USH2A Gene Mutations in Rabbits Lead to Progressive Retinal Degeneration and Hearing Loss.

Purpose: Mutations in USH2A gene are responsible for the greatest proportion of the Usher Syndrome (USH) population, among which more than 30% are frameshift mutations on exon 13. A clinically relevant animal model has been absent for USH2A-related vision loss. Here we sought to establish a rabbit model carrying USH2A frameshift mutation on exon 12 (human exon 13 equivalent). Methods: CRISPR/Cas9 reagents targeting the rabbit USH2A exon 12 were delivered into rabbit embryos to produce an USH2A mutant rabbit line. The USH2A knockout animals were subjected to a series of functional and morphological analyses, including acoustic auditory brainstem responses, electroretinography, optical coherence tomography, fundus photography, fundus autofluorescence, histology, and immunohistochemistry. Results: The USH2A mutant rabbits exhibit hyper-autofluorescent signals on fundus autofluorescence and hyper-reflective signals on optical coherence tomography images as early as 4 months of age, which indicate retinal pigment epithelium damage. Auditory brainstem response measurement in these rabbits showed moderate to severe hearing loss. Electroretinography signals of both rod and cone function were decreased in the USH2A mutant rabbits starting from 7 months of age and further decreased at 15 to 22 months of age, indicating progressive photoreceptor degeneration, which is confirmed by histopathological examination. Conclusions: Disruption of USH2A gene in rabbits is sufficient to induce hearing loss and progressive photoreceptor degeneration, mimicking the USH2A clinical disease. Translational Relevance: To our knowledge, this study presents the first mammalian model of USH2 showing the phenotype of retinitis pigmentosa. This study supports the use of rabbits as a clinically relevant large animal model to understand the pathogenesis and to develop novel therapeutics for Usher syndrome.

Usefulness of Gemcabene in Homozygous Familial Hypercholesterolemia (from COBALT-1).

Homozygous familial hypercholesterolemia (HoFH) is a rare genetic disorder characterized by severely elevated plasma low-density lipoprotein-cholesterol (LDL-C), and premature atherosclerotic cardiovascular disease. Depending on residual LDL receptor (LDLR) function, most HoFH patients respond modestly to statins, ezetimibe, and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors. However, LDL-C typically remains markedly elevated necessitating additional therapies, including apheresis. Gemcabene is a novel lipid-lowering agent with a mechanism of action independent of the LDLR, which has previously demonstrated the ability to reduce levels of LDL-C on top of maximally tolerated statins. The present study (COBALT-1) assessed efficacy, tolerability, and safety of gemcabene as an adjunctive therapy to current lipid-lowering treatment for familial hypercholesterolemia patients. Eight patients with either a clinical or genetic diagnosis of HoFH on stable standard of care, including statins, ezetimibe, and PCSK9 inhibitors, were treated with gemcabene in an open-label study for 12 weeks. DNA analysis for mutations in the LDLR, apolipoprotein B, and PCSK9 genes was performed. Patients received 300 mg gemcabene for the first 4 weeks, 600 mg for the next 4 weeks, and 900 mg for the final 4 weeks. All patients completed the 12-week study. Mean change from baseline in LDL-C was -26% (p = 0.004) at Week 4 (300 mg), -30% (p = 0.001) at Week 8 (600 mg), and -29% (p = 0.001) at Week 12 (900 mg). In conclusion, the COBALT-1 study demonstrates gemcabene has potential to significantly reduce LDL-C levels when used as an adjunctive therapy to current lipid-lowering treatment for familial hypercholesterolemia patients.

Gemcabene, a First-in-Class Hypolipidemic Small Molecule in Clinical Development, Attenuates Osteoarthritis and Pain in Animal Models of Arthritis and Pain.

