Factor XII full and partial null in rat confers robust antithrombotic efficacy with no bleeding.

This report aims at exploring quantitatively the relationship between FXII inhibition and thromboprotection. FXII full and partial null in rats were established via zinc finger nuclease-mediated knockout and siRNA-mediated knockdown, respectively. The rats were subsequently characterized in thrombosis and hemostasis models. Knockout rats exhibited complete thromboprotection in both the arteriovenous shunt model (∼100% clot weight reduction) and the FeCl3-induced arterial thrombosis model (no reduction in blood flow), without any increase in cuticle bleeding time compared with wild-type control rats. Ex-vivo aPTT and the ellagic acid-triggered thrombin generation assay (TGA) exhibited anticoagulant changes. In contrast, ex-vivo PT or high tissue factor-triggered TGA was indistinguishable from control. Rats receiving single doses (0, 0.01, 0.03, 0.1, 0.3, 1 mg/kg) of FXII siRNA exhibited dose-dependent knockdown in liver FXII mRNA and plasma FXII protein (95 and 99%, respectively, at 1 mg/kg) at day 7 post dosing. FXII knockdown was associated with dose-dependent thromboprotection (maximal efficacy achieved with 1 mg/kg in both models) and negligible change in cuticle bleeding times. Ex-vivo TGA triggered with low-level (0.5 µmol/l) ellagic acid tracked best with the knockdown levels and efficacy. Our findings confirm and extend literature reports of an attractive benefit-to-risk profile of targeting FXII for antithrombotic therapies. Titrating of FXII is instructive for its pharmacological inhibition. The knockout rat is valuable for evaluating both mechanism-based safety concerns and off-target effects of FXII(a) inhibitors. Detailed TGA analyses will inform on optimal trigger conditions in studying pharmacodynamic effects of FXII(a) inhibition.

Improved Stability of Proline-Derived Direct Thrombin Inhibitors through Hydroxyl to Heterocycle Replacement.

Modification of the previously disclosed (S)-N-(2-(aminomethyl)-5-chlorobenzyl)-1-((R)-2-hydroxy-3,3-dimethylbutanoyl)pyrrolidine-2-carboxamide 2 by optimization of the P3 group afforded novel, low molecular weight thrombin inhibitors. Heterocycle replacement of the hydroxyl functional group helped maintain thrombin in vitro potency while improving the chemical stability and pharmacokinetic profile. These modifications led to the identification of compound 10, which showed excellent selectivity over related serine proteases as well as in vivo efficacy in the rat arteriovenous shunt. Compound 10 exhibited significantly improved chemical stability and pharmacokinetic properties over 2 and may be utilized as a structurally differentiated preclinical tool comparator to dabigatran etexilate (Pro-1) to interrogate the on- and off-target effects of oral direct thrombin inhibitors.

Titrating haemophilia B phenotypes using siRNA strategy: evidence that antithrombotic activity is separated from bleeding liability.

Haemophilia A and B are characterised by a life-long bleeding predisposition, and several lines of evidence suggest that risks of atherothrombotic events may also be reduced. Establishing a direct correlation between coagulation factor levels, thrombotic risks and bleeding propensity has long been hampered by an inability to selectively and specifically inhibit coagulation factor levels. Here, the exquisite selectivity of gene silencing combined with a gene knockout (KO) approach was used to define the relative contribution of factor IX (fIX) to thrombosis and primary haemostasis in the rat. Using a lipid nanoparticle (LNP) formulation, we successfully delivered fIX siRNAs to the liver by intravenous administration. The knockdown (KD) of target gene mRNA was achieved rapidly (within 24 hour post-siRNA dosing), sustained (maintained for at least 7 days post dosing) and not associated with changes in mRNA expression levels of other coagulation factors. We found that intermediate levels of liver fIX mRNA silencing (60-95 %) translating into a 50-99 % reduction of plasma fIX activity provided protection from thrombosis without prolonging the cuticle bleeding time. Over 99 % inhibition of fIX activity was required to observe increase in bleeding, a phenotype confirmed in fIX KO rats. These data provide substantial evidence of a participation of fIX in the mechanisms regulating thrombosis prior to those regulating primary haemostasis, therefore highlighting the potential of fIX as a therapeutic target. In addition, hepatic mRNA silencing using LNP-encapsulated siRNAs may represent a promising novel approach for the chronic treatment and prevention of coagulation-dependent thrombotic disorders in humans.

