Lack of predictability of classical animal models for hypolipidemic activity: a good time for mice?

Atherosclerosis 140 (1998) 15 – 24

Review article

Lack of predictability of classical animal models for hypolipidemic activity: a good time for mice? Brian R. Krause a,*, Hans M.G. Princen b a

Parke-Da6is Pharmaceutical Research, Warner-Lambert Company, 2800 Plymouth road, Ann Arbor, MI 48105, USA b Gaubius Laboratory TNO-PG, Leiden, The Netherlands Received 22 January 1998; received in revised form 11 May 1998; accepted 18 May 1998

Abstract Hypolipidemic drugs that are efficacious in man are not always active in classical animal models of dyslipidemia. Inhibitors of HMG-CoA reductase (statins) do not lower plasma cholesterol in rats, but yet this species was alone in providing activity for fibrate-type drugs. Nicotinic acid possesses many desirable features with regard to clinical use, but most of these actions are lacking in rats and monkeys. The metabolism of low density lipoproteins in hamsters is widely thought to be similar to that in humans, yet neither statins or fibrates lower plasma lipids in these species. With the advent of mouse models expressing specific human genes (or disruption of genes) it is now possible to re-examine the effect of established drugs and to characterize new hypolipidemic compounds with respect to site and mechanism of action. Drug responses observed in humans are now being seen in such mouse models (e.g. HDL elevation with fenofibrate in mice with the human apo A-I gene). Moreover, mice are now being screened for compounds that lower plasma (human) Lp(a), or lower plasma cholesterol in the absence of LDL receptors. It is proposed that these new genetic mouse models may afford a more focused examination of drug action and provide, for new compounds, better prediction of the human response. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Classic models; Hypolipidemic activity; HMG-CoA reductase

1. Introduction Views differ as to what is the best animal model for evaluating new or marketed lipid-lowering drugs in experimental animals. In 1962 Steinberg stated that ‘a drug which lowers serum lipid levels in experimental animals on a conventional dietary regiment is clearly a drug worthy of attention.’ [1]. But yet most animal models since then have focused on various dietary-induced forms of hyperlipidemia, using diets which are anything but conventional when compared to human diets, in an attempt to elevate plasma cholesterol to * Corresponding author. Tel.: + 1 734 6227677; fax: +1 313 9963135; e-mail: [email protected]

levels approaching the human dyslipidemias. With regard to choice of animal species, several have been touted as being ‘human-like’, yet many types of drugs which lower lipids in man are either inactive or poorly tolerated in such animals. Evaluation of the major classes of lipid-lowering drugs in various animal models has led us to several conclusions or generalizations, reviewed herein and listed in Table 1. In general, the concept that each animal model ‘will only yield positive results with compounds showing particular modes of action’ [1] is still valid, but there have been some surprises, and it may be that a more focused approach using genetically engineered mice will offer better predictability for the human response for future hypolipidemic (or HDL-elevating) drugs. This brief overview is

0021-9150/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0021-9150(98)00141-5

B.R. Krause, H.M.G. Princen / Atherosclerosis 140 (1998) 15–24

16

largely limited to drug effects on plasma lipids/lipoproteins. A more detailed review on the effects of hypolipidemic drugs on atherosclerosis in various animal models has recently appeared [2].