Our clinical studies have demonstrated that gemcabene, a small molecule in late-stage clinical development, lowers pro-inflammatory acute-phase protein, C-reactive protein (CRP). This observation was further confirmed in a cell-based study showing inhibition of cytokine-induced CRP production. Based on these observations, in the present study, we tested the hypothesis that gemcabene may possess anti-inflammatory activities in animal models of inflammatory disease. Efficacy of gemcabene was investigated in rat models of carrageenan-induced thermal hyperalgesia (CITH), monosodium iodoacetate (MIA)-induced osteoarthritis (OA), and IL-6/IL-6sR-induced inflammation. We also evaluated efficacy of gemcabene in collagen antibody-induced joint swelling and arthritis in BALB/c mice. In CITH rat model, gemcabene administration attenuated paw withdrawal latency (60% at 30 mg/kg/d and 97% at 100 mg/kg/d) and showed improvement in joint swelling (-50% at 30 mg/kg/d) in MIA model of OA. These findings were further corroborated by IL-6/IL-6sR knee injection model in rat, showing 63 and 71% reduction in hind paw weight distribution at 10 and 30 mg/kg/d doses, respectively. In mouse model of monoclonal antibody-induced arthritis, a dose-dependent attenuation of joint swelling was observed. These results demonstrate that the anti-inflammatory activity of gemcabene previously observed in cell-based and in clinical studies also occurred in animal models of inflammation-induced arthritis and hyperalgesia. Thus, in addition to hypolipidemic efficacy, the anti-inflammatory activity of gemcabene may have additional benefits to patients with elevated vascular inflammation.

Gemcabene downregulates inflammatory, lipid-altering and cell-signaling genes in the STAM™ model of NASH.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) can advance, if untreated, to liver fibrosis, cirrhosis, hepatocellular carcinoma, liver failure and liver-related death. In the United States, NASH affects approximately 2-5% of the population and an additional 10-30% have NAFLD. The number of drugs in development for NASH is growing steadily, along with nonclinical models to support prediction of clinical success. Here we evaluate gemcabene, a first-in-class clinical candidate for dyslipidemia, for its potential utility, based on its combined lipid-lowering and anti-inflammatory efficacy in clinical trials, in a preclinical model of NASH. METHODS: Gemcabene was evaluated in the STAM™ murine model of NASH. Gemcabene intervention in mice made diabetic with streptozotocin and fed a high fat high-caloric diet was assessed for changes in plasma, and hepatic histological and mRNA markers of lipid metabolism and inflammation. RESULTS: Gemcabene significantly downregulated hepatic mRNA markers of inflammation (TNF-α, MCP-1, MIP-1ß, CCR5, CCR2, NF-κB), lipogenesis and lipid modulation (ApoC-III, ACC1, ADH-4, Sulf-2), and fibrosis (TIMP-1 and MMP-2). These effects are important for the prevention of steatosis, inflammation, and hepatocyte ballooning (i.e., the components of the NAFLD Activity Score or NAS), and inhibition of fibrosis progression, and were observed following treatment with gemcabene. CONCLUSIONS: These non-clinical findings corroborate with existing clinical data to support the clinical evaluation of gemcabene as a potential new treatment for NASH.

Comparative Evaluation of Gemcabene and Peroxisome Proliferator-Activated Receptor Ligands in Transcriptional Assays of Peroxisome Proliferator-Activated Receptors: Implication for the Treatment of Hyperlipidemia and Cardiovascular Disease.