Proof-of-concept Studies for siRNA-mediated Gene Silencing for Coagulation Factors in Rat and Rabbit.

The present study aimed at establishing feasibility of delivering short interfering RNA (siRNA) to target the coagulation cascade in rat and rabbit, two commonly used species for studying thrombosis and hemostasis. siRNAs that produced over 90% mRNA knockdown of rat plasma prekallikrein and rabbit Factor X (FX) were identified from in vitro screens. An ionizable amino lipid based lipid nanoparticle (LNP) formulation for siRNA in vivo delivery was characterized as tolerable and exerting no appreciable effect on coagulability at day 7 postdosing in both species. Both prekallikrein siRNA-LNP and FX siRNA-LNP resulted in dose-dependent and selective knockdown of target gene mRNA in the liver with maximum reduction of over 90% on day 7 following a single dose of siRNA-LNP. Knockdown of plasma prekallikrein was associated with modest clot weight reduction in the rat arteriovenous shunt thrombosis model and no increase in the cuticle bleeding time. Knockdown of FX in the rabbit was accompanied with prolongation in ex vivo clotting times. Results fit the expectations with both targets and demonstrate for the first time, the feasibility of targeting coagulation factors in rat, and, more broadly, targeting a gene of interest in rabbit, via systemic delivery of ionizable LNP formulated siRNA.

Factor XIIa inhibition by Infestin-4: in vitro mode of action and in vivo antithrombotic benefit.

Coagulation factor XII (FXII) plays a central role in initiating the intrinsic cascade of blood coagulation. Purified recombinant Human Albumin-tagged Infestin-4 (rHA-Infestin-4) is a recently described FXIIa inhibitor that displayed strong anticoagulant activity without compromising haemostasis in several animal models. We pursued detailed in vitro characterisation of rHA-Infestin-4 and demonstrated that it is a competitive inhibitor of FXIIa with slow on and off rate constants for binding (kon=5x105 M⁻¹s⁻¹, koff=6x10⁻4 s⁻¹), it can block FXIIa activation of its physiological substrates (plasma prekallikrein and FXI), and it can inhibit ellagic acid-triggered thrombin generation in plasma. Potency and selectivity profiling in enzyme assays suggest that rHA-Infestin-4 is indeed highly potent on FXIIa (IC50=0.3 ± 0.06, 1.5 ± 0.06, 1.2 ± 0.09 nM, for human, rat, and rabbit FXIIa, respectively) with at least >100-fold selectivity against factors IIa, Xa, IXa, XIa, VIIa, and plasma kallikrein in all three species. rHA-Infestin-4 dose-dependently and markedly reduced clot weight in the arteriovenous shunt thrombosis model in rats and rabbits, accompanied with minimal increase in cuticle bleeding times in either species. rHA-Infestin-4 treatment at 5 mg/kg in rabbit resulted in a 13% reduction in ex vivo FXa activity, demonstrating a modest off-target effect. In summary, our findings confirmed and extended previous reports that inhibition of FXIIa by rHA-Infestin-4 can produce strong antithrombotic efficacy while preserving haemostasis. Our comprehensive selectivity profiling, mode of action, and kinetic studies of rHA-Infestin-4 reveal limitations of this molecule and offer new perspectives on any potential effort of discovering novel FXIIa inhibitors.

Atorvastatin increases intestinal expression of NPC1L1 in hyperlipidemic men.