2. Inhibitors of HMG-CoA reductase (statins) The first major conclusion reached over the last few years is that, despite their enormous clinical success, inhibitors of HMG-CoA reductase (statins) lower plasma triglycerides, not cholesterol, in chow-fed rats [3 – 6] and are inactive in cholesterol-fed rats [7]. In fact, several companies showed little interest initially in compactin and related compounds because they failed to lower plasma cholesterol in cholesterol-fed rats, which was the most widely-used animal model in the late 1970s. Only at high doses (\100-fold that required to inhibit cholesterol synthesis) did cholesterol lowering occur in chow-fed rats with statins, usually coincident with body weight loss. This species was nonetheless used by Alberts et al. [8] for screening purposes by injecting radiolabeled acetate IP and measuring plasma 14 C-digitonin precipitable sterols. Thus, statins clearly inhibit cholesterol synthesis in rats. But the hypocholesterolemic response does not occur because in rats (which have little or no LDL) these drugs affect VLDL assembly and secretion, and this is evidenced by a reduction in triglycerides rather than cholesterol since VLDL is triglyceride-rich. Most likely, the availability of cholesterol (or cholesteryl esters) is decreased by the statins, which increases the degradation of apo B, thereby decreasing VLDL secretion [9]. This mechanism is probably active even in animals which, unlike the rat, convert most VLDL to LDL (‘LDL animals’). Thus, statins that are more efficacious in guinea pigs or rabbits are also more effective at lowering triglycerides in chow-fed rats [10]. Lastly, triglycerides may decrease in rats also because triglyceride synthesis (phosphatidate phosphohydrolase) and cholesterol synthesis (HMG-CoA reductase) appear to be coordinately regulated [11].

Unlike cholesterol-fed rats, statins lower plasma cholesterol in cholesterol-fed rabbits, as first shown by Kritchevsky et al. [12]. Despite this finding, the group at Merck, and later Parke-Davis, used the more difficult casein-fed rabbit model to characterize statins [3,13]. In the latter model, an endogenous hypercholesterolemia (EH) develops since the diet is cholesterol-free. Mechanisms for the hypercholesterolemia include decreased LDL clearance [14,15], increased hepatic secretion of ACAT-derived cholesteryl esters [16,17], and a marked reduction in bile acid excretion [18] and expression of 7a-hydroxylase in the liver [17]. In this model atorvastatin is more potent and efficacious than lovastatin for lowering LDL-cholesterol [19], whereas in cholesterolfed rabbits these drugs are probably equipotent [20]. Thus, the EH rabbit model may be somewhat more predictive of the human response, at least with statins. The cholesterol-fed rabbit model was taken advantage of by Bocan and colleagues to provide what we believe is the first comprehensive evaluation of the antiatherosclerotic activity of statins in animals [20]. Clearly, in cholesterol-fed rabbits some statins alter lesion development (e.g. atorvastatin, simvastatin) while others do not (e.g. pravastatin), even though they all lower plasma cholesterol. It is still unknown, however, why statins would lower plasma cholesterol in a model in which cholesterol synthesis is most certainly already inhibited by 90% or more (by the dietary cholesterol). For this reason, it has been generally assumed that the cholesterol-fed rabbit is ‘quite inappropriate for identifying compounds that inhibit cholesterol biosynthesis’ [1]. Moreover, it is unclear why statins would lower plasma cholesterol in cholesterol-fed rabbits but not cholesterol-fed rats. In chow-fed rabbits, we [10] and others [21] find no change in VLDL lipid secretion (production) with statin treatment, but there is decreased production and enhanced clearance of LDL [22]. These mechanisms may be operating in cholesterol-fed rabbits treated with statins, but to date lipoprotein kinetics have not been reported. It seems that efficacy might be partly independent of cholesterol synthesis inhibition for the reason cited above. For example, inhibition of cholesterol absorption may also

Table 1 Qualitative assessment of cholesterol-lowering activities for hypolipidemic drugs Drug

Statins Fibrates Resin Nicotinic acid Avasimibe(ACAT inhibitor)

Rat

Rabbit

Hamster

Dog

Monkey

Hyperlipidemic human

Chow

CF

Chow

CF

EH

Chow

CF

Chow

CF

Chow

CF

-+ − -+

-+ + + +

+ -+ ? NT

+ -+ −/+ +

+ -+ -+

--+ ? −/+

--+ ? +

+ -+ + +

--+ ? +

−/+ -+ + +

--+ ? +

+ + + + NT

Chow, animals consuming a normal chow diet; CF, cholesterol-fed; EH, endogenous hypercholesterolemia (casein-fed); +, active, --, not active; −/+, active at very high doses only; ?, no data found or available; NT, not tested.