Gemcabene, a late-stage clinical candidate, has shown efficacy for LDL-C, non-HDL cholesterol, apoB, triglycerides, and hsCRP reduction, all risk factors for cardiovascular disease. In rodents, gemcabene showed changes in targets, including apoC-III, apoA-I, peroxisomal enzymes, considered regulated through peroxisome proliferator-activated receptor (PPAR) gene activation, suggesting a PPAR-mediated mechanism of action for the observed hypolipidemic effects observed in rodents and humans. In the current study, the gemcabene agonist activity against PPAR subtypes of human, rat, and mouse were compared with known lipid lowering PPAR activators. Surprisingly, gemcabene showed no or little PPAR-α transactivation compared with reference agonists, which showed concentration-dependent transactivation against human PPAR-α of 2.4- to 30-fold (fenofibric acid), 17-fold (GW590735), and 2.3- to 25-fold (WY-14643). These agents also showed robust transactivation of mouse and rat PPAR-α in a concentration-dependent manner. The known PPAR-δ agonists, GW1516, L165041, and GW0742, showed potent agonist activity against human, mouse, and rat receptors (ranging from 165- to 396-fold). By contrast, gemcabene at the highest concentration tested (300 µM) showed no response in mouse and rat and a marginal response against human PPAR-δ receptors (3.2-fold). For PPAR-γ, gemcabene showed no agonist activity against all 3 species at 100 µM and marginal activity (3.6- to 5-fold) at 300 µM. By contrast, the known agonists, rosiglitazone, indomethacin, and muraglitazar showed strong activation against the mouse, rat, and human PPAR-γ receptors. No clear antagonist activity was observed with gemcabene against any PPAR subtypes for all 3 species over a wide range of concentrations. In summary, the transactivation studies rule out gemcabene as a direct agonist or antagonist of PPAR-α, PPAR-γ, and PPAR-δ receptors of these 3 species. These data suggest that the peroxisomal effects observed in rodents and the lipid regulating effects observed in rodents and humans are not related to a direct activation of PPAR receptors by gemcabene.

Gemcabene, a first-in-class lipid-lowering agent in late-stage development, down-regulates acute-phase C-reactive protein via C/EBP-δ-mediated transcriptional mechanism.

Inflammation plays a key role in setting the stage leading to atherosclerosis progression, and high-sensitivity C-reactive protein (CRP) has been recognized as a predictor of cardiovascular risk. As a monotherapy and in combination with statins, gemcabene markedly reduced CRP in humans. Present investigation was undertaken to understand the mechanism of CRP reduction. In human hepatoma cells, gemcabene inhibited IL-6 plus IL-1ß-induced CRP production in a concentration-dependent manner, reaching 70% inhibition at 2 mM. In TNF-α-stimulated primary human coronary artery endothelial cells, both CRP and IL-6 productions were reduced by 70% at 2 mM gemcabene concentration. To investigate the mechanism of gemcabene-mediated reduction of CRP, transfection studies were performed with human CRP regulatory sequences in luciferase/ß-gal system that showed 25-fold increase in IL-6- and IL-6 plus IL-1ß-stimulated CRP transcription. Luciferase activity was reduced by 50% by gemcabene, suggesting transcriptional down-regulation of CRP. Site-directed mutagenesis of human CRP promoter revealed that the overlapping downstream C/EBP and NF-κB binding sites are important for gemcabene-mediated CRP transcription. Gel shift assays identified the transcription factor that binds to the downstream CRP promoter as C/EBP-δ. In conclusion, gemcabene decreases CRP by C/EBP-δ and NF-κB-mediated transcriptional mechanism and suppresses IL-6 and IL-1ß-induced CRP production.

Efficacy and safety of gemcabene as add-on to stable statin therapy in hypercholesterolemic patients.

BACKGROUND: Ezetimibe added to statin therapy further reduces LDL-C and clinical atherosclerotic cardiovascular disease compared to statin alone. However, the number of effective and safe oral agents for patients not at LDL-C goal is limited. In prior clinical trials, gemcabene reduced LDL-C and was generally well-tolerated in nearly 900 patients treated for up to 12 weeks. OBJECTIVE: To evaluate the LDL-C lowering and safety of gemcabene as add-on to stable statin therapy in hypercholesterolemic patients. METHODS: This was an 8-week, double-blind, placebo-controlled, randomized, phase 2 study in men and postmenopausal women ≥18 and ≤65 years of age with LDL-C ≥130 mg/dL (3.4 mmol/L) while on low-intensity to high-intensity stable statin (the majority on moderate intensity) therapy. Sixty-six patients were randomized 1:1:1 to gemcabene 300 mg, 900 mg, or placebo QD. RESULTS: Gemcabene 300 mg and 900 mg produced a mean percent change in LDL-C of -23.4 ± 4.7% (P = .005) and -27.7 ± 4.3% (P < .001), respectively, vs -6.2 ± 4.3% for placebo. The median percent change in CRP was -26.1% (P = .196) and -53.9% (P < .001) for gemcabene 300 mg and 900 mg, respectively, vs -11.1% for placebo. Gemcabene 300 mg and 900 mg were well-tolerated with no significant difference in AEs compared to placebo. CONCLUSIONS: Gemcabene as add-on to stable statin therapy demonstrated additional dose-dependent and statistically significant reductions in LDL-C of >20% and CRP >40% compared to placebo. The results support gemcabene-continued development for patients requiring LDL-C lowering beyond that provided by background statin therapy.

