Synthetic LXR agonists increase LDL in CETP species

Abstract

Liver X receptor (LXR) nuclear receptors regulate the expression of genes involved in whole body cholesterol trafficking, including absorption, excretion, catabolism, and cellular efflux, and possess both anti-inflammatory and antidiabetic actions. Accordingly, LXR is considered an appealing drug target for multiple indications. Synthetic LXR agonists demonstrated inhibition of atherosclerosis progression in murine genetic models; however, these and other studies indicated that their major undesired side effect is an increase of plasma and hepatic triglycerides. A significant impediment to extrapolating results with LXR agonists from mouse to humans is the absence in mice of cholesteryl ester transfer protein, a known LXR target gene, and the upregulation in mice but not humans of cholesterol 7α-hydroxylase. To better predict the human response to LXR agonism, two synthetic LXR agonists were examined in hamsters and cynomolgus monkeys. In contrast to previously published results in mice, neither LXR agonist increased HDL-cholesterol in hamsters, and similar results were obtained in cynomolgus monkeys. Importantly, in both species, LXR agonists increased LDL-cholesterol, an unfavorable effect not apparent from earlier murine studies.These results reveal additional problems associated with current synthetic LXR agonists and emphasize the importance of profiling compounds in preclinical species with a more human-like LXR response and lipoprotein metabolism. Liver X receptor (LXR) nuclear receptors regulate the expression of genes involved in whole body cholesterol trafficking, including absorption, excretion, catabolism, and cellular efflux, and possess both anti-inflammatory and antidiabetic actions. Accordingly, LXR is considered an appealing drug target for multiple indications. Synthetic LXR agonists demonstrated inhibition of atherosclerosis progression in murine genetic models; however, these and other studies indicated that their major undesired side effect is an increase of plasma and hepatic triglycerides. A significant impediment to extrapolating results with LXR agonists from mouse to humans is the absence in mice of cholesteryl ester transfer protein, a known LXR target gene, and the upregulation in mice but not humans of cholesterol 7α-hydroxylase. To better predict the human response to LXR agonism, two synthetic LXR agonists were examined in hamsters and cynomolgus monkeys. In contrast to previously published results in mice, neither LXR agonist increased HDL-cholesterol in hamsters, and similar results were obtained in cynomolgus monkeys. Importantly, in both species, LXR agonists increased LDL-cholesterol, an unfavorable effect not apparent from earlier murine studies. These results reveal additional problems associated with current synthetic LXR agonists and emphasize the importance of profiling compounds in preclinical species with a more human-like LXR response and lipoprotein metabolism. The liver X receptors LXRα and LXRβ (1Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. LXR, a nuclear receptor that defines a distinct retinoid response pathway.Genes Dev. 1995; 9: 1033-1045Google Scholar) are ligand-activated transcription factors of the nuclear receptor superfamily that control the expression of genes involved in cholesterol homeostasis and fatty acid metabolism (2Repa J.J. Mangelsdorf D.J. The liver X receptor gene team: potential new players in atherosclerosis.Nat. Med. 2002; 8: 1243-1248Google Scholar, 3Edwards P.A. Kast H.R. Anisfeld A.M. BAREing it all: the adoption of LXR and FXR and their roles in lipid homeostasis.J. Lipid Res. 2002; 43: 2-12Google Scholar). LXRα is highly expressed in liver (hence its name) but is also prevalent in adipose tissue, gut, kidney, and macrophages. LXRβ is more widely expressed and found in most tissues. Natural ligands for LXRs are oxidized derivatives of cholesterol, such as 24S- and 25-epoxycholesterol and 24S- and 22R-hydroxycholesterol (4Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha.Nature. 1996; 383: 728-731Google Scholar, 5Lehmann J.M. Kliewer S.A. Moore L.B. Smith-Oliver T.A. Oliver B.B. Su J.L. Sundseth S.S. Winegar D.A. Blanchard D.E. Spencer T.A. et al.Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway.J. Biol. Chem. 1997; 272: 3137-3140Google Scholar, 6Forman B.M. Ruan B. Chen J. Schroepfer Jr., G.J. Evans R.M. The orphan nuclear receptor LXRalpha is positively and negatively regulated by distinct products of mevalonate metabolism.Proc. Natl. Acad. Sci. USA. 1997; 94: 10588-10593Google Scholar). LXRs are intracellular cholesterol sensors that upregulate key enzymes and transporters of cholesterol metabolism and transport, such as ABCA1 and ATP binding cassette protein G1 (ABCG1) (7Venkateswaran A. Laffitte B.A. Joseph S.B. Mak P.A. Wilpitz D.C. Edwards P.A. Tontonoz P. Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXR alpha.Proc. Natl. Acad. Sci. USA. 2000; 97: 12097-12102Google Scholar, 8Wang N. Lan D. Chen W. Matsuura F. Tall A.R. ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins.Proc. Natl. Acad. Sci. USA. 2004; 101: 9774-9779Google Scholar, 9Nakamura K. Kennedy M.A. Baldan A. Bojanic D.D. Lyons K. Edwards P.A. Expression and regulation of multiple murine ATP-binding cassette transporter G1 mRNAs/isoforms that stimulate cellular cholesterol efflux to high density lipoprotein.J. Biol. Chem. 2004; 279: 45980-45989Google Scholar), ABCG5 and ABCG8 (10Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters.Science. 2000; 290: 1771-1775Google Scholar, 11Yu L. Hammer R.E. Li-Hawkins J. Bergmann K. Von Lutjohann D. Cohen J.C. Hobbs H.H. Disruption of Abcg5 and Abcg8 in mice reveals their crucial role in biliary cholesterol secretion.Proc. Natl. Acad. Sci. USA. 2002; 99: 16237-16242Google Scholar), apolipoprotein E (apoE) in adipocyte and macrophages (12Laffitte B.A. Repa J.J. Joseph S.B. Wilpitz D.C. Kast H.R. Mangelsdorf D.J. Tontonoz P. LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes.Proc. Natl. Acad. Sci. USA. 2001; 98: 507-512Google Scholar), and cholesteryl ester transfer protein (CETP) (13Luo Y. Tall A.R. Sterol upregulation of human CETP expression in vitro and in transgenic mice by an LXR element.J. Clin. Invest. 2000; 105: 513-520Google Scholar). In mice but not in primates, hepatic cholesterol 7α-hydroxylase (cyp7a) is also upregulated by LXR (5Lehmann J.M. Kliewer S.A. Moore L.B. Smith-Oliver T.A. Oliver B.B. Su J.L. Sundseth S.S. Winegar D.A. Blanchard D.E. Spencer T.A. et al.Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway.J. Biol. Chem. 1997; 272: 3137-3140Google Scholar, 14Peet D.J. Turley S.D. Ma W. Janowski B.A. Lobaccaro J.M. Hammer R.E. Mangelsdorf D.J. Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR alpha.Cell. 1998; 93: 693-704Google Scholar, 15Menke J.G. Macnaul K.L. Hayes N.S. Baffic J. Chao Y.S. Elbrecht A. Kelly L.J. Lam M.H. Schmidt A. Sahoo S. et al.A novel liver X receptor agonist establishes species differences in the regulation of cholesterol 7alpha-hydroxylase (CYP7a).Endocrinology. 2002; 143: 2548-2558Google Scholar). LXR also affects triacylglycerol metabolism by stimulating lipogenesis and triglyceride synthesis attributable to the upregulation of sterol-regulatory element binding protein 1c (SREBP1c) and the FAS complex (16Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. et al.Role of LXRs in control of lipogenesis.Genes Dev. 2000; 14: 2831-2838Google Scholar, 17Joseph S.B. Laffitte B.A. Patel P.H. Watson M.A. Matsukuma K.E. Walczak R. Collins J.L. Osborne T.F. Tontonoz P. Direct and indirect mechanisms for regulation of fatty acid synthase gene expression by liver X receptors.J. Biol. Chem. 2002; 277: 11019-11025Google Scholar). In addition, LXRs also have direct anti-inflammatory effects by downregulation of several proinflammatory genes in macrophages (18Joseph S.B. Castrillo A. Laffitte B.A. Mangelsdorf D.J. Tontonoz P. Reciprocal regulation of inflammation and lipid metabolism by liver X receptors.Nat. Med. 2003; 9: 213-219Google Scholar, 19Castrillo A. Joseph S.B. Marathe C. Mangelsdorf D.J. Tontonoz P. Liver X receptor-dependent repression of matrix metalloproteinase-9 expression in macrophages.J. Biol. Chem. 2003; 278: 10443-10449Google Scholar, 20Fowler A.J. Sheu M.Y. Schmuth M. Kao J. Fluhr J.W. Rhein L. Collins J.L. Willson T.M. Mangelsdorf D.J. Elias P.M. et al.Liver X receptor activators display anti-inflammatory activity in irritant and allergic contact dermatitis models: liver-X-receptor-specific inhibition of inflammation and primary cytokine production.J. Invest. Dermatol. 2003; 120: 246-255Google Scholar). Based on these data, LXR has been considered an attractive antiatherosclerosis target. Using the potent synthetic LXR agonist GW3965, our group collaborated in a study in which antiatherosclerotic effects of LXR agonism were demonstrated in two murine models of atherogenesis, the apoE knockout and the LDL receptor knockout models (21Joseph S.B. McKilligin E. Pei L. Watson M.A. Collins A.R. Laffitte B.A. Chen M. Noh G. Goodman J. Hagger G.N. et al.Synthetic LXR ligand inhibits the development of atherosclerosis in mice.Proc. Natl. Acad. Sci. USA. 2002; 99: 7604-7609Google Scholar). Antiatherosclerotic effects of LXR agonists in mice were also seen by others (22Terasaka N. Hiroshima A. Koieyama T. Ubukata N. Morikawa Y. Nakai D. Inaba T. T-0901317, a synthetic liver X receptor ligand, inhibits