Patent Grant
8455523
High plasma lipids and lipoproteins are risk factors for various cardiovascular and metabolic disorders. Statins enhance the removal of apolipoprotein B (apoB)-lipoproteins and lower plasma cholesterol. Another approach to lower plasma lipids is to inhibit the biosynthesis of apoB-lipoproteins, a process critically dependent on an endoplasmic reticulum (ER) resident chaperone, microsomal triglyceride transfer protein (MTP). MTP inhibitors decrease apoB-lipoprotein secretion and lower plasma cholesterol. However, they increase plasma aminotransferases, such as ALT and AST, indicating liver injury. Therefore, what is needed is an improved method for treating high plasma lipids and lipoproteins increases in microsomal free cholesterol, without causing an increase in induction of Endoplasmic Reticulum stress and cell death.
An estimated 35.4 million Americans in the United States aged 20 or over have total blood cholesterol levels of >240 mg/dl (normal <200 mg/dl). Hyperlipidemic states (total blood cholesterol >200 mg/dl; LDL Cholesterol >100 mg/dl) have been implicated as a major risk factor for cardiovascular disease (CVD), the leading cause of death in the United States for the past 80 years. Numerous clinical trials and outcome studies have demonstrated that improving such dyslipidemia lowers the progression of atherosclerosis as well as the resulting adverse cardiovascular (CV) events. Statins that remove apolipoprotein B (apoB)-containing lipoproteins from plasma have long been the cornerstone of lowering plasma cholesterol.
Despite their acclaim as the “miracle drug”, approximately 60% of statin-treated patients continue to have adverse coronary events. Furthermore, many patients cannot achieve current target levels for cholesterol owing either to intolerance or an inadequate response to conventional statin therapy. Increasing dosage of statins to try and reach target levels result in an increased likelihood of encountering statin related side effects such as, rhabdomyolysis. Therefore, there is a need to formulate new approaches or regimens to treat hyperlipidemia. One possible approach is to lower plasma lipids at the stage of lipoprotein biosynthesis. Lipoprotein biosynthesis depends on the carrier protein, apolipoprotein B (apoB), and the chaperone, microsomal triglyceride transfer protein (MTP). MTP transfers triglycerides, phospholipids and cholesterol esters to nascent apoB, which readies the protein for secretion as either a chylomicron from the intestine or as VLDL from the liver.
The present invention is directed to a therapeutic combination and a method for lowering the high plasma lipids and lipoproteins in the blood of a patient without the negative effects of many of the existing drugs/procedures available on the market today.
a and 4b. Effect of MTP inhibitor (CP-34086) on plasma lipids and lipoproteins in C57Bl/6J mice fed western diet ad libitum for 8 weeks. MTP inhibitor solubilzed in DMSO was administered orally for one week (15 mg/kg/day) along with either DMSO (MTPi), lovastatin (50 mg/kg/day), or WY14643 (10 mg/kg/day). Control mice were gavaged with DMSO only. Stars on MTPi+Lovastatin and MTPiwy14643 represent individual comparisons with MTPi groups. *, p, 0.05, **P, 0.01, ***, p<0.001.
a and 5b. Liver samples from
a and 6b. Liver samples from
a and 7b. Mice were fed western diet for five weeks. In the last week, they either received DMSO (control, n+3), MTPi, MTPi+pioglitazone, or MTPi+lovastatin. Livers were collected and used to measure MTP activity (A), triglyceride (B), total cholesterol (C), free cholesterol (D), esterified cholesterol (E) and free fatty acids (F).
a and 8b. Plasma was obtained from animals described in
a and 9b. C57B/6J mice were fed a western diet for 8 weeks and then treated with MTP inhibitor and other indicated compounds for 8 days as described in
The present invention provides a combination comprising at least one Microsomal Triglyceride transfer Protein (MTP) inhibitor and at least one lipid lowering agent, both in an amount effective to treat hyperlipidemias.
It has been surprisingly found that administering this combination avoids increases in plasma AST/ALT, hepatic triglyceride/free cholesterol, and ER stress. Accordingly, the composition and method of the present invention provide a way to treat hyperlipidemias without evidence of hepatic disease by using MTP antagonists along with agents that avoid accumulation of cellular lipids.
Examples of lipid lowering agent include PPARα/PPARγ agonists or statins. An example of a PPARα/PPARγ agonist is (RS)-5-(4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl)thiazolidine-2,4-dione. An examples of a statin is 1S,3R,7S,8S,8aR)-8-{2-[(2R,4R)-4-hydroxy-6-oxooxan-2-yl]ethyl}-3,7-dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl (2S)-2-methylbutanoate.
Also provided is a method of treating hyperlipidemias in a subject by administering a combination of at least one Microsomal Triglyceride transfer Protein (MTP) inhibitor and at least one lipid lowering agent to said subject. Preferably, the MTP inhibitor is administered in an amount of about 50-100 mg per day and the lipid-lowering agent is administered in an amount of about 50-100 mg per day. The MTP inhibitor and the lipid lowering agent may be administered together as a pharmaceutical composition or as part of the same, unitary dosage form. The MTP inhibitor and the lipid lowering agent may also be administered separately, but as part of the same therapeutic regimen.
Abetalipoproteinemia is characterized by the absence of plasma apoB-lipoproteins, extremely low plasma cholesterol, and lipid soluble vitamin deficiencies. Using genetic approaches it has been shown that afflicted individuals have mutations in the mttp gene. Several mutations in the mttp gene have since been documented in abetalipoproteinemia. Reconstitution of MTP activity in heterologous systems rescued apoB secretion, while tissue specific liver knockout models recreated the apoB and lipid deficiencies present in abetalipoproteinemia. Furthermore, cell culture studies showed that wild type MTP can rescue apoB secretion but mutated proteins cannot. MTP is required during the early stages of assembly to prevent the aberrant folding of apoB and its degradation by proteasomes.
MTP consists of two polypeptides, a 97 kDa MTP subunit and a 55 kDa PDI subunit. The PDI (protein disulfide isomerase) subunit is ubiquitously expressed ER resident enzyme. Based on sequence homology with lipovitellin, the MTP subunit is proposed to contain three domains (
MTP can transfer several lipids in vitro. In order to study the importance of different lipid transfer activities of MTP, a Drosophila homolog of human MTP was cloned. The Drosophila MTP transferred phospholipids but did not transfer triacylglycerols. Even though it lacked the neutral lipid transfer activity, the Drosophila MTP was able to assist in the secretion of apoB-lipoproteins indicating that the phospholipid transfer activity was the most ancient activity of MTP and that this activity was sufficient to support apoB-lipoprotein assembly. The phospholipid transfer activity of MTP is also necessary for the lipidation during the biosynthesis of CD1d, a glycolipid antigen-presenting molecule, and in the NKT cell development. We have proposed that phospholipid and triglyceride transfer activities of MTP play different roles during the first step lipidation of nascent apoB. The triglyceride transfer activity may enhance “nucleation” sites for the assembly of apoB-lipoproteins on the ER membrane. The phospholipid transfer activity may play a critical role by adding phospholipids and completing the synthesis of newly detached “primordial” lipoprotein particles and rendering them secretion competent.