B.R. Krause, H.M.G. Princen / Atherosclerosis 140 (1998) 15–24

contribute [23]. Lastly, although lovastatin has been shown to lower plasma cholesterol in chow-fed hamsters at high doses [24] as well as in cholesterol-fed hamsters [25], we must conclude from our experience that statins are poorly tolerated by hamsters regardless of diet or mode of drug administration (i.e. gavage or diet-admix). Interestingly, others have reported that statins transiently increase plasma cholesterol in hamsters [26] or induce liver toxicity at relatively low doses [27]. Therefore, the rodent species thought to resemble humans with regard to regulation of LDL metabolism [28] has contributed little to our understanding of statin pharmacology.

3. Fibric acid derivatives (fibrates) Another major conclusion concerning animal models that is not widely appreciated is that fibrates lower plasma cholesterol only in rats. On a mg/kg basis, the doses required to produce lipid-lowering are remarkably similar in rats and in humans [29]. In rats the effects of fibrates are greater in males versus females [30], and usually absent if animals are fasted [31]. We recently dosed female Sprague-Dawley rats (cholesterolfed) with gemfibrozil and found no changes in plasma total, HDL- and nonHDL-cholesterol at doses up to 100 mg/kg (Krause, unpublished results) whereas in male rats these parameters change significantly at only 10 mg/kg under the same conditions [7]. It is also of note that in chow-fed rats gemfibrozil is unique since, unlike clofibrate, bezafibrate or fenofibrate, it elevates HDL-cholesterol and plasma apo E levels [7,32–35]. Despite the lack of effect of gemfibrozil on plasma cholesterol in rabbits, anti-atherosclerotic activity has been reported. [36]. The mechanism for this remains to be explained, if it is indeed reproducible. At very high doses it has been shown that gemfibrozil can lower plasma triglycerides in rabbits by increasing VLDL-receptor activity in the periphery [37]. It would be important to determine drug blood levels in such experiments to compare to clinical data. Unlike statins, which reportedly lower cholesterol in chow-fed dogs and monkeys at very high doses (\ 50 mg/kg), fibrates generally do not lower cholesterol in dog and monkey models. The responses to fibrate treatment in hamsters are also unlike humans or other species (e.g. plasma and liver triglycerides increase rather than decrease) [38]. We have found very inconsistent effects of fibrates on plasma lipids in hamsters fed chow or cholesterol-containing diets, and fibrates are also inactive in guinea pigs [3,7]. In summary, for fibrate-like compounds only the male rat has provided data which seems to reliably predict the human response. It is now recognized that in rats and probably in man fibrates alter the expression of many genes by activating

17

nuclear, ligand-activated transcription factors termed peroxisome proliferator-activated receptors (PPARs) [39,40]. One particularly important PPAR target gene is apo C-III, which is downregulated at the transcriptional level by fibrates [41]. However, apo C-III expression or plasma apo C-III levels are not the only determinants of plasma triglyceride concentration in rats, and the best overall index predicting the extent of triglyceride lowering by fibrates in rats may be the apo E/apo C-II +apo C-III ratio [33]. All of these genes are all involved in lipoprotein clearance. Fibrates do not reduce VLDL-triglyceride production in chow-fed rats [32,33] but this does occur in hypertriglyceridemic rats [32]. It would seem that the new, unifying mechanism for fibrates (e.g. PPAR) would eventually have to take into account or explain the marked species (and gender) differences noted above and exemplified by the opposite regulation of human versus mouse apo A-I [42]. It might be hypothesized that there exists different PPARs in different species, thus accounting for the lack of efficacy in certain animal models noted above. Further knowledge may also explain the variability of responses to fibrate treatment in hyperlipidemic patients, or provide a rationale for combination treatment with other PPAR-activating drugs (e.g. thiazolidinediones) [43–45].