Inhibition of cholesterol synthesis by 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR) inhibitors has been associated with an increase in intestinal cholesterol absorption. This study examined how HMG-CoAR inhibition by atorvastatin modulates expression of key genes involved in intestinal cholesterol metabolism. A crossover study was conducted in which 22 hyperlipidemic men received atorvastatin, 40 mg/day, or placebo, each for 12 weeks. Gene expression was assessed by real-time PCR using duodenal biopsy samples obtained at the end of each phase of treatment. Treatment with atorvastatin was associated with a 76% reduction in lathosterol and significant increases in sitosterol (70%). Atorvastatin significantly increased intestinal mRNA levels of HMG-CoAR (59%), LDL receptor (LDLR) (52%), PCSK9 (187%), SREBP-2 (44%), and HNF-4α (13%). Furthermore, atorvastatin significantly increased intestinal mRNA levels of NPC1L1 by 19% and decreased mRNA levels of both ABCG5 and ABCG8 by 14%. Positive correlations were observed between changes in SREBP-2 and HNF-4α expression and concurrent changes in the intestinal mRNA levels of HMG-CoAR, LDLR, and NPC1L1. These results indicate that HMG-CoAR inhibition with atorvastatin stimulates the intestinal expression of NPC1L1, LDLR, and PCSK9; increases cholesterol absorption; and reduces expression of ABCG5/8; these effects are most likely mediated by upregulation of the transcription factors SREBP-2 and HNF-4α.

Gender-dependent effect of Gpbar1 genetic deletion on the metabolic profiles of diet-induced obese mice.

G-protein-coupled bile acid receptor 1 (GPBAR1/TGR5/M-Bar/GPR131) is a cell surface receptor involved in the regulation of bile acid metabolism. We have previously shown that Gpbar1-null mice are resistant to cholesterol gallstone disease when fed a lithogenic diet. Other published studies have suggested that Gpbar1 is involved in both energy homeostasis and glucose homeostasis. Here, we examine the functional role of Gpbar1 in diet-induced obese mice. We found that body weight, food intake, and fasted blood glucose levels were similar between Gpbar1-null mice and their wild-type (WT) littermates when fed a chow or high-fat diet (HFD) for 2 months. However, insulin tolerance tests revealed improved insulin sensitivity in male Gpbar1(-/-) mice fed chow, but impaired insulin sensitivity when fed a HFD. In contrast, female Gpbar1(-/-) mice exhibited improved insulin sensitivity when fed a HFD compared with their WT littermates. Female Gpbar1(-/-) mice had significantly lower plasma cholesterol and triglyceride levels than their WT littermates on both diets. Male Gpbar1(-/-) mice on HFD displayed increased hepatic steatosis when compared with Gpbar1(+)(/)(+) males and Gpbar1(-/-) females on HFD. These results suggest a gender-dependent regulation of Gpbar1 function in metabolic disease.

Lack of FFAR1/GPR40 does not protect mice from high-fat diet-induced metabolic disease.

OBJECTIVE: FFAR1/GPR40 is a G-protein-coupled receptor expressed predominantly in pancreatic islets mediating free fatty acid-induced insulin secretion. However, the physiological role of FFAR1 remains controversial. It was previously reported that FFAR1 knockout (Ffar1(-/-)) mice were resistant to high-fat diet-induced hyperinuslinemia, hyperglycemia, hypertriglyceridemia, and hepatic steatosis. A more recent report suggested that although FFAR1 was necessary for fatty acid-induced insulin secretion in vivo, deletion of FFAR1 did not protect pancreatic islets against fatty acid-induced islet dysfunction. This study is designed to investigate FFAR1 function in vivo using a third line of independently generated Ffar1(-/-) mice in the C57BL/6 background. RESEARCH DESIGN AND METHODS: We used CL-316,243, a beta3 adrenergic receptor agonist, to acutely elevate blood free fatty acids and to study its effect on insulin secretion in vivo. Ffar1(+/+) (wild-type) and Ffar1(-/-) (knockout) mice were placed on two distinct high-fat diets to study their response to diet-induced obesity. RESULTS: Insulin secretion was reduced by approximately 50% in Ffar1(-/-) mice, confirming that FFAR1 contributes significantly to fatty acid stimulation of insulin secretion in vivo. However, Ffar1(+/+) and Ffar1(-/-) mice had similar weight, adiposity, and hyperinsulinemia on high-fat diets, and Ffar1(-/-) mice showed no improvement in glucose or insulin tolerance tests. In addition, high-fat diet induced comparable levels of lipid accumulation in livers of Ffar1(+/+) and Ffar1(-/-) mice. CONCLUSIONS: FFAR1 is required for normal insulin secretion in response to fatty acids; however, Ffar1(-/-) mice are not protected from high-fat diet-induced insulin resistance or hepatic steatosis.

Cholesterol homeostasis by the intestine: lessons from Niemann-Pick C1 Like 1 [NPC1L1).