development of atherosclerosis in LDL receptor-deficient mice.FEBS Lett. 2003; 536: 6-11Google Scholar). For a more comprehensive summary of LXR, the reader is referred to several excellent reviews (2Repa J.J. Mangelsdorf D.J. The liver X receptor gene team: potential new players in atherosclerosis.Nat. Med. 2002; 8: 1243-1248Google Scholar, 3Edwards P.A. Kast H.R. Anisfeld A.M. BAREing it all: the adoption of LXR and FXR and their roles in lipid homeostasis.J. Lipid Res. 2002; 43: 2-12Google Scholar, 23Laffitte B.A. Tontonoz P. Orphan nuclear receptors find a home in the arterial wall.Curr. Atheroscler. Rep. 2002; 4: 213-221Google Scholar, 24Jaye M. LXR agonists for the treatment of atherosclerosis.Curr. Opin. Investig. Drugs. 2003; 4: 1053-1058Google Scholar, 25Joseph S.B. Tontonoz P. LXRs: new therapeutic targets in atherosclerosis?.Curr. Opin. Pharmacol. 2003; 3: 192-197Google Scholar, 26Lund E.G. Menke J.G. Sparrow C.P. Liver X receptor agonists as potential therapeutic agents for dyslipidemia and atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1169-1177Google Scholar, 27Cao G. Liang Y. Jiang X.C. Eacho P.I. Liver X receptors as potential therapeutic targets for multiple diseases.Drug News Perspect. 2004; 17: Scholar, effects of the liver X receptor 2004; Scholar). the results obtained in mice, it is of LXRs a for the of human dyslipidemia and The upregulation by LXR of genes that stimulate cholesterol as as the anti-inflammatory effects of LXR agonism are to increased lipogenesis and triglyceride synthesis A. B.M. P.J. T. T. et of lipogenesis by of the liver X receptor to of density lipoprotein Biol. Chem. 2002; 277: Scholar), as seen in mice and hamsters with the synthetic LXR agonist highly to are the of an LXRβ and the development of an LXR with more effects on target gene expression with upregulation of M. LXR agonists for the treatment of atherosclerosis.Curr. Opin. Investig. Drugs. 2003; 4: 1053-1058Google Scholar, 26Lund E.G. Menke J.G. Sparrow C.P. Liver X receptor agonists as potential therapeutic agents for dyslipidemia and atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1169-1177Google Scholar, P.A. Kennedy M.A. Mak P.A. LXRs: nuclear receptors that regulate genes lipid Pharmacol. 2002; Scholar). For the of models that are of the response to LXR agonists in humans is are to models the LXR target gene which is in human lipoprotein and their gene is upregulated by of CETP in humans an additional for LXR as CETP triglyceride in for HDL-cholesterol and in a more lipoprotein and possess a CETP gene, and in neither of these species to an LXR target. these species are to more mice of the human response to LXR is a potent LXR agonist that is similar to P. et on the effects of both and on plasma and lipoprotein cholesterol and in hamsters and cynomolgus monkeys. In addition, the expression of LXR target genes in and a fatty acid of and fatty are The results in to an increase of plasma both LXR agonists increase plasma and LXR agonists LXR to antiatherosclerosis mechanisms from effects on plasma obtained with current GW3965, previously D.E. B.M. Edwards P.A. of the gene by and Biol. Chem. 1998; Scholar), and are similar synthetic LXR agonists is as potent as in LXRα in LXRβ and in and cholesterol efflux in not were and hamsters were obtained from were for with of with the LXR agonist and in were on and of the the on the hamsters were and were obtained were by on a with as the an with The cholesterol of the cholesterol the and were were a and J. M. A for the and of from Biol. Chem. Scholar) from a of a major liver and triglyceride of For for hamsters treatment group were with significant to significant hamsters were by macrophages were by of the with of and by for in a were in with and were of the to the of the and liver of a major were and to and of to were of were in in and with and by and liver were the to the from from treatment group were two for the and LXR agonist treatment of with the of and murine to the in an in with of and of the and in and for ATP binding cassette protein cholesteryl ester transfer density lipoprotein A sterol-regulatory element binding protein in a new ATP binding cassette protein cholesteryl ester transfer density lipoprotein A sterol-regulatory element binding protein For gene and tissue, expression to the gene as in for target and genes and the of these two for the two from hamsters to For of the treatment the for gene and and the to from the by the as by with significant to significant hamsters were with with in for the hamsters were and were obtained of plasma from and LXR were and the were to lipid The lipid of cynomolgus examined for to and to to treatment An of these for as a for studies were treatment to with For were