituitary–thyroid axis. This has not been found to be a significant problem yet. Only when NZW rabbits were treated with a 10-fold dose of T-0681 (~360 nmol/kg/day) did they exhibit signs of overt biochemical and clinical hypothyroidism, reducing oral intake and developing constipation (Tancevski et al., 2009). At lower doses of 36 nmol/kg/day, although reduction in plasma free T4 (fT4) (75%) but not in fT3 was evident, no differences in heart rate or rectal temperature to suggest relevant thyroid dysfunction were observed (Tancevski et al., 2009). Mild elevations in transaminases have been observed with T-0681 but not with GC-1 or KB-141 (Grover et al., 2003, 2004; Tancevski et al., 2009). However, these studies were relatively short in duration and thus, longer term evaluation of the adverse events from thyromimetics is warranted. Regardless, the results from preclinical trials of these agents have been promising, particularly since adverse events seem limited. For eprotirome, significant decreases in fT4 levels but not TSH or fT3 have been noted (Berkenstam et al., 2008). In the trial by Ladenson et al., dose-dependent reductions of 12 to 21% in fT4 and 22 to 34% in total T4 levels were observed, with these values remaining within or at the lower limit of the reference range (Ladenson et al., 2010a). Importantly, no changes in serum TSH, fT3 or total T3 were evident. No changes in cardiac parameters have been noted (Berkenstam et al., 2008; Ladenson et al., 2010a). Furthermore, no changes in bone turnover were noted (Ladenson et al., 2010a). Although there was a dose-dependent increase in sex-hormone binding globulin, no adverse effects on levels of serum free testosterone in men and estradiol in women were noted (Ladenson et al., 2010a). Mild increases in transaminases have been noted with eprotirome therapy, but these appear to be reversible (Berkenstam et al., 2008; Ladenson et al., 2010a). These results are thus very promising for a

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future application of thyromimetics (Table 2), particularly as a potential addition to current LDL-C-lowering treatments available. 6. Conclusions The strong relation between LDL-C levels and CVD risk has prompted the search for novel therapeutic agents focused on LDL-C reduction. The therapeutic agents discussed here have been primarily developed as a consequence of observed changes in lipid levels with clinical disorders such as hyperthyroidism (thyromimetics), abetalipoproteinemia (MTP inhibitors) or FH (due to APOB or PCSK9 mutations) (apo B or PCSK9 inhibitors, respectively). Although LDL-C lowering seems intuitively beneficial to reducing CVD risk, the utility of the current agents in development requires a careful balance between the risks and benefits. The ideal method for PCSK9 inhibition, the uncertainties surrounding the long-term disposition of ASOs within the body, the search for intestine-specific MTP inhibitors to avoid hepatic steatosis, and the development of highly specific thyromimetics all pin on reducing the adverse effects of these therapies while still demonstrating clinical benefit. It is difficult to determine which of these agents will be most beneficial, although data for significant efficacy with low incidence of adverse effects are currently in favor of antibody-mediated and siRNA-mediated PCSK9 inhibitors. However, these agents are early in development and realistically, further long-term data in larger populations is required for all of these agents. Hopefully also, the results of these trials will be supported by evidence of cardiovascular benefit. 7. Conflicts of interest/disclosures Dr. Joy has – received speakers' honoraria from: Novo Nordisk, Merck, Eli Lilly, GSK, Sepracor – been HCP consultant for: Novo Nordisk, Sanofi, Merck – served on advisory board for: Sepracor – been designated a co-investigator on other investigators' grants funded by Amgen, Aegerion, Genzyme, and Sanofi. References Abifadel, M., Varret, M., Rabes, J. P., Allard, D., Ouguerram, K., Devillers, M., et al. (2003). Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet 34(2), 154–156. Aegerion Pharmaceuticals Inc (2011). Quarterly Report. (2011, May 16, 2011). (Retrieved June 7, 2011, 2011, from). http://ir.aegerion.com/secfiling.cfm?filingID=119312511-141595&CIK=1338042 (Accessed on) Aggarwal, D., West, K. L., Zern, T. L., Shrestha, S., Vergara-Jimenez, M., & Fernandez, M. L. (2005). JTT-130, a microsomal triglyceride transfer protein (MTP) inhibitor lowers plasma triglycerides and LDL cholesterol concentrations without increasing hepatic triglycerides in guinea pigs. BMC Cardiovasc Disord 5, 30. Akdim, F., Stroes, E. S., Sijbrands, E. J., Tribble, D. L., Trip, M. D., Jukema, J. W., et al. (2010). Efficacy and safety of mipomersen, an antisense inhibitor of apolipoprotein B, in hypercholesterolemic subjects receiving stable statin therapy. J Am Coll Cardiol 55(15), 1611–1618. Akdim, F., Tribble, D. L., Flaim, J. D., Yu, R., Su, J., Geary, R. S., et al. (2011). Efficacy of apolipoprotein B synthesis inhibition in subjects with mild-to-moderate hyperlipidaemia. Eur Heart J 32(21), 2650–2659. Akdim, F., Visser, M. E., Tribble, D. L., Baker, B. F., Stroes, E. S., Yu, R., et al. (2010). Effect of mipomersen, an apolipoprotein B synthesis inhibitor, on low-density lipoprotein cholesterol in patients with familial hypercholesterolemia. Am J Cardiol 105(10), 1413–1419. Alnylam Pharmaceuticals (2012). Alnylam Reports Positive Preliminary Clinical Results for ALN-PCS, an RNAi Therapeutic Targeting PCSK9 for the Treatment of Severe Hypercholesterolemia. (Retrieved January 6, 2012, from). http://phx.corporate-ir. net/phoenix.zhtml?c=148005&p=irol-newsArticle&ID=1644329&highlight= Bennett, C. F., & Swayze, E. E. (2010). RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu Rev Pharmacol Toxicol 50, 259–293. Berkenstam, A., Kristensen, J., Mellstrom, K., Carlsson, B., Malm, J., Rehnmark, S., et al. (2008). The thyroid hormone mimetic compound KB2115 lowers plasma LDL cholesterol and stimulates bile acid synthesis without cardiac effects in humans. Proc Natl Acad Sci U S A 105(2), 663–667.

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