ApoA-IMilano phospholipid complex (ETC-216) infusion in human volunteers. Insights into the phenotypic characteristics of ApoA-IMilano carriers.

Epidemiological studies support an inverse correlation between HDL-C and cardiovascular disease. However, low HDL-C levels do not always segregate with premature disease. These include, LCAT deficiency and the apolipoproteinA-IMilano (AIM) variant. AIM has a cysteine for arginine at position 173 in the otherwise cysteine free protein permitting AIM homodimerization and apoA-II heterodimerization. We relate the biochemical characteristics of low HDL-C phenotype AIM carriers to lipoprotein changes in humans administered recombinant dimeric AIM/palmitoyl-oleoyl phosphatidyl choline (ETC-216). Pharmacokinetic analysis of infused ETC-216 suggest a slow distribution of AIM into peripheral tissue and an extremely long terminal half-life in plasma. Following ETC-216 administration to normal human volunteers, an initial dose-dependent HDL-C elevation was observed. Thereafter, subjects transiently acquired a lipoprotein profile similar to that of AIM carriers, including reduced HDL-C and mild hypertriglyceridemia. The time-dependent changes in plasma lipids/lipoproteins may support an increased tissue cholesterol removing capacity of ETC-216. These findings provide mechanistic insight into the rapid removal of atheromatous plaques observed in humans, possibly linked to enhanced cholesterol removal capacity of ETC-216.

Development and antitumor activity of a BCL-2 targeted single-stranded DNA oligonucleotide.

PNT100 is a 24-base, chemically unmodified DNA oligonucleotide sequence that is complementary to a region upstream of the BCL-2 gene. Exposure of tumor cells to PNT100 results in suppression of proliferation and cell death by a process called DNA interference. PNT2258 is PNT100 that is encapsulated in protective amphoteric liposomes developed to efficiently encapsulate the PNT100 oligonucleotide, provide enhanced serum stability, optimized pharmacokinetic properties and antitumor activity of the nanoparticle both in vivo and in vitro. PNT2258 demonstrates broad antitumor activity against BCL-2-driven WSU-DLCL2 lymphoma, highly resistant A375 melanoma, PC-3 prostate, and Daudi-Burkitt's lymphoma xenografts. The sequence specificity of PNT100 was demonstrated against three control sequences (scrambled, mismatched, and reverse complement) all encapsulated in a lipid formulation with identical particle characteristics, and control sequences did not demonstrate antiproliferative activity in vivo or in vitro. PNT2258 is currently undergoing clinical testing to evaluate safety and antitumor activity in patients with recurrent or refractory non-Hodgkin's lymphoma and additional studies are planned.

A phase 1 study of the BCL2-targeted deoxyribonucleic acid inhibitor (DNAi) PNT2258 in patients with advanced solid tumors.

PURPOSE: Maximum tolerated dose, safety, pharmacokinetics, and pharmacodynamics were assessed in this phase 1 study of PNT2258, a BCL-2-targeted liposomal formulation of a 24-base DNA oligonucleotide called PNT100. METHODS: Patients with malignant solid tumors were assigned sequentially to one of ten dose-escalation cohorts of PNT2258 at 1, 2, 4, 8, 16, 32, 64, 85, 113, and 150 mg/m(2) administered intravenously on days 1 through 5 of each 21-day cycle. Pharmacokinetics were determined on days 1 and 5 of the first cycle. Lymphocyte and platelets concentrations were measured for evidence of BCL2-targeted effect. CT scans were used to identify radiologic evidence of anti-tumor effect. RESULTS: Twenty-two subjects received PNT2258, and the maximum tolerated dose for PNT2258 was not reached. Doses at or above 32 mg/m(2) resulted in exposure to PNT2258 above the exposure level required for anti-tumor activity in preclinical xenograft testing of 22,377 ng h/ml (PK analysis 2012). Fatigue was the most commonly reported adverse event. Dose-limiting toxicity, manifesting as a transient increase in aspartate aminotransferase, occurred at 150 mg/m(2), the highest dose tested. Four subjects, two each with diagnosis of non-small-cell lung cancer and sarcoma, treated at doses of 64 mg/m(2) or higher, remained on study for 5-8 cycles. CONCLUSIONS: PNT2258 was safe and well tolerated at the doses tested up to 150 mg/m(2). Exposure to PNT2258 resulted in clinically manageable decreases in lymphocyte and platelet concentrations.