MTP in Cholesteryl Ester Biosynthesis
Apart from its lipid transfer activity, we have recently shown that MTP also regulates cholesteryl ester biosynthesis in the liver and intestinal cells. Genetic deletion and chemical inhibition of MTP causes accumulation of cellular triglycerides. It has been assumed that cells would also accumulate cholesteryl esters along with triglycerides. However, we found that inhibition or loss of MTP decreased cellular cholesteryl esters and increased free cholesterol levels. Mechanistic studies revealed that MTP relieves product inhibition of cholesteryl ester synthesis and enhances their biosynthesis.
Increases in cellular free cholesterol might cause tissue damage and enhance the release of hepatic enzymes into the plasma. It is known that high cellular free cholesterol levels damage extra-hepatic tissues. For example, excess amounts of cellular free cholesterol induce apoptosis, especially in the arterial wall macrophages contributing to atherogenesis. Enhanced free cholesterol levels in the ER of macrophages induce ER stress and apoptosis. On the contrary, hepatic free cholesterol is not usually considered a problem since liver can either excrete it as such or after its conversion to bile acids. This assumption may not be totally true. The success of statin therapy is due to the inhibition of hepatic HMG CoA reductase (HMGR) activity present in the ER membranes. Therefore, a more plausible explanation is that free cholesterol in the ER membrane is critical and perturbations that lead to increases in microsomal free cholesterol may cause injury. Furthermore, it has been suggested that the progression of hepatosteatosis to steatohepatitis might be related to free cholesterol, not free fatty acid or triglyceride, accumulation in the liver. The relationship between free microsomal cholesterol assimilation, MTP inhibition, and increases in plasma transaminases can be evaluated by lowering cellular free cholesterol.
A feasible approach to lower cellular cholesterol might be to inhibit HMG CoA reductase. Statins inhibit HMG CoA reductase, increase hepatic LDL receptor expression, and decrease plasma cholesterol. Therefore, it is tempting to suggest that a combined inhibition of MTP and HMG CoA reductase may be useful in lowering plasma cholesterol and avoiding cellular cholesterol accumulation. Similarly, potent inhibitors of squalene synthase can be used with MTP inhibitors to achieve these goals.
Another combinatorial approach to avoid free cholesterol accumulation is to enhance its efflux. In this regards, liver X receptor (LXR) agonists appear promising. LXRs are nuclear hormone receptors that control expression of genes involved in cholesterol efflux in macrophages, hepatic bile acid synthesis, and intestinal cholesterol absorption. LXR agonists increase expression of ABCA1 in macrophages, enhance cholesterol efflux, and decrease atherosclerosis in apoe−/− and Idlr−/− mice. They increase hepatic bile acid synthesis and reduce hepatic cholesterol levels. These agonists up regulate ABCG5 and ABCG8 in the intestine and reduce cholesterol absorption. Unfortunately, a major side effect of LXR agonist is hypertriglyceridemia. MTP inhibitors reduce hypertriglyceridemia. Therefore, it is worth examining whether LXR agonist and MTP antagonists can be used in combination to prevent hypertriglyceridemia, increases in plasma transaminases, and steatosis.
Several animal studies also indicate a relationship between decreased MTP expression and steatosis. Chronic alcohol feeding causes fatty liver and is associated with decreases MTP. Transgenic expression of Hepatitis C virus core protein inhibits MTP activity, reduces VLDL secretion, and causes steatosis. We have shown that steatosis induced by carbon tetrachloride rapidly involves post-translational degradation of MTP. Similarly, inhibition of MTP in cells also results in triglyceride accumulation. Therefore, significant reduction in MTP activity is usually associated with steatosis.
MTP and Steatosis:
There are several reports in humans indicating cellular fat accumulation (steatosis) with decreased MTP expression. Intestinal steatosis is uniformly observed in all abetalipoproteinemia patients. Hepatic steatosis has also been reported with MTP deficiency. An abetalipoproteinemia infant with substantial hepatomegaly and persistent elevated levels of serum aminotransferases but normal bilirubin levels was described in the literature. Microscopic examination revealed large fat droplets in hepatocytes. In addition to genetic defects, there is an association between low MTP expression due to a polymorphism in the promoter sequence and liver steatosis in type 2 diabetes patients. Hepatitis C virus type 3 infected subjects show reduced MTP activity and mRNA levels and high degree of steatosis.
Inhibitors of MTP have been sought as possible treatment options for hyperlipidemia. However, uses of these inhibitors have demonstrated increases in hepatic fat and the plasma transaminases, Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST). Our lab has demonstrated that inhibition of MTP results in elevations in hepatic triglycerides and free cholesterol. It has been illustrated in non-hepatic tissue that cellular free cholesterol has damaging effects e.g., an excess amount of cellular free cholesterol induces apoptosis of arterial wall macrophages contributing to atherosclerotic plaque formation. In these tissue macrophages, the elevated free cholesterol localizes to the ER and induces the Endoplasmic Reticulum stress (ER stress) pathway to either maintain survival or signal apoptosis. Free cholesterol is not normally considered a problem in the liver due to the many metabolic pathways in which the organ can deal with free cholesterol; (1) it can be converted into cholesterol esters and secreted with VLDL particles via MTP, (2) it can be converted into bile acids and secreted into the bile canaliculus or (3) it can be effluxed out of the hepatocyte. However, with loss of MTP the ability of the liver to cycle free cholesterol into the esterification pathway diminishes. As a result, accumulation of free cholesterol ensues and may be too great for the liver to compensate with other arms of the cholesterol metabolic pathway. With this in mind, a plausible explanation for plasma transaminase elevation. In the present study we will establish that loss of hepatic MTP results in elevations in cellular free cholesterol and plasma ALT/AST. It is also demonstrated that by alleviating free cholesterol accumulation in the liver, we can lower plasma ALT/AST elevations due to loss of MTP can be lowered. It is shown that the induction of ER stress in the liver as a result of MTP inhibition and that by lowering free cholesterol ER stress can be alleviated. Finally, we will demonstrate that induction of ER stress in the liver can lead to plasma AST/ALT elevations in MTP deficient models and that by inhibiting ER stress we can abrogate or curb the rise in AST/ALT seen with MTP inhibition.