4. Nicotinic acid and resins Although widely advocated prior to the introduction of statins, nicotinic acid is now only prescribed in about 11% of hyperlipidemic patients in the US [46]. Identifying improved analogs of nicotinic acid without the common side effects has been hampered in part by the lack of suitable animal models. Specifically, it is of note that nicotinic acid does not lower plasma cholesterol in normal, chow-fed rats [47], or does so to a very minor extent [29,48]. It is also generally inactive in cholesterolfed rats [49], or at best produces minor changes which are not dose-dependent (Krause, unpublished results). The elevation of HDL-cholesterol seen clinically has also not been observed in chow-fed rats [48]. Nicotinic acid will lower plasma cholesterol in monkeys when administered subcutaneously [50] but not orally [51], and it is hypocholesterolemic in cholesterol-fed rabbits only at high doses [52]. We have found no data on the effect of nicotinic acid in hamsters and can only assume that it is poorly tolerated, like statins. Unlike nicotinic acid, the hypocholesterolemic effect that results from bile acid sequestration appears to represent one mechanism that is common to most animals, regardless of diet (Table 1), prompting the long, unfulfilled search for more effective resins. Interestingly, the response in the liver is also similar across species: the activities of both HMG-CoA reductase and cholesterol 7a-hydroxylase

18

B.R. Krause, H.M.G. Princen / Atherosclerosis 140 (1998) 15–24

increase in rats, rabbits, hamsters and guinea pigs treated with cholestyramine [53]. However, HDLcholesterol elevation, as reported in most human studies with resins, does not occur in all animal models. Increases in HDL-cholesterol have been observed in cholesterol-fed rats treated with cholestyramine [54], but not in chow-fed [55,56] or cholesterol-fed hamsters [57]. In chow-fed rats cholestyramine has little or no effect on plasma total cholesterol [58] or HDL-cholesterol [48]. These data on HDL again underscore the lack of ‘human-like’ responses in animals for lipid-lowering drugs.

5. Avasimibe (ACAT inhibitor) Avasimibe (CI-1011, PD 148515) is an example of an experimental drug which, like resins, has shown cholesterol-lowering activity in most animal models examined to date. In some models the hypocholesterolemic activity of avasimibe exceeds that of the statins [19,59]. Preclinical data for avasimibe is consistent with inhibition of intestinal and hepatic acyl-CoA:cholesterol acyltransferase (ACAT) activity as being the primary mechanism of action, resulting in decreased cholesterol absorption and hepatic VLDL-cholesteryl ester secretion, respectively [60]. However, other mechanisms might be operative since the compound enhances triglyceride clearance in chow-fed rats [61], lowers Lp(a) in monkeys [62] and is also more potent than gemfibrozil or atorvastastin at lowering triglycerides in hypertriglyceridemic rats (Krause, unpublished data). At the present time these biological activities are not easily ascribed to ACAT inhibition. But the question now is whether any of this preclinical data predict the human response. This is difficult to answer since it is uncertain whether ACAT is an important regulatory enzyme in human lipoprotein metabolism as it appears to be in animals. The data from cholesterol-fed rabbits feeds this uncertainty since both statins and previous ACAT inhibitors (e.g. CL 277082) are hypocholesterolemic in this model yet to date no ACAT inhibitor has shown significant efficacy in humans. Moreover, statins do not lower plasma cholesterol in the most sensitive animal model for ACAT inhibitors (i.e. cholesterol-fed rats). However, the failure of first-generation ACAT inhibitors to lower plasma cholesterol in humans (e.g. CL 277082) [63] may also be a function of suboptimal potency or pharmacokinetics. It will be of interest whether a structurally novel inhibitor like avasimibe, which attains low micromolar blood levels and possesses a prolonged half-life in healthy volunteers [64], is efficacious for the treatment of human dyslipidemia. Regardless, it should be noted that avasimibe may have the added benefit of inhibiting ACAT in arterial wall macrophages, resulting in less

cholesteryl ester accumulation and foam cell formation. In this way avasimibe would directly alter the atherosclerotic process independent of changes in plasma lipids. This anti-atherosclerotic mechanism for bioavailable ACAT inhibitors has been previously discussed by us [65,66] and others [67] and has been specifically demonstrated for avasimibe in cholesterolfed rabbits [68] and hamsters [69].