PON1 and oxidative stress in human sepsis and an animal model of sepsis.

Sepsis is the leading cause of death in critically ill patients. The pathophysiological mechanisms implicated in the development of sepsis and organ failure are complex and involve activation of systemic inflammatory response and coagulation together with endothelial dysfunction. Oxidative stress is a major promoter and mediator of the systemic inflammatory response. Serum PON1 has been demonstrated in multiple clinical and animal studies to protect against oxidative stress, but also to undergo inactivation upon that condition. We found decreased plasma PON1 activity in patients with sepsis compared to healthy controls or critically ill patients without sepsis; furthermore, in sepsis patients PON1 activity was lower and remained lower in the course of sepsis in the non-survivors compared to the survivors. Plasma PON1 activity was positively correlated with high-density lipoprotein cholesterol and negatively correlated with markers of lipid peroxidation. In an experimental animal model of sepsis, murine cecal ligation and puncture, the time course of plasma PON1 activity was very similar to that found in sepsis patients. Persistently low PON1 activity in plasma was associated with lethal outcome in human and murine sepsis.

Dose-related effects of repeated ETC-216 (recombinant apolipoprotein A-I Milano/1-palmitoyl-2-oleoyl phosphatidylcholine complexes) administrations on rabbit lipid-rich soft plaques: in vivo assessment by intravascular ultrasound and magnetic resonance imaging.

OBJECTIVES: This study sought to evaluate in vivo the minimal dose of apolipoprotein (apo) A-I(Milano) phospholipid complex (recombinant apoA-I(Milano) and 1-palmitoyl-2-oleoyl phosphatidylcholine complexes [ETC-216]) able to induce atherosclerosis regression in a rabbit model of lipid-rich plaques. BACKGROUND: A single high dose of recombinant apoA-I(Milano) has promoted atherosclerosis regression in animal models. More recently, regression of atherosclerosis was achieved in coronary patients by repeated infusions of ETC-216. METHODS: Thirty-six rabbits underwent perivascular injury at both carotid arteries, followed by a 1.5% cholesterol diet. After 90 days, rabbits were randomly divided into 6 groups and treated 5 times with vehicle or ETC-216 at 5, 10, 20, 40, or 150 mg/kg dose every 4 days. Carotid plaque changes were evaluated in vivo by intravascular ultrasound (IVUS) and magnetic resonance imaging (MRI), performed before and at the end of treatments. Magnetic resonance imaging scans were also recorded after administration of the second dose for rabbits infused with vehicle 40 or 150 mg/kg. RESULTS: Atheroma volume in vehicle-treated rabbits increased dramatically between the first and the second IVUS analyses (+26.53%), whereas in ETC-216-treated animals, a reduced progression at the lower doses and a significant regression at the higher doses, up to -6.83%, was detected. Results obtained by MRI analysis correlated significantly with those at IVUS (r = 0.706; p < 0.0001). The MRI evaluations after the second infusion established that a significant regression was achieved with only 2 administrations of the highest dose. CONCLUSIONS: These results confirm the efficacy of ETC-216 for atherosclerosis treatment and provide guidance for dose selection and frequency to obtain a significant reduction of plaque volume.

Apolipoprotein A-IMilano/POPC complex attenuates post-ischemic ventricular dysfunction in the isolated rabbit heart.