Accordingly, MTP is a possible therapeutic target in treating hyperlipidemias; but its inhibition is associated with side effects. Lowering cellular free cholesterol and inhibiting endoplasmic reticulum (ER) stress avoid toxicities associated with the inhibition of Microsomal Triglyceride Transfer Protein (MTP).
In the development of the present invention it has been found that the genetic ablation of MTP in Reversa mice and its chemical inhibition in western diet fed C57Bl/6J mice decreased plasma triglyceride and cholesterol by 50% and increased hepatic triglyceride by 2-fold consistent with other studies. These mice, however, had 50% increased hepatic free cholesterol and 2 to 3-fold higher plasma AST/ALT. Daily administration of PPARα/PPARγ agonists to MTP ablated or inhibited mice reduced hepatic triglyceride/free cholesterol and avoided increases in plasma AST/ALT. Lovastatin reduced hepatic free cholesterol without affecting triglyceride and abrogated increases in plasma AST/ALT. Therefore, implying that increases in plasma AST/ALT were most likely due to increases in hepatic free cholesterol. Mechanistic studies showed that MTP inhibition and hepatic free cholesterol accumulation increase hepatic ER stress. Inhibition of ER stress with 4-phenyl butyric acid did not reduce hepatic lipids in MTP inhibited mice but spared increases in plasma AST/ALT. Moreover, acute induction of ER stress by tunicamycin increased plasma AST/ALT by 2.5-fold. This research leads to a conclusion that increases in plasma AST/ALT after MTP inhibition might be due to hepatic free cholesterol accretion and induction of ER stress.
In an effort to maximize the affect of MTP while minimizing the potentially harmful side effects associated with MTP therapy, the present invention provide % a pharmaceutical combination and method for treating hyperlipidemias using the combination. The combination comprises at least one lipid lowering agent and at least one Microsomal Triglyceride Transfer Protein (MTP) inhibitor.
It has been surprisingly found that administering this combination in mice avoids increases in plasma AST/ALT, hepatic triglyceride/free cholesterol, and ER stress. The same was evaluated in primates using Western-diet fed bonnet macaques that were fed MTP antagonists with or without lovastatin or pioglitazone. MTP antagonists decreased plasma lipids but increased AST/ALT. Animals that also received pioglitazone did not show elevated plasma AST/ALT. Accordingly, the composition and method of the present invention provides a way to treat hyperlipidemias without evidence of hepatic disease by using MTP antagonists along with agents that avoid accumulation of cellular lipids.
Example of MTP inhibitors which may be used in the compositions and methods of the present invention, include but are not limited to Ire1β, CP-346086, JTT-130, BMS-201038, AEGR-733 (Lomitapide), SLx-4090 and benzothiazole derivatives such as triamide derivatives bearing a benzothiazole core as described by Vu C B et al., in Bioorg Med Chem Lett 2009 Mar. 1; 19(5):1416-20, Epub 2009 Jan. 19, which is incorporated by reference herein as if fully set forth.
Examples of lipid lowering agents include but are not limited to statins such as e.g., atorvastatin, resovastatin, fluvastatin, pravastatin. lovastatin and simvastatin. Examples of PPARα/PPARγ agonists include but are not limited to Glitazars such as Muraglitazar, Tesaglitazar, Aleglitazar and the compound (RS)-5-(4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl)thiazolidine-2,4-dione (pioglitazone).
As used herein, the term “combination” means that the components, i.e., the MTP inhibitor and lipid lowering agent, may be administered together as a pharmaceutical composition or as part of the same, unitary dosage form. A combination also includes administering the MTP inhibitor and lipid lowering agent each separately, but as part of the same therapeutic regimen. The components, if administered separately, need not necessarily be administered at essentially the same time, although they can if so desired. Thus, the MTP inhibitor and lipid lowering agent may be administered as separate dosages or dosage forms, but at the same time. As used herein, a combination also includes separate administration at different times and in any order.
Daily dosages for the MTP inhibitor and lipid lowering agent will, of course, vary depending on a variety of factors, for example, the particular compound chosen. In general, however, satisfactory results are achieved on administration of both MTP inhibitor and lipid lowering agent in the order of 1-100 mg per day, with a preferred dosage of 50-100 mg per day. Thus for example, an PPARα/PPARγ agonist may be administered at a dosage of 1-100 mg per day and a statin may be administered at a dosage of 1-100 mg per day, as a single dose or in divided doses. The MTP inhibitor and lipid lowering agent may be administered by any conventional route, in particular enterally, e.g., orally, e.g., in the form of tablets, capsules, drink solutions, or parenterally, e.g., in the form of injectable solutions or suspensions.
MTP, a Target to Treat Hyperlipidemias:
Hyperlipidemias are major risk factors for atherosclerosis. There are two metabolic abnormalities that could lead to hyperlipidemias, over production or decreased catabolism. Significant progress has been made with statins in lowering plasma lipids by increasing their catabolism. However, attempts to control lipoprotein production have not yet been successful. ApoB and MTP are prime candidates to curb lipoprotein production. Since apoB does not have a biochemical activity amenable to through put screening, siRNA technology has been used to lower its production.
In contrast to apoB, due to its lipid transfer activity, MTP has been a favorite target to identify small molecule inhibitors and to lower plasma lipids. Indeed several MTP antagonists have been identified that decrease lipoprotein production and plasma lipids. Unfortunately, these drugs exhibit two types of tissue specific side effects. The first side effect is related to the inhibition of chylomicron assembly by enterocytes and manifests as gastrointestinal disturbances such as steatorrhea and diarrhea. These disturbances have been successfully avoided by administering MTP inhibitors 4 h after the supper.
Recently, Ire1β has been shown to down regulate intestinal MTP indicating that its up-regulation might be a viable target for lowering intestinal MTP. The second side effect is related to the inhibition of hepatic lipoprotein assembly and secretion. In about 10-30% of the individuals, MTP inhibitors increase plasma levels of liver enzymes, mainly AST and ALT. Thus, there is a critical need for novel approaches to inhibit MTP without causing steatosis. It is proposed that intestine-specific inhibition of MTP might be beneficial because of the inherent property of the intestine to self-renew. In fact, intestine-specific MTP inhibitor, JTT-130, has been shown to lower plasma triglyceride and LDL cholesterol in guinea pigs without increasing hepatic triglyceride. Similarly, another intestine-specific compound, SLx-4090, has been shown to lower plasma lipids. Thus, an intestine-specific inhibition of MTP might avoid hepatic toxicity. Here, we propose an alternate possibility.