6. Future utility of mouse models The traditional animal models listed in Table 1 have provided insights into mechanism, but do little to inspire confidence in clinical outcomes. Transgenic and knock-out mice (via transgenesis and gene targeting using homologous recombination techniques) now appear to allow a new, possibly more predictive approach towards the testing of novel hypolipidemic drugs. Although used fairly extensively to examine the effect of specific genes on atherosclerosis [70,71], few studies have been reported thus far assessing hypolipidemic drugs in transgenic or knockout mice. We propose that in the future it may be desirable to characterize the major hypolipidemic drug classes as well as newer compounds in such mouse models to gain further insights into mechanisms of action and/or to confirm suspected mechanisms based on the animal models discussed above. Assessment of anti-atherosclerotic activity is an added benefit which also presumably reflects potential clinical benefit. The major reason for such work is that mice can be genetically engineered to provide ‘human-like’ responses which otherwise are not elicited. Limited data is available on the effects of hypolipidemic drugs in these new mouse models (Table 2). For example, fibrates like fenofibrate and gemfibrozil increase plasma apo A-I both in humans and in mice overexpressing the human apo A-I gene [72], whereas in rats or nontransgenic mice these drugs are without effect or even decrease plasma levels of apo A-I [72,73] (Table 2). The drug probucol also shows similar responses in humans and in the apo A-I transgenics. Specifically, it decreased HDL-cholesterol (HDL2) by increasing HDL-cholesteryl ester fractional catabolic rate and decreasing apo A-I transport (synthesis) rate [74]. Thus, these mice provide a model in which to study the in vivo regulation of apo A-I expression by drugs in which the responses mimic those in humans. It would be of interest, for example, to study the effect of cholestyramine on apo A-I levels and metabolism in this model since in man it is thought to increase apo A-I by enhancing apo A-I synthesis [75]. Hopefully, such models will contribute towards the discovery of better drugs for elevating HDL since at the present time there is probably no single animal model that adequately predicts HDL elevation in man.

Human apo A-I transgenic Human apo A-I transgenic Human apo AI transgenic Apo E-KO Apo E-KO Apo E3-leiden Apo E3-leiden Apo E3-leiden LDL receptor-KO LDL receptor-KO LDL receptor-KO

Fenofibrate

0.500 2.000 0.100 0.200 0.200 0.168 0.168 0.168

0.200

0.500

0.500

Dose (%)

12 4 2 2 2 2 2 2

2

1

1

Duration (weeks)

Total cholesterol (%change)

NA

Chow CF CF CF CF Chow Chow Chow −39 −37 −20 −5 −29 −49 (−63)* −3 (−4)* −37 (−44)*

Chow −36

Chow

Chow +162

Diet

−43 NA −18 +10 −20 −32 −4 −68

−33

+73

+202

HDL-cholesterol (%change)

Chow, animals consuming a normal chow diet; CF, cholesterol-fed; NA, data not available; KO, knockout. * Values in parentheses are changes in LDL-cholesterol (%); SB204990, inhibitor of ATP-citrate lyase (SmithKline Beecham).