Irreversible myocardial injury is a potential consequence of coronary artery revascularization. Reperfusion leads to the production of oxidized products that can damage myocardium. High-density lipoproteins (HDL) are effective at removing oxidized lipids. We hypothesized that a synthetic HDL preparation, comprising recombinant apolipoprotein A-I(Milano) (apoA-I(M)) complexed with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) (apoA-I(M)/POPC) would protect the heart from reperfusion injury. The ex vivo model consisted of rabbit hearts perfused by the Langendorff method. Hearts were equilibrated with Krebs-Henseleit buffer (10 min), pretreated with either apoA-I(M)/POPC (0.45 mg/mL) or vehicle (10 min), subjected to global ischemia (30 min) and reperfused for 60 min. ApoA-I(M)/POPC (n=7) prevented the left ventricular end-diastolic pressure elevation observed in the vehicle group (n=6) at the end of reperfusion (p<0.05). During reperfusion, coronary artery perfusion pressure increased in the controls (p<0.001), but not with apoA-I(M)/POPC. ApoA-I(M)/POPC reduced the release of creatine kinase at the end of the ischemic period (p<0.001). It also reduced cardiac left ventricle muscle lipid hydroperoxides by 46% (p<0.05). Direct comparison of the antioxidant potential indicated that recombinant apoA-I(M) was much more potent than apoA-I in attenuating low-density lipoprotein oxidation. Electron microscopy showed that apoA-I(M)/POPC prevented mitochondrial granulation, disorganization and sarcomere contraction band formation indicative of reperfusion injury. The apoA-I(M)/POPC complex thus appears to reduce reperfusion injury under global ischemic conditions, and may therefore have therapeutic application in the reduction of myocardial ischemia.

Peroxisome proliferator-activated receptor-alpha selective ligand reduces adiposity, improves insulin sensitivity and inhibits atherosclerosis in LDL receptor-deficient mice.

Fenofibrate, a selective (1)PPAR-alpha activator, is prescribed to treat human dyslipidemia. The aim of this study was to delineate the mechanism of fenofibrate-mediated reductions in adiposity, improvements in insulin sensitivity, and lowering of triglycerides (TG) and free fatty acids (FFA) and to investigate if these favorable changes are related to the inhibition of lipid deposition in the aorta. To test this hypothesis we used male LDLr deficient mice that exhibit the clinical features of metabolic syndrome X when fed a high fat high cholesterol (HF) diet. LDLr deficient mice fed HF diet and simultaneously treated with fenofibrate (100 mg/kg body weight) prevented development of obesity, lowered serum triglycerides and cholesterol, improved insulin sensitivity, and prevented accumulation of lipids in the aorta. Lowering of circulating lipids occurred via down-regulation of lipogenic genes, including fatty acid synthase, acetyl CoA carboxylase and diacyl glycerol acyl transferase-2, concomitant with decreased liver TG and cholesterol, and TG output rate. Fenofibrate also suppressed liver apoCIII mRNA levels and markedly increased lipoprotein lipase mRNA levels, known to enhance serum TG catabolism. In addition, fenofibrate profoundly reduced epididymal fat and mesenteric fat mass to the levels seen in lean mice. The reductions in body weight were associated with elevation of hepatic uncoupling protein 2 (UCP2) mRNA, a concomitant increase in the ketone body formation, and improved insulin sensitivity associated with tumor necrosis factor-alpha reductions and phosphoenol pyruvate carboxykinase down-regulation. These results demonstrate that fenofibrate improves lipid abnormalities partly via inhibition of TG production and partly via clearance of TG-rich apoB particles by elevating LPL and reduced apoCIII. The prevention of obesity development occurred via energy expenditure. Fenofibrate-mediated hypolipidemic effects together with improved insulin sensitivity and loss of adiposity led to the reductions in the aortic lipid deposition by inhibiting early stages of atherosclerosis possibly via vascular cell adhesion molecule-1 (VCAM-1) modulation. These results suggest that potent PPAR-alpha activators may be useful in the treatment of syndrome X.

Influence of various central moieties on the hypolipidemic properties of long hydrocarbon chain diols and diacids.