As MTP primarily transfers neutral lipids in vitro and helps in the transport of neutral lipids in vivo by assembling apoB-lipoproteins, it has been assumed that toxicities associated with MTP inhibition and genetic ablation are due to the accumulation of neutral lipids. We should, however, realize that synthesis and storage of neutral lipids (triglycerides and cholesterol esters) is beneficial in avoiding toxicities associated with excess free fatty acids and free cholesterol. The question then arises why MTP inhibition increases plasma hepatic enzymes. A common explanation provided is that MTP inhibition leads to higher, possibly toxic, amounts of neutral lipids in the liver. However, there are reports indicating that, at least, short-term use of MTP inhibitors does not always lead to an overt accumulation of hepatic lipids. We are not aware of studies describing a clear-cut relationship between the accumulation of fat in the liver and the appearance of hepatic enzymes in the plasma. Assuming that the toxicity due to MTP antagonists is associated with hepatic fat accumulation, it can conceivably be avoided by upregulating mitochondrial and peroxisomal oxidation of fatty acids. PPARs are nuclear hormone receptors that enhance fatty acid oxidation by peroxisomes. Potent PPARα agonists decrease hypercholesterolemia and atherosclerosis in Idlr−/− mice. A combined use of MTP inhibitors and PPARα activators has been speculated before. Still, experimental evidence for their beneficial use is lacking. Hence, there is a need to question the fundamental paradigm that MTP inhibitors increase plasma hepatic enzymes by augmenting cellular concentrations of triglycerides and to find new ways to circumvent hepatosteatosis.
Cellular triglyceride buildup is a key feature of MTP inhibition. Triglyceride synthesis involves fatty acid uptake, intracellular transport to microsomes by fatty acid binding proteins, and acylation with glycerol by several monoacylglycerol and diacylglycerol acyltransferases. Inhibition of these steps will likely reduce cellular triglyceride levels. In this respect, repression of liver fatty acid binding protein along with MTP inhibition has been shown to lessen steatosis. Several studies have shown that flavonoids inhibit triglyceride transfer activity of MTP. These compounds affect several other biological pathways and have pleiotropic effects. For example, Taxifolin, a plant flavonoid, inhibits triglyceride synthesis and MTP activity without increasing cellular lipids. Therefore, combined inhibition of triglyceride synthesis and MTP activity might avoid triglyceride accumulation. This is supported by observations that DGAT1 deficient mice do not develop hepatic steatosis, demonstrate increased energy expenditure and have significantly lower levels of triglycerides in lipogenic tissues. Thus, DGAT inhibition with MTP antagonism offers an attractive opportunity for therapeutic interventions in obese and diabetic patients. Here, we are hypothesizing that the liver injury might be due to high free cellular cholesterol levels and are proposing new approaches to avoid toxicities associated with MTP inhibition.
The ER Stress Response:
In vertebrates, three major integral membrane proteins [Inositol requiring enzyme 1 (IRE1), protein kinase-like ER kinase (PERK), and activating transcription factor 6 (ATF6)] monitor the ER stress. These three proteins act in parallel to transmit information across the ER membrane to decrease protein synthesis and induce expression of transcription factors to enhance the synthesis of chaperones. IRE1 and PERK have similar lumenal stress-sensing domains, which normally bind to an ER chaperone Bip. When the amounts of misfolded proteins increase, Bip dissociates from IRE1 and PERK. This in turn results in the oligomerization and activation of these kinases. Activation of IRE1 unmasks an endoribonuclease activity. This activity mediates a unique splicing event generating a distinct shorter, functional form of XBP1. XBP1 is a transcription factor and activates several stress responsive genes. Activated PERK phosphorylates a subunit of eukaryotic translation initiation factor 2 (eIF2α) in the cytosol and inhibits general protein synthesis, thereby inhibiting further synthesis of toxic misfolded proteins. Phosphorylated eIF2α also mediates a specific and selective enhancement of ATF4 translation. This transcription factor up-regulates a number of UPR-regulated genes, such as C/EBP-homologous protein (CHOP) and Bip. In contrast to the IRE1 and PERK activation, ER stress facilitates the egress of ATF6 from the ER to the Golgi. In the Golgi, ATF6 is cleaved sequentially by the site-1 and site-2 proteases releasing an active ATF6, which also up-regulates a group of genes encoding ER resident molecular chaperones and folding enzymes.
It has been shown that ER stress is induced in macrophages with high levels of intracellular free cholesterol. In the case of cholesterol-loaded macrophages, upstream ER stress signaling molecules are required for early macrophage viability, although a distal branch involving the ER stress response effector CHOP triggers apoptosis. In view of these findings, the Applicants of the present invention explored whether free cholesterol accumulation after gene deletion induces ER stress in other tissues.
Plasma AST/ALT, Markers of Hepatic Injury:
Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are hepatic enzymes involved in intermediary metabolism. AST and ALT catalyze reversible reactions that transfer an amino group from the amino acids aspartate and alanine to the tri-carboxylic acid (TCA) cycle intermediate α-ketoglutarate to form oxaloacetate and pyruvate, respectively. These enzymes allow for the regeneration of TCA cycle and glycolytic intermediates in times of low glucose availability. Two isoforms are present in humans for each enzyme; AST-1, AST-2, ALT-1, ALT-2. AST-1 is a cytosolic enzyme that localizes to the heart and red blood cells, but it is also found in the liver. AST-2 is a mitochondrial enzyme that is only found in the liver. It is assumed that this form of AST is the major form found in serum. ALT-1 is a cytosolic enzyme that is present in the liver, kidney and skeletal muscle. It is thought that this form of ALT is the major form found in serum. ALT-2 mRNA have been found in muscle, adipose tissue and liver; conflicting data exists as to whether the ALT-2 protein is found in hepatocytes.
The measurements of serum AST and ALT are part of a normal liver function panel and are the most commonly used test to ascertain liver function. They are considered to be sensitive markers for parenchymal damage and/or inflammation. It is currently held that serum aminotransferase elevations are a result of the release of intracytosolic contents from dying hepatocytes, but many recent publications have shown significant elevations in serum ALT with little to no indication of parenchymal inflammation/injury. Several recent reports have also shown that serum AST/ALT values are predictive of the progression of non-alcoholic fatty liver disease and the metabolic syndrome, however, mechanisms for their release in these disorders are unknown.
The present invention provides compositions and methods of treatments that alters mechanisms that lead to augmented release of AST/ALT into the plasma of animals exposed to MTP inhibitors. This leads to the avoidance of toxicities associated with MTP inhibition by lowering hepatic free cholesterol would indicate that MTP inhibitors could be used to treat different forms of hyperlipidemias, such as familial combined hyperlipidemia, familial hypercholesterolemia. Using the compositions and methods of the present invention makes MTP targeting a viable approach to lowering plasma lipids.