Ezetimibe is a selective cholesterol absorption inhibitor, which potently inhibits the uptake and absorption of biliary and dietary cholesterol from the small intestine without affecting the absorption of fat-soluble vitamins, triglycerides or bile acids. Identification and characterization of Niemann-Pick C1 Like 1 (NPC1L1) has established NPC1L1 as an essential protein in the intestinal cholesterol absorption process. While otherwise phenotypically normal, Npc1l1 null mice exhibit a significant reduction in the intestinal uptake and absorption of cholesterol and phytosterols. Characterization of the NPC1L1 pathway revealed that ezetimibe specifically binds to NPC1L1 and inhibits its sterol transport function. Npc1l1 null mice were resistant to diet-induced hypercholesterolemia, and when crossed with apoE null mice, were completely resistant to the development of atherosclerosis. In Npc1l1/apoE null mice or apoE null mice treated with ezetimibe plasma cholesterol levels were reduced primarily in the apoB48 containing chylomicron remnant lipoproteins relative to untreated apoE null mice. SR-B1 has been proposed to play a role in intestinal cholesterol uptake, but in Npc1l1/SR-B1 double null mice intestinal cholesterol absorption was not different than Npc1l1 null alone mice. Therefore, NPC1L1 is the critical intestinal sterol transporter which influences whole body cholesterol homeostasis, and is the molecular target of ezetimibe.

Ezetimibe improves high fat and cholesterol diet-induced non-alcoholic fatty liver disease in mice.

Ezetimibe is a novel cholesterol and plant sterol absorption inhibitor that reduces plasma low-density lipoprotein-cholesterol by selectively binding to the intestinal cholesterol transporter, Niemann-Pick C1-Like 1. Mice deficient in Niemann-Pick C1-Like 1 are protected from high fat/cholesterol diet-induced fatty liver as well as hypercholesterolemia. The object of the present study was to determine whether ezetimibe treatment could reduce hepatic steatosis in diet-induced obese mice. C57BL/6J mice were fed a high fat/cholesterol containing semi-purified diet (45% Kcal fat and 0.12% cholesterol) for 7 months after weaning. These mice were not only obese, but also developed hepatomegaly and hepatic steatosis, with varying degrees of liver fibrosis and steatohepatitis. About 87% of the mice on the high fat/cholesterol diet for 7 months had elevated plasma alanine aminotransferase activity, a biomarker for non-alcoholic fatty liver disease. Chronic administration of ezetimibe for 4 weeks significantly reduced hepatomegaly by decreasing hepatic triglyceride, cholesteryl ester and free cholesterol in diet-induced obese mice fed high fat/cholesterol diet for 7 months. Chronic ezetimibe treatment also significantly decreased plasma alanine aminotransferase activity. These results suggest that ezetimibe may be a novel treatment for high fat/cholesterol-induced non-alcoholic fatty liver disease.

Deficiency of Niemann-Pick C1 Like 1 prevents atherosclerosis in ApoE-/- mice.

OBJECTIVE: The objective of this study was to determine whether the deficiency of Niemann-Pick C1 Like 1 (Npc1l1) prevents atherosclerosis in apoE null mice. METHODS AND RESULTS: Npc1l1(-/-)/apoE null-/- mice were generated and found to have a significant reduction in cholesterol absorption (-77%) compared with wild-type or apoE-/- mice. Npc1l1/apoE-/- mice were fed a chow or Western diet for 24 weeks, then lipoprotein, hepatic, and biliary cholesterol, and atherosclerosis development was compared with apoE-/-, Npc1l1-/-, wild-type, and ezetimibe-treated apoE-/- mice. Chylomicron remnant/VLDL cholesterol levels were reduced 80% to 90% in both chow and Western diet-fed Npc1l1/apoE-/- mice relative to apoE-/- mice. Male Npc1l1-/- and Npc1l1/apoE-/- mice were completely resistant to diet induced hypercholesterolemia, and both male and female mice were completely resistant to increases in hepatic and biliary cholesterol levels. Atherosclerosis was reduced 99% in aortic lesion surface area, 94% to 97% in innominate artery intimal lesion area, and >90% in aortic root lesion area in both male and female Npc1l1/apoE-/- mice relative to apoE-/- mice. CONCLUSIONS: Lack of Npc1l1, the molecular target of the cholesterol absorption inhibitor ezetimibe, in apoE-/- mice results in a significant reduction in cholesterol absorption and plasma cholesterol levels, and causes a nearly complete protection from the development of atherosclerosis, under both cholesterol-fed and non-cholesterol-fed conditions.