A series of long (11-15) hydrocarbon chain diols and diacids with various central functional groups and terminal gem-dimethyl or -methyl/aryl substituents was synthesized and evaluated in both in vivo and in vitro assays for its potential to favorably alter lipid disorders including metabolic syndrome. Compounds were assessed for their effects on the de novo incorporation of radiolabeled acetate into lipids in primary cultures of rat hepatocytes, as well as for their effects on lipid and glycemic variables in obese female Zucker fatty rats, Crl:(ZUC)-faBR. The most active compounds were hydroxyl-substituted symmetrical diacids and diols with a 13-atom chain and terminal gem-dimethyl substituents. Furthermore, biological activity was enhanced by central substitution with O, C=O, S, S=O compared to the methylene analogues and was diminished for compounds with central functional groups such as carbamate, ester, urea, acetylmethylene, and hydroxymethylene.

alpha-Cycloalkyl-substituted omega-keto-dicarboxylic acids as lipid regulating agents.

A series of cycloalkyl-substituted oxo-alkanedicarboxylic acids have been prepared by the TosMIC methodology departing from haloalkyl-substituted cycloalkylcarboxylic esters. cyclopropyl derivatives showed IC(50) activity in the 0.3-1.0 microM range on the de novo incorporation of radiolabeled acetate into lipids in primary cultures of rat hepatocytes, and they showed lipid-regulating properties when tested in vivo in female obese Zucker fatty rats.

Long hydrocarbon chain keto diols and diacids that favorably alter lipid disorders in vivo.

Keto-substituted hydrocarbons with 11-19 methylene and bis-terminal hydroxyl and carboxyl groups have been synthesized and evaluated in both in vivo and in vitro assays for their potential to favorably alter lipid disorders including metabolic syndrome. Compounds were assessed for their effects on the de novo incorporation of radiolabeled acetate into lipids in primary cultures of rat hepatocytes as well as for their effects on lipid and glycemic variables in obese female Zucker fatty rats [Crl:(ZUC)-faBR] following 1 and 2 weeks of oral administration. The most active compounds were found to be symmetrical with four to five methylene groups separating the central ketone functionality and the gem dimethyl or methyl/aryl substituents. Furthermore, biological activity was found to be greatest in both in vivo and in vitro assays for the tetramethyl-substituted keto diacids and diols (e.g., 10c, 10g, 14c), and the least active were shown to be the bis(arylmethyl) derivatives (e.g., 10e, 10f, 14f). Compound 14c dose-dependently elevated HDL-cholesterol, reduced triglycerides, and reduced NEFA, with a minimum effective dose of 30 mg/kg/day. Compound 1 g dose-dependently modified non-HDL-cholesterol, triglycerides, and nonesterified fatty acids, with a minimum effective dose of 10 mg/kg/day. At this dose, compound 10g elevated HDL-cholesterol levels 2-3 times higher than pretreatment levels, and a dose-dependent reduction of fasting insulin and glucose levels was observed.

Long hydrocarbon chain ether diols and ether diacids that favorably alter lipid disorders in vivo.

Long hydrocarbon chain ethers with bis-terminal hydroxyl or carboxyl groups have been synthesized and evaluated for their potential to favorably alter lipid disorders including metabolic syndrome. Compounds were assessed for their effects on the de novo incorporation of radiolabeled acetate into lipids in primary cultures of rat hepatocytes as well as for their effects on lipid and glycemic variables in female obese Zucker fatty rats following 1 and 2 weeks of daily oral administration. The most active compounds were found to be symmetrical with four to five methylene groups separating the central ether functionality and the gem dimethyl or methyl/aryl substituents. Biological activity was found to be greatest for tetramethyl-substituted ether diols (e.g., 28 and 31), while bis(arylmethyl) derivatives (e.g., 10, 11, and 27), diethers (e.g., 49, 50, and 56), and diphenyl ethers (e.g., 35 and 36) were the least active. For the most biologically active compound 28, we observed as much as a 346% increase in serum HDL-cholesterol and a 71% reduction in serum triglycerides at the highest dose administered (100 mg/kg) after 2 weeks of treatment. For compound 31 we observed a 69% reduction in non-HDL-cholesterol, accompanied by a 131% increase in HDL-cholesterol and an 84% reduction in serum triglycerides under the same treatment conditions.