That is, data obtained and discussed herein highlights a significant relationship between MTP inhibition and aberrant elevations in plasma transaminases. These studies demonstrate that MTP inhibition leads to enhanced cellular free cholesterol and that toxicities associated with MTP inhibition can be avoided by reducing hepatic free cholesterol. Furthermore, the present invention indicates that MTP inhibitors could be used in combination with cholesterol lowering agents to treat hyperlipidemias.
Avoiding toxicities associated with MTP inhibition by lowering cellular free cholesterol levels challenges the current paradigm that toxicities observed in hepatosteatosis are mainly due to the accumulation of neutral lipids and provide a new paradigm, that other lipids, besides triglycerides, must be taken into account when treating and explaining toxicities associated with hepatic lipid accumulation.
MTP antagonists are currently used only for limited purposes, such as, lowering lipids in familial hypercholesterolemia and controlling obesity in dogs. However, in view of the present invention, the side effects associated with MTP inhibition can be avoided by lowering free hepatic cholesterol. Accordingly, the combination of the present invention includes not only MTP but at least one lipid lowering agent, both in an amount effective to treat hyperlipidemias.
As discussed further below and in the examples, the lipid lowering agents including statins, successfully lower plasma lipid levels and decrease in plasma cholesterol. Unexpectedly, several beneficial effects of statins have been observed that are, most likely, unrelated to reduction in plasma lipids and are usually referred to as “pleiotropic effects.” The present application indicates that statins not only reduce plasma lipids but they also decrease cellular free cholesterol. Accordingly, most likely some of the pleiotropic effects of statins might be related to reductions in cellular free cholesterol. The composition of the present invention has lead to the understanding how in vivo exposure of MTP inhibitor and other effective lipid-lowering agents in attenuating dyslipidemia. The present invention provides mechanistic data that allows determining how MTP inhibitor-linked excess free cholesterol contributes to hepatic injury. MTP inhibition might be particularly useful for individuals with inherited dyslipidemia who are unable to reach an adequate LDL cholesterol goal.
The idea that hepatic toxicities associated with MTP inhibition are due to cellular free cholesterol accumulation is in itself novel. Moreover, the present invention provides a composition and method for lowering of cellular free cholesterol so as to avoid toxicities associated with MTP antagonists and to treat hyperlipidemias.
The following examples are meant to further illustrate the invention and are not intended in any way to limit the scope of the invention.
To find out if mttp gene ablation enhances plasma AST/ALT levels, we used mice that were only deficient in hepatic MTP. MTPfl/fl (WT) mice were crossed with Alb-Cre mice to obtain liver-specific MTP deletion (L-MTP−/−). These L-MTP−/− mice were injected with adenoviruses expressing luciferase (Ad-Luc) or human MTP (Ad-hMTP-FLAG) to re-express MTP. Western blotting (
Next, we measured the effect of mttp gene ablation and hMTP expression on apoB-lipoprotein assembly and secretion (
We also studied the effect of hepatic mttp gene deletion and expression of human MTP on plasma lipids and hepatic enzymes (
The changes in hepatic lipids in these mice were then studied and the results are provided below. Deletion of mttp significantly enhanced hepatic triglyceride (
Analysis of plasma AST/ALT indicated that the absence of hepatic MTP increases plasma ALT/AST levels compared to MTPfl/fl (WT) and expression of human MTP, but not luciferase, reduces these levels (
The effect of MTP inhibitors in C57Bl/6J mice fed western diet for 8 weeks was studied and the results are shown in
The effect of MTP inhibition and co-administration of other drugs on MTP activity is shown in
Changes in the expression of different genes involved in lipid metabolism and the results are shown in
In the above studies lovastatin reduced free cholesterol but had no effect on hepatic triglyceride. By contrast, WY14643 decreased hepatic triglyceride and had no effect on free cholesterol. These studies showed that increases in AST/ALT could be avoided by lowering hepatic free cholesterol but not by triglyceride. Nevertheless, accumulation of hepatic triglyceride will not be tolerated over a long term. Therefore, we considered the possibility of combining lovastatin and WY14643 with MTPi. However, combination of three drugs is not going to be a favorable therapeutic approach. Therefore, we sought to find another agent that could reduce both free cholesterol and triglyceride in the liver and that could be used in combination with MTPi. In preliminary studies, we found that omega 3 fatty acids that act as agonists of PPARα/PPARγ reduced both hepatic free cholesterol and triglyceride. Therefore, we evaluated pioglitazone, which is known to activate both PPARα and PPARγ, for its efficacy in decreasing hepatic lipids and its possible use in combination with MTPi.
As expected, MTPi significantly reduced MTP activity (
Changes in plasma lipids and AST/ALT levels were studied and the results are shown in
From the above studies the mechanisms that lead to release of cellular AST/ALT after MTP inhibition was beginning to be understood. To understand mechanisms that might contribute to increases in plasma aminotransferases, we measured genes involved in ER stress and inflammation. MTPi and other compounds had no significant effect on mRNA levels of PERK and ATF-6 (
To determine whether ER stress itself would increase plasma AST/ALT, C57Bl/6J mice were injected with tunicamycin (1 mg/kg) as described before. Plasma AST/ALT levels were measured after 36 h (
The above experiments show that MTP ablation or its inhibition in mice and hepatoma cells increases cellular triglyceride, total and free cholesterol, Ire1α mRNA, and plasma/media AST/ALT levels. Cellular free cholesterol is decreased after treating with lovastatin and T-0901317 but not with WY14647. Peritoneal injections of Ω-3 FA (PPARα/PPARγ agonists) decreased both free cholesterol and triglyceride, and reduced plasma AST/ALT levels (data not shown). We, therefore, hypothesize that MTP ablation/inhibition leads to free cholesterol accumulation. In the absence of apoB-lipoprotein assembly and secretion this free cholesterol cannot be removed from ER. Thereafter, increases in free cholesterol induce ER stress leading to enhanced release of AST/ALT from liver cells. The buildup of hepatic free cholesterol can be avoided by inhibiting cholesterol synthesis using lovastatin or enhancing free cholesterol efflux/bile acid secretion by LXR agonists. Reduction in cellular free cholesterol diminishes Ire1α mRNA and reduces release of AST/ALT. Therefore; toxicities associated with MTP inhibition can be avoided by reducing cellular free cholesterol and triglyceride. These hypotheses will be tested in this proposal.