obtained on and and on and and of and were on an and were as by the and cholesterol were from and were with LDL and Direct For the from the of For treatment study and the treatment contrast for These and their were with The were for the multiple a and treatment study The of has been previously J.L. A.M. Watson M.A. Moore L.B. D.J. J.G. J.G. et of a liver X receptor agonist synthesis of Med. Chem. 2002; Scholar), and has been in a of in and in vitro studies. from in a on the with GW3965, is as potent in LXRα in LXRβ and in and cholesterol efflux in not of hamsters on a for with the synthetic LXR agonist increased plasma to and the effect on plasma however, and plasma were increased to hamsters In contrast to the effects on plasma effect on plasma cholesterol with high of of increased plasma cholesterol To the effect of LXR agonist treatment on the cholesterol of plasma by The effects of LXR agonists on plasma with the in plasma triglycerides. in the two treatment to that of the of and increased by and to The increase in by the effects of the two of which the effects of these on plasma of LXR agonists on the of plasma lipoprotein cholesterol in on a were for with obtained the by and the cholesterol of The cholesterol of the LDL and are differences and LXR agonist treatment are as with significant density increased by both LXR agonists increased by to with hamsters, increased to of the treatment group In contrast to the effects of LXR agonists on significant in were the of both and with the group by and effect on body development not of the the of the study an increase in triglyceride in the of and triglyceride not increased in of hamsters with GW3965, with the of with and the effects of these compounds on hepatic gene expression The expression of LXR target genes in and examined to with the in and the and examined and upregulation of LXR target genes by both LXR agonists in as seen earlier in mice M. LXR agonists for the treatment of atherosclerosis.Curr. Opin. Investig. Drugs. 2003; 4: 1053-1058Google Scholar). attributable in to the of expression of LXR target genes to genes in with other examined not ABCA1 and were upregulated in the by and by ABCG5 and ABCG8 were also upregulated by both compounds but to a the ABC genes and A were upregulated by the LXR agonists in in FAS expression were LXR target genes expression upregulated by both LXR agonists in CETP and a LXR target gene a protein that inhibits activity T. M. M. N. N. Y. H. R. M. protein by the liver X Biol. Chem. 2003; 278: Scholar, T. M. M. K. Y. R. K. Inaba T. H. T. et density lipoprotein triglyceride by inhibition of lipoprotein Biol. Chem. 2002; 277: of LXR agonists on gene expression in liver and macrophages of on a were for with expression by as in and differences and LXR agonist treatment are as with significant the of expression In the effects of LXR agonist treatment on LXR target gene expression were more with in The effects were for which upregulated by and with to a of the in hepatic ABCA1 and expression were obtained with and with the upregulated to a of the genes examined in with the high hepatic expression of LXRα and the of for on other including density lipoprotein and were not were not considered In expression of and increased by and by The upregulation of by both synthetic LXR agonists and its with with in and plasma in LXR the of as a of gene is both an LXR Y. M. Y. Liang C.P. C. Tall A.R. inhibits ATP-binding cassette transporter cholesterol efflux and Biol. Chem. 2003; 278: Scholar) and D.E. B.M. Edwards P.A. of the gene by and Biol. Chem. 1998; Scholar, D.E. B.M. Edwards P.A. of and transcription factors for transcription of and Biol. Chem. Scholar) target gene, and as in it upregulated by LXR agonists in the in the synthesis of fatty The of is acid by the of acid To the upregulation of in hamsters were with LXR agonists and both and plasma were to fatty acid For hamsters were with with for and a from group with the upregulation of an increased of acid in both plasma fatty with both the of and with In with the increased of fatty acid in the plasma fatty acid and triglyceride in LXR hamsters, the of the fatty acid acid by LXR The effects of LXR agonist treatment on the of acid in both fatty and the effects with The increased of the fatty with in the of both and fatty for an increase of fatty acid synthesis and by LXR To the effects of synthetic LXR agonists on and in hamsters the of LXR agonism in species, were in cynomolgus for with synthetic LXR agonist increased plasma both compounds increased cholesterol treatment The increase in plasma cholesterol by both compounds is attributable to an increase of plasma increased in but not with treatment In with the increase of plasma by both were also