Apolipoprotein A-IMilano and 1-palmitoyl-2-oleoyl phosphatidylcholine complex (ETC-216) protects the in vivo rabbit heart from regional ischemia-reperfusion injury.

Ex vivo studies demonstrated that a synthetic high-density lipoprotein (HDL) comprised of a complex of recombinant apolipoprotein A-IMilano and 1-palmitoyl-2-oleoyl phosphatidylcholine protects the isolated rabbit heart from reperfusion injury. Therefore, we sought to determine whether a pharmaceutical preparation of this complex, ETC-216, was cardioprotective in an in vivo model of left anterior descending artery (LAD) occlusion and reperfusion. Initially, ETC-216 (100 mg/kg) was tested in acute (one-treatment) and chronic (two-treatment) i.v. administrations. ETC-216-treated rabbits developed smaller infarcts expressed as percentage of area at risk (p <0.01) compared with vehicle treatments. No differences were noted between chronic and acute administration. Therefore, ETC-216 (10, 3, or 1 mg/kg) or equivalent vehicle volumes were acutely infused. Compared with vehicle, ETC-216 reduced infarct size as a percentage of the area at risk at 10 (p <0.0005) and 3 mg/kg (p <0.05). No significant differences occurred at 1 mg/kg. To determine whether ETC-216 could protect the heart after initiation of ischemia, the synthetic HDL (10 mg/kg) was infused intravenously beginning 5 min before the end of 30 min of LAD occlusion. Infarct size as percentage of the area at risk was 31.6 +/- 3.0 (ETC-216) versus 49.5 +/- 2.5 (vehicle) (p <0.001), and as percentage of left ventricle was 19.7 +/- 1.6 (ETC-216) versus 34.1 +/- 2.3 (vehicle) (p <0.0005). Electron microscopy demonstrated that ETC-216 prevented irreversible cardiac damage as assessed by mitochondrial granulation and sarcomere contraction band formation. These findings suggest ETC-216 reduces reperfusion injury and may have utility for coronary artery revascularization procedures.

Aging, gender and APOE isotype modulate metabolism of Alzheimer's Abeta peptides and F-isoprostanes in the absence of detectable amyloid deposits.

Aging and apolipoprotein E (APOE) isoform are among the most consistent risks for the development of Alzheimer's disease (AD). Metabolic factors that modulate risk have been elusive, though oxidative reactions and their by-products have been implicated in human AD and in transgenic mice with overt histological amyloidosis. We investigated the relationship between the levels of endogenous murine amyloid beta (Abeta) peptides and the levels of a marker of oxidation in mice that never develop histological amyloidosis [i.e. APOE knockout (KO) mice with or without transgenic human APOEepsilon3 or human APOEepsilon4 alleles]. Aging-, gender-, and APOE-genotype-dependent changes were observed for endogenous mouse brain Abeta40 and Abeta42 peptides. Levels of the oxidized lipid F2-isoprostane (F2-isoPs) in the brains of the same animals as those used for the Abeta analyses revealed aging- and gender-dependent changes in APOE KO and in human APOEepsilon4 transgenic KO mice. Human APOEepsilon3 transgenic KO mice did not exhibit aging- or gender-dependent increases in F2-isoPs. In general, the changes in the levels of brain F2-isoPs in mice according to age, gender, and APOE genotype mirrored the changes in brain Abeta levels, which, in turn, paralleled known trends in the risk for human AD. These data indicate that there exists an aging-dependent, APOE-genotype-sensitive rise in murine brain Abeta levels despite the apparent inability of the peptide to form histologically detectable amyloid. Human APOEepsilon3, but not human APOEepsilon4, can apparently prevent the aging-dependent rise in murine brain Abeta levels, consistent with the relative risk for AD associated with these genotypes. The fidelity of the brain Abeta/F2-isoP relationship across multiple relevant variables supports the hypothesis that oxidized lipids play a role in AD pathogenesis, as has been suggested by recent evidence that F2-isoPs can stimulate Abeta generation and aggregation.