As stated above, MTP inhibitors have long been regarded as a possible alternative to the widely used statins due to their ability to target lipoprotein assembly. However, adverse effects in the liver i.e., tissue accumulation of lipids and elevations in plasma AST/ALT, have kept them from becoming a mainstay treatment for hyperlipidemia. Triglyceride and cholesterol, in particular free cholesterol, accumulation and subsequent plasma AST/ALT elevations are the hallmark adverse effects in the liver. The magnitude of the lipid-accumulation typifies hepatosteatosis, a growing public health concern due to its link to steatohepatitis and possibly cirrhosis. The studies discussed below focused on ameliorating the side effects associated with MTP inhibitors. To accomplish this, mice and monkeys treated with MTP inhibitors were also administered a combined PPARα/agonist (Picglitazone) or a statin (Lovastatin). PPAR α agonists act to increase p-oxidation of fatty acids, thereby metabolizing the accumulated triglycerides in the liver of MTP inhibitor treated animals. PPAR y agonists also act to increase p-oxidation of fatty acids, but have the added property of promoting cholesterol efflux from hepatocytes. Statins are HMG-coA reductase inhibitors and therefore, block the endogenous synthesis of cholesterol. Statins, thereby, act to lower the accumulated cholesterol in hepatocytes. In both mice and monkeys, we observed a decrease in the accumulation of the appropriate lipids in the liver. The combined treatment of MTP inhibitor with a PPAR α/γ agonist prevented the accumulation of both triglycerides and cholesterol in the liver and curbed the elevations in plasma AST/ALT seen with the MTP inhibitor treatment alone. Similarly, statins curbed the rise in plasma AST/ALT but, only prevented the accumulation of cholesterol in the liver. The results of our studies advocate the use of a combination comprising at least one MTP inhibitor and at least one lipid lowering agent to treat the hyperlipidemias associated with metabolic diseases.
Additional experiments conducted confirm the conclusion that a combination of at least one Microsomal Triglyceride transfer Protein (MTP) inhibitor and at least one lipid lowering agent both in an amount effective to treat hyperlipidemias is effective in treating hyperlipidemias.
Animals: Mttptm2Sgy LdIrtm1Her Apobtm2Sgy Tg(Mx1-cre)1Cgn/J mice (Reversa) (stock number 004192) were obtained from Jackson Laboratories. Male reversa mice were used to study the effect of mttp gene deletion on plasma and tissue lipids. These animals are transgenic for apolipoprotein B, LDLR−/− and floxed at exon 1 of the mttp locus. In addition, these mice express cre recombinase under the control of the Mx1 promoter, which is induced after intraperitoneal injection of polyinosinic polycytidylic ribonucleic acid (plpC—Sigma) in the liver, spleen and intestine. Injection of plpC activates the cre recombinase and deletes the mttp gene in the liver and intestines. Reversa mice were injected with either PBS or plpC. Mice injected with plpC were orally gavaged with DMSO (Sigma-Aldrich D8418), DMSO+Lovastatin (50 mg/kg/day) (Calbiochem 438186), DMSO+T0901317 (50 mg/kg/day) (Calbiochem 575310), or DMSO+WY14643 (10 mg/kg/day) (Calbiochem 681725). Mice injected with plpC were also injected with either PBS or Ω-3 fatty acids (SUNY Downstate Medical Center). All animals were given supplemental treatments for 30 days.
C57BL/6J Black 6 mice (stock number 000664) were obtained from Jackson Laboratory and used for chow fed and western diet fed experiments. MTP inhibitor (BMS 2122122-01) (1 mg/kg/day), Lovastatin, Pioglitazone (Toronto Research Chemicals P471000) (25 mg/kg/day) and 4-phenylbutyric acid (Calbiochem 820986) (1 g/kg/day) were administered by oral gavage. Chow fed animals were treated for one week with either DMSO or MTP inhibitor. Western diet fed animals were fed DMSO, MTP inhibitor, Pioglitazone, MTP inhibitor+Pioglitazone, Lovastatin, MTP inhibitor+Lovastatin, 4-phenylbutyric acid, and MTP inhibitor+4-phenylbutyric acid. Chow fed animals were also injected with either PBS or Tunicamycin and animals were sacrificed at 4, 8, 12 and 24 hours.
Eleven (11) Male bonnet macaque monkeys were fed western diet for 40 days and treated with MTP inhibitor (BMS 2122122-01) with or without either Pioglitazone or Lovastatin for one month. Plasma samples were obtained at Day 0, 20, 40, 47, 54, 61 and 68 days. Liver biopsies were obtained on Day 40 and Day 68. Biopsies were conducted over two days; 2 animals from each group were biopsied per day.
All animals were kept in 07:00-19:00 h lighting schedule. All animals had free access to water and standard laboratory chow. Food was withdrawn 16 h before the sacrifice of the mice. On the day of the experiment, mice were anesthetized and blood was collected from the heart. Liver was collected, washed in ice-cold PBS, cut into small pieces and used for lipid extraction, histological staining, to measure different protein activities/quantities and for mRNA analysis. Lipids were extracted from the tissue homogenates following the Bligh and Dyer method. Triglyceride (Infinity TM Triglyceride, TR22421) and total cholesterol (Infinity TM Cholesterol, TR13421) levels in the tissues were determined using commercial kits (Thermo Scientific). Free cholesterol and free fatty acid levels were measured using kits from Wako Chemicals (Germany). Esterified cholesterol was calculated by subtracting the free cholesterol from the total cholesterol.
Determination of MTP Activity in Tissues:
After extensive washes with ice-cold PBS, small pieces (0.1 g) of liver and ˜1-cm segments of proximal small intestine were homogenized with 1 ml of ice-cold 1 mM Tris-HCl, pH 7.6, 1 mM EGTA, and 1 mM MgCl2 buffer in a glass homogenizer. The homogenates were centrifuged (SW55 Ti rotor, 50,000 rpm, 10° C., 1 h), and supernatants were used for MTP assay as described before (41; 42) using a kit (Chylos, Inc.).
mRNA quantifications and primers used: Total RNA from tissues and cells were isolated using TriZol™ (Invitrogen). The purity and integrity of RNA were assessed by the A260/A280 ratio and 1% agarose gel electrophoresis, respectively. Only the RNAs with ratios more than 1.7 were used for cDNA synthesis. The first strand cDNA was synthesized using Omniscript RT (Qiagen) kit. Briefly, 2 μg of total RNA, 1 μM random primers (Invitrogen), 0.5 mM dNTP solution, and 0.5 U/μl Omniscript Reverse Transcriptase were incubated at 37° C. for 1 h in 20 μl of RT buffer and the reaction was terminated by incubating at 95° C. for 5 minutes. Each reaction of quantitative RT-PCR was carried out in a volume of 20 μl consisting of 5 μl cDNA sample (1:100 dilution of the first strand cDNA sample) and 15 μl of PCR master mix solution containing 1×PCR reaction buffer, 6 mM MgCl2, 200 nM primer pair, 0.025 U/μl HotStar Gold™ DNA polymerase, 200 μM dNTP solution, and 0.3 μl SYBR Green I solution (gPCR™ Core Kit for SYBR Green I, Eurogentec). The PCR was carried out by incubating the reaction mixture first for 10 minute at 95° C. followed by 40 cycles of 15 sec incubations at 95° C. and 1 min at 60° C. in the ABI 7000 SDS PCR machine. The data were analyzed using ΔΔCT method according to manufacturer's instruction and presented as arbitrary units. The primers used in this study were designed with the PrimerExpress 3.0 software (Applied Biosystems, CA) and are presented as Table 1.