Targeted deletion of Gpbar1 protects mice from cholesterol gallstone formation.

The Gpbar1 [G-protein-coupled BA (bile acid) receptor 1] is a recently identified cell-surface receptor that can bind and is activated by BAs, but its physiological role is unclear. Using targeted deletion of the Gpbar1 gene in mice, we show that the gene plays a critical role in the maintenance of bile lipid homoeostasis. Mice lacking Gpbar1 expression were viable, developed normally and did not show significant difference in the levels of cholesterol, BAs or any other bile constituents. However, they did not form cholesterol gallstones when fed a cholic acid-containing high-fat diet, and liver-specific gene expression indicated that Gpbar1-deficient mice have altered feedback regulation of BA synthesis. These results suggest that Gpbar1 plays a critical role in the formation of gallstones, possibly via a regulatory mechanism involving the cholesterol 7alpha-hydroxylase pathway.

Characterization of the putative native and recombinant rat sterol transporter Niemann-Pick C1 Like 1 (NPC1L1) protein.

The exact mechanistic pathway of cholesterol absorption in the jejunum of the small intestines is a poorly understood process. Recently, a relatively novel gene, Niemann-Pick C1 Like 1 (NPC1L1), was identified as being critical for intestinal sterol absorption in a pathway which is sensitive to sterol absorption inhibitors such as ezetimibe. NPC1L1 is a multi-transmembrane protein, with a putative sterol sensing domain. Very little else is known about the NPC1L1 protein. In this report, we characterize the native and recombinant rat NPC1L1 protein. We show that NPC1L1 is a 145 kDa membrane protein, enriched in the brush border membrane of the intestinal enterocyte and is highly glycosylated. In addition, sequential detergent extraction of enterocytes result in highly enriched preparations of NPC1L1. An engineered Flag epitope tagged rat NPC1L1 cDNA was expressed as recombinant protein in CHO cells and demonstrated cell surface expression, similar to the native rat protein. These biochemical data indicate that NPC1L1 exists as a predominantly cell surface membrane expressed protein, consistent with its proposed role as the putative intestinal sterol transporter.

Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis.

Niemann-Pick C1 Like 1 (NPC1L1) is a protein localized in jejunal enterocytes that is critical for intestinal cholesterol absorption. The uptake of intestinal phytosterols and cholesterol into absorptive enterocytes in the intestine is not fully defined on a molecular level, and the role of NPC1L1 in maintaining whole body cholesterol homeostasis is not known. NPC1L1 null mice had substantially reduced intestinal uptake of cholesterol and sitosterol, with dramatically reduced plasma phytosterol levels. The NPC1L1 null mice were completely resistant to diet-induced hypercholesterolemia, with plasma lipoprotein and hepatic cholesterol profiles similar to those of wild type mice treated with the cholesterol absorption inhibitor ezetimibe. Cholesterol/cholate feeding resulted in down-regulation of intestinal NPC1L1 mRNA expression in wild type mice. NPC1L1 deficiency resulted in up-regulation of intestinal hydroxymethylglutaryl-CoA synthase mRNA and an increase in intestinal cholesterol synthesis, down-regulation of ABCA1 mRNA, and no change in ABCG5 and ABCG8 mRNA expression. NPC1L1 is required for intestinal uptake of both cholesterol and phytosterols and plays a major role in cholesterol homeostasis. Thus, NPC1L1 may be a useful drug target for the treatment of hypercholesterolemia and sitosterolemia.

Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption.

Dietary cholesterol consumption and intestinal cholesterol absorption contribute to plasma cholesterol levels, a risk factor for coronary heart disease. The molecular mechanism of sterol uptake from the lumen of the small intestine is poorly defined. We show that Niemann-Pick C1 Like 1(NPC1L1) protein plays a critical role in the absorption of intestinal cholesterol. NPC1L1 expression is enriched in the small intestine and is in the brush border membrane of enterocytes. Although otherwise phenotypically normal, NPC1L1-deficient mice exhibit a substantial reduction in absorbed cholesterol, which is unaffected by dietary supplementation of bile acids. Ezetimibe, a drug that inhibits cholesterol absorption, had no effect in NPC1L1 knockout mice, suggesting that NPC1L1 resides in an ezetimibe-sensitive pathway responsible for intestinal cholesterol absorption.