increased in LXR multiple in plasma lipoprotein cholesterol, and apolipoprotein in cynomolgus with the LXR agonist were for with were obtained and of and a The of the from the of in differences from were not have in plasma lipoprotein cholesterol, and apolipoprotein in cynomolgus with the LXR agonist were for with were obtained and of and a The of the from the of in differences from were not have in plasma lipoprotein cholesterol, and in cynomolgus with the LXR agonist were for with were obtained and of and a The of the from the of have in plasma lipoprotein cholesterol, and in cynomolgus with the LXR agonist were for with were obtained and of and a The of the from the of have in plasma lipoprotein cholesterol, and in cynomolgus with the LXR agonist were for with were obtained and of and a The of the from the of have In hamsters, the novel agonist increased plasma and with but significant in plasma were also increased in plasma were seen with GW3965, the of These that LXR agonists in increase hepatic triglyceride and as previously in increase in hepatic triglyceride synthesis by and in hepatic triglyceride with and of with a increase in for and in and macrophages. An increase in is to in an increased of to and of fatty acid species in the triglyceride and fatty acid in plasma demonstrated a increase in the of with a in of and fatty with an upregulation and increased activity of a increase in for CETP in also to and the increase in plasma and in plasma by the of plasma and CETP a and CETP and the associated effects on LDL and are known to in human and other CETP species a P.J. Jr., D.J. Tall A.R. ester transfer a novel target for and atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2003; 23: Scholar). upregulation of CETP by LXR agonists the of and These unfavorable in lipoprotein in hamsters and with agonists of their in on a LXR agonists are to upregulate ABCA1 and other ABC transporters that cellular cholesterol efflux and hepatic and In our and upregulated the ABC transporters ABCA1 and in and macrophages as as ABCG5 and ABCG8 in these cholesterol and an increase in excretion, their effect on plasma not in of an of the effects on synthesis and To the complex LXR and plasma and were in a CETP species, cynomolgus monkeys. In studies in with compounds both LXR agonists increased plasma cholesterol, and the of increased a in the increase more the of LDL cholesterol of is that in neither study increased the it is that the increase is effects of LXR agonists on plasma in the mouse have been (21Joseph S.B. McKilligin E. Pei L. Watson M.A. Collins A.R. Laffitte B.A. Chen M. Noh G. Goodman J. Hagger G.N. et al.Synthetic LXR ligand inhibits the development of atherosclerosis in mice.Proc. Natl. Acad. Sci. USA. 2002; 99: 7604-7609Google Scholar). our that both compounds increased in hamsters, neither increased plasma in cynomolgus monkeys. attributable to the more metabolism and of in as the hamsters were not the A study with in cynomolgus the of the effects increased of treatment in however, that is not attributable to the LXR agonist for the by LXR and in are significant in the and increase with the neither LXR agonist increased in hamsters in in CETP by in with not from the study were not in the of CETP an increased from LXR agonism to for the results results in two CETP species, cynomolgus and hamsters, the that agonists increase plasma and and the of agents of as for the treatment of and atherosclerosis in the antiatherosclerosis of LXR agonists in mice and their anti-inflammatory and cholesterol in a of species, it is to on LXR as a therapeutic target for human To LXR agonists of upregulation of cholesterol genes such as ABC transporters genes An additional of such compounds the anti-inflammatory seen with current LXR agonists have been for the development of such LXR LXRβ and LXR The that LXRβ agonists the of LXR agonists is on from knockout mice, which plasma and hepatic gene expression in LXRα but not LXRβ knockout mice D.J. 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K. mechanisms of ligands and receptor Biol. 2000; Scholar, and regulation of the human 2002; Scholar). it demonstrated that is in mice and transcription in vitro B. S. S. D. J. R. cholesterol hepatic and by a LXR Lipid Res. 2004; Scholar). with the these that an more that of is for human therapeutic The to possess an LXR in vitro and in in mice D.A. A.R. Chen L. C.P. P. by a synthetic oxysterol ligand of the liver X Lipid Res. 2004; Scholar). additional of such LXR in species that more human lipoprotein such as hamsters and to that the LXR agonists and increase in CETP species, emphasize the importance of such models for the of the of LXR The for fatty acid of plasma A. Watson for and T. for of