Statistical analyses: Data are presented as mean±S.D. Unless noted otherwise, n=3 for each group or condition. Statistical significance (P<0.05) was determined using the Student's t-test (GraphPad Prism).
Mttp Gene Deletion Lowers Plasma and Tissue Lipids and Elevates Transaminases:
Male reversa mice were used to study the effect of mttp gene deletion on plasma and tissue lipids. Reversa mice were injected with either PBS or plpC. PipC treatment reduced hepatic MTP activity by ˜75% compared to controls (
Lowering Hepatic Free Cholesterol Decreases Plasma AST/ALT in MTP Deficient Mice:
To determine whether accumulation of hepatic triglyceride and free cholesterol is related to increases in plasma AST/ALT, we sought to reduce these lipids using different chemical compounds of known physiologic effects. Mice injected with plpC were orally gavaged with DMSO, DMSO+Lovastatin (50 mg/kg/day), DMSO+T0901317 (50 mg/kg/day), or DMSO+WY14643 (10 mg/kg/day). Lovastatin (Lova), an HMG-CoA reductase (HMGR) inhibitor, and T0901317 (T0), an LXR agonist, are expected to decrease hepatic free cholesterol by inhibiting endogenous cholesterol synthesis and increasing cholesterol efflux, respectively. WY14643 (WY), a PPARα agonist, increases β-oxidation of fatty acids and lowers hepatic triglycerides. These compounds had no significant effect on the residual hepatic MTP activity in Reversa mice subjected to conditional mttp gene deletion (
Since, WY treatment only modestly reduced hepatic triglycerides we sought a better method for their reduction and in determining their role in plasma AST/ALT elevations. In data not shown, we demonstrated that intraperitoneal injection of Ω-3 fatty acids (Ω-3 FA), an agonist for both PPARα and PPARγ, can significantly reduce hepatic triglycerides and free cholesterol. To avoid the necessity of injections, we searched for an oral agent with similar properties. Pioglitazone, a known agonist of PPARα and PPARγ, was selected to replace Ω-3 FA injections and was used for all subsequent experiments in animals.
Since, WY treatment only modestly reduced hepatic triglycerides we sought a better method for their reduction and in determining their role in plasma AST/ALT elevations. Ω-3 fatty acids (Ω-3 FA), an agonist for both PPARα and PPARγ, was selected based on triglyceride reduction capabilities (data not shown). To evaluate the efficacy of Ω-3 FA in reducing hepatic triglyceride, plpC injected mice were treated with intraperitoneal injection of Ω-3 FA (3 mg/kg/2 day) or Ω-3 FA and Lova. These treatments had no effect on the residual MTP activity in mttp deficient animals (
Increases in Plasma ALT/AST Due to Chemical Inhibition of MTP can be Avoided by Reducing Hepatic Free Cholesterol In Vivo.
We next studied the effects of chemical inhibition of MTP in C57Bl/6J mice fed either a chow or western diet (WD) for 5 weeks. Chow fed animals were orally gavaged with either DMSO or MTP inhibitor BMS-212122-01 (MTPi; 1 mg/kg/day) for one week. WD fed animals were treated with DMSO, MTP inhibitor with or without lovastatin (50 mg/kg/day) or pioglitazone (25 mg/kg/day) daily by oral gavage for one week. As expected, treatment with MTPi significantly reduced MTP activity and was not further affected by lovastatin or pioglitazone (
Besides the liver, MTP is also expressed in the intestine and heart. Therefore, we also studied changes in lipids in these tissues following MTP inhibition and co-administration of lovastatin and pioglitazone. As in the liver, accumulation of triglycerides and free cholesterol ensued in the intestine (Table 2). Furthermore, administration of pioglitazone in conjunction with MTPi attenuated the rise in intestinal free cholesterol seen with MTPi alone (Table 1). As expected, lovastatin had no effect on intestinal lipids (Table 1). Cardiac tissue exhibited no changes in lipid profiles with MTPi or MTPi+pioglitazone or lovastatin treatment (Table 2).
In order to ascertain if the effects seen were a species-specific effect, we studied the treatment of MTP inhibitors either alone or in conjunction with Pioglitazone or Lovastatin in hyperlipidemic bonnet macaques. These animals were fed ad libitum with a WD for 40 days and then subsequently treated with MTP inhibitors for one month. There were no significant differences in fasting plasma triglycerides in the three groups at the start of the trial, after 40 days of western diet or after treatment suggesting that neither the diet or drug intervention had any significant effect on fasting plasma triglycerides (
Plasma AST/ALT levels increased slightly after feeding WD for 40 days; however, they were not significantly different from baseline levels (
Hepatic triglycerides rose significantly upon treatment with MTP inhibitor (
Induction of ER Stress Increases Plasma AST/ALT After MTP Inhibition:
Attempts were then made to identify mechanisms that lead to the release of AST/ALT after MTP inhibition. There were no significant differences in various cytokines in the plasma of control and MTP inhibitor treated mice excluding inflammation as a cause for the release of AST/ALT (
To establish further that ER stress enhances plasma AST/ALT, normal C57/BL6J were treated with tunicamycin for different time points. Tunicamycin had no effect on hepatic MTP activity, lowered plasma cholesterol and triglycerides and increased hepatic lipids (
To examine whether ER stress is involved in the release of AST/ALT in MTP deficient models, animals treated with MTP inhibitor were co-treated mice with an ER stress inhibitor, 4-phenyl-butyric acid (PBA). Treatment of mice with MTP inhibitors reduced MTP activity (
Reports of AST/ALT elevations in MTP deficient models have been varied. In a clinical trial of Familial Hypercholesterolemia (FH) patients treated with MTP inhibitors, increases in both plasma aminotransferases and hepatic fat in four out of the six patients were reported. Samaha et al described, in a clinical trial of MTP inhibitor+Ezetimibe (intestinal cholesterol absorption inhibitor) treated hyperlipidemic patients, that nine of the fifty-six patients had elevations of ALT/AST at least 2 times the upper limit of the normal range. In a review by Joy and Hegele, it was suggested that MTP deficiency associated hepatic fat accumulation might be correlated with AST/ALT elevations. In case studies of patients with ABL (or Familal Hypobetalipoproteinemia (mutation in apolipoprotein B) it has been reported that a majority of patients had significantly increased hepatic fat and plasma AST/ALT. As varied as reports have been in human studies, so has it been in animal models. In one mouse model it was suggested that MTP deficiency sensitizes the liver to toxin mediated injury and alone has no effect on plasma AST/ALT. However, in another model of MTP deficiency plasma AST/ALT elevations were seen in female rats at higher doses and were correlated with increased hepatic fat]. Despite the varied reports of plasma AST/ALT elevations in MTP deficient models, one consistency seems to be that elevations in plasma AST/ALT are associated with elevations in hepatic fat content. This may be a reflection of the differential capacities of the liver to accommodate lipid accumulation. Furthermore, in many of the clinical trials studying MTP inhibitors individual basal AST/ALT levels were never compared to post-treatment AST/ALT levels. In all trials elevations were reported as factor X times the upper limit of the normal ranges of these enzymes. Therefore, it is entirely plausible that AST/ALT levels may be within normal ranges or slightly above normal ranges and still is elevated when compared to individual basal levels.