Ezetimibe potently inhibits cholesterol absorption but does not affect acute hepatic or intestinal cholesterol synthesis in rats.

1. Ezetimibe (1-(4-fluorophenyl)-(3R)-[3-(4-fluorophenyl)-(3S)-hydroxypropyl]-(4S)-(4-hydroxyphenyl)-2-azetidinone) and its analog SCH48461 are potent and selective cholesterol absorption inhibitors that inhibit the transport of cholesterol across the intestinal wall, thereby lowering plasma cholesterol. 2. After a dose response for ezetimibe in rats was established, experiments were conducted to determine whether acute administration could alter hepatic or intestinal cholesterol synthesis. To determine whether this class of intestinal cholesterol absorption inhibitors could discriminate between newly synthesized cholesterol in the intestine versus exogenously administered cholesterol, rats were intraduodenally dosed with (14)C-cholesterol and (3)H-mevalonate, and mesenteric lymph was analyzed for radiolabeled cholesterol and cholesteryl ester content. 3. Ezetimibe attenuated diet-induced hypercholesterolemia 60-94% at doses of 0.1-3 mg x kg(-1) in rats. A single administration of ezetimibe did not have a direct effect on intestinal or hepatic cholesterol synthesis, while ketoconazole significantly inhibited cholesterol synthesis after a single dose. The ezetimibe analog, SCH48461, inhibited the movement of exogenously administered cholesterol into lymph, but did not affect the appearance of newly synthesized cholesterol into lymph. 4. These data suggest that this class of cholesterol absorption inhibitors does discriminate by blocking the movement of exogenous cholesterol in the enterocyte before it reaches the intracellular cholesterol pool to be incorporated into intestinal lipoproteins, without affecting the incorporation of newly synthesized cholesterol into intestinal lipoproteins.

The identification of intestinal scavenger receptor class B, type I (SR-BI) by expression cloning and its role in cholesterol absorption.

The molecular mechanisms of cholesterol absorption in the intestine are poorly understood. With the goal of defining candidate genes involved in these processes a fluorescence-activated cell sorter-based, retroviral-mediated expression cloning strategy has been devised. SCH354909, a fluorescent derivative of ezetimibe, a compound which blocks intestinal cholesterol absorption but whose mechanism of action is unknown, was synthesized and shown to block intestinal cholesterol absorption in rats. Pools of cDNAs prepared from rat intestinal cells enriched in enterocytes were introduced into BW5147 cells and screened for SCH354909 binding. Several independent clones were isolated and all found to encode the scavenger receptor class B, type I (SR-BI), a protein suggested by others to play a role in cholesterol absorption. SCH354909 bound to Chinese hamster ovary (CHO) cells expressing SR-BI in specific and saturable fashion and with high affinity (K(d) approximately 18 nM). Overexpression of SR-BI in CHO cells resulted in increased cholesterol uptake that was blocked by micromolar concentrations of ezetimibe. Analysis of rat intestinal sections by in situ hybridization demonstrated that SR-BI expression was restricted to enterocytes. Cholesterol absorption was determined in SR-B1 knockout mice using both an acute, 2-h, assay and a more chronic fecal dual isotope ratio method. The level of intestinal cholesterol uptake and absorption was similar to that seen in wild-type mice. When assayed in the SR-B1 knockout mice, the dose of ezetimibe required to inhibit hepatic cholesterol accumulation induced by a cholesterol-containing 'western' diet was similar to wild-type mice. Thus, the binding of ezetimibe to cells expressing SR-B1 and the functional blockade of SR-B1-mediated cholesterol absorption in vitro suggest that SR-B1 plays a role in intestinal cholesterol metabolism and the inhibitory activity of ezetimibe. In contrast studies with SR-B1 knockout mice suggest that SR-B1 is not essential for intestinal cholesterol absorption or the activity of ezetimibe.