The studies presented here provide evidence that MTP gene ablation and its chemical inhibition decreases plasma non-HDL triglyceride and cholesterol as well as hepatic cholesteryl esters; elevates hepatic triglyceride and free cholesterol; and enhances plasma AST and ALT levels. Reductions in non-HDL lipids can be explained by the well-known role of MTP in the assembly and secretion of triglyceride-rich apoB-containing lipoproteins. Reductions in hepatic cholesteryl esters are consistent with the studies of Iqbal et al who showed that MTP plays a regulatory role in their biosynthesis. They showed that MTP removes cholesteryl esters and relieves product inhibition. Therefore, in the absence of MTP activity cholesterol ester biosynthesis is curtailed leading to accumulation of free cholesterol in the liver. Increases in hepatic triglyceride can be explained by the inhibition of apoB-lipoprotein biosynthesis. Similarly, free cholesterol accumulation can be secondary to inhibition of cholesteryl ester biosynthesis. Data presented here provides evidence that these increases might be secondary to accumulation of free cholesterol.
To try and alleviate the burden of hepatic lipid accumulation, we co-administered either cholesterol or triglyceride lowering drugs with genetic ablation or chemical inhibition of MTP. Lovastatin, an HMGR inhibitor, and T-0901317, a LXR agonist, treatment in cells or animals without functional MTP resulted in a decrease in accumulated cellular free cholesterol and more importantly, a depressed rise in plasma ALT/AST. Conversely, treatment with triglyceride lowering drugs, such as WY14643, a PPARα agonist, lowered cellular triglycerides but had no effect free cholesterol levels and the rise in plasma transaminases. It was only when a cholesterol lowering agent was supplemented with a triglyceride lowering treatment, or when treatment involved a single agent with triglyceride and cholesterol lowering properties, such as Ω-3 fatty acids or Pioglitazone (combined PPARα/γ agonists), did a triglyceride lowering agent have an effect on plasma ALT/AST. Collectively this data provides compelling evidence that reductions in free cholesterol can abrogate the changes in plasma ALT/AST seen in MTP deficient models.
Genetic expression of endoplasmic reticulum stress effectors was also elevated in MTP deficient models, suggesting that the accumulation of free cholesterol in the liver induces stress in the ER. The role of ER stress in toxicities associated with MTP deficiency has not been fully elucidated. Liao et al reported that genetic ablation and chemical inhibition of MTP interferes with apoB secretion without causing retention or stress in the ER. However, when considering the massive lipid accumulation in the liver with MTP inhibition and the role free cholesterol plays in the induction of ER stress in arterial wall macrophages, it does not seem plausible that ER stress plays no role in MTP inhibitor associated toxicities. ER stress has been implicated in numerous models of hepatic lipid perturbations. Collision et al reports that high fructose corn syrup leads to elevations in hepatic cellular triglycerides and elevations in ER stress markers. In a review by Kaplowitz et al, it is suggested that there is sufficient evidence for an important role of the endoplasmic reticulum (ER) stress response in the pathogenesis of chronic viral hepatitis, insulin resistance and nonalcoholic fatty liver disease, genetic disorders of protein malfolding, and alcoholic liver disease. Basseri et al and Lee and Glimcher, both implicate intersections between the ER stress pathway and hepatic lipid metabolism. Lee and Glimcher, go as far to suggest that it is unlikely that ER stress is not active in a model of MTP deficiency and owes the inability of its recognition to inadequate means of assaying for ER stress. Furthermore, we demonstrate that ER stress induced by Tunicamycin can result in the release of these enzymes into the plasma. More importantly, we demonstrate that by inhibiting ER stress with the molecular chaperone PBA, we can curb the ER stress response and the rise in plasma AST/ALT seen with MTP inhibition.
MTP has been a target of therapeutic intervention for almost 20 years. Several pharmaceutical companies have heavily invested in identifying MTP antagonists to lower plasma cholesterol levels. However, therapeutic use of all currently developed compounds results in elevated plasma transaminases and hepatic lipid accumulation. As a consequence, MTP antagonists are only used for limited purposes, such as, lowering lipids in patients with familial hypercholesterolemia or controlling obesity in dogs. It is also currently being evaluated as a possible alternative to bariatric surgery to control blatant obesity, and liver transplantation to lower hyperlipidemias in familial hypercholesterolemia. The idea that hepatic toxicities associated with MTP inhibition are due to free cholesterol is novel. These studies introduce a new concept for avoiding the side effects associated with MTP antagonists and advocates novel combinatorial approaches to treat hyperlipidemias and lead to new therapeutic modalities for the treatment of various hyperlipidemias and have immediate potential for translational use.
While the above description contains many specifics, these specifics should not be construed as limitations of the invention, but merely as exemplifications of preferred embodiments thereof. Those skilled in the art will envision many other embodiments within the scope and spirit of the invention as defined by the claims appended hereto.
The present application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/292,992, filed Jan. 7, 2010.
This invention was funded, at least in part, under grants from the National Institutes of Health: NIH DK-46700. The Government may therefore have certain rights in the invention.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20110217293 | Fujise et al. | Sep 2011 | A1 |
| Number | Date | Country | |
|---|---|---|---|
| 20120029028 A1 | Feb 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61292992 | Jan 2010 | US |