Probucol Plant sterols Lovastatin Gemfibrozil SB204990 Atorvastatin Pravastatin Simvastatin

Probucol

Gemfibrozil

Model

Drug

Table 2 Evaluation of lipid-lowering agents in genetic mouse models

−50 NA NA NA NA NA NA NA

−46

+72

+758

Apo A-I (%change)

NA 0 0 −53 −42 +6 −36 −31

NA

NA

NA

Triglycerides (%change)

87 90 98 98 98 101 101 101

74

72

72

Ref

B.R. Krause, H.M.G. Princen / Atherosclerosis 140 (1998) 15–24 19

20

B.R. Krause, H.M.G. Princen / Atherosclerosis 140 (1998) 15–24

Another example constitutes the generation of YAC (yeast artificial chromosome) transgenic mice containing the entire human apo(a) gene surrounded by a high amount of flanking DNA [76], and transgenic mice containing both the human apo(a) and apo B-100 genes, resulting in formation of Lp(a)[77,78]. These mice can be used to study the effects of drugs which may decrease apo(a) gene expression as well as secretion by the hepatocyte or extracellular assembly of the Lp(a) particle. Only a few animal models exist, such as Old World monkeys or hedgehogs, which possess this atherogenic lipoprotein. Thus, these transgenic mice represent valuable and more approachable alternatives, especially in terms of availability, cost and amount of drugs required. To date the effects of sex hormones have been described [76,79], and these results tend to mimic the human situation [80]. But the effects of lipid-lowering drugs such as nicotinic acid and gemfibrozil, which have been reported to decrease Lp(a) in humans [81,82] and monkeys [51,83], have not been tested in this mouse model. This model might be ideal since it has been postulated that gemfibrozil, like estrogen and tamoxifen [79], may act at the transcriptional level to lower Lp(a) [83]. Transgenic animal models have especially highlighted the potential importance of apo E in lipoprotein metabolism. In mice made deficient in apo E by gene targeting the plasma cholesterol is elevated on a chow diet due to the absence of the ligand for clearance pathways of chylomicron and VLDL remnants [84–86]. The advanced, fibroproliferative arterial lesions that develop due to the hypercholesterolemia are similar in many respects to human atherosclerotic lesions [70,71] and no doubt also result from the absence of apo E in arterial wall macrophages. Few studies with hypolipidemic drugs have been reported in apo E-deficient mice (Table 2). Probucol efficiently decreased plasma total cholesterol in apo E-deficient mice, and hence did so by apo E-independent pathways [87]. Surprisingly, probucol enhanced atherosclerosis despite the cholesterollowering [87], unlike other antioxidants which are anti-atherosclerotic but have no effect on plasma cholesterol in apo E-deficient mice [88]. This lack of anti-atherosclerotic activity of probucol could not be easily explained by the expected decrease in HDLcholesterol [87]. It is possible that in order for probucol to be anti-atherosclerotic it requires an apo E-mediated mechanism, such as enhanced secretion of apo E from macrophages [89], which would be lacking in the apo E-deficient mice. In other studies, a plant sterol preparation was found to lower plasma cholesterol by 3040% over a span of 18 weeks, resulting in decreased lesion size and complexity [90]. Although it is well established that plant sterols inhibit cholesterol absorption and lower plasma cholesterol [91], it was also hypothesized that the plant sterols may be absorbed to

a small extent, transported on LDL, and thereby render LDL less susceptible to oxidation [90]. Estrogen and tamoxifen, although not lipid-lowering drugs per se, have been considered to be cardioprotective in part because they have beneficial effects on plasma lipoproteins. Hence, they were tested in apo E-deficient mice. In ovariectomized apo E-deficient mice both estrogen (12 mg/day) and tamoxifen (80 mg/day) treatment (subdermal) decreased plasma cholesterol modestly (B 20%), and lowered triglycerides (approx. 50%), but yet only estrogen decreased atherosclerotic lesion size [92]. These results, and the fact that ovariectomy itself decreased lesion size but not plasma cholesterol, suggested to the authors that the anti-atherosclerotic activity of estrogen cannot be fully explained by changes in plasma lipids. Estrogen also decreased plasma cholesterol (30%) and lesion size (43%) in male apo E-deficient mice [92]. In contrast, tamoxifen reduced plasma cholesterol in males by 17% but had no effect on lesion size. In another study in which tamoxifen (approx. 50 mg/day) was administered orally to male apo E-deficient mice greater changes in plasma cholesterol (86%) were observed, and the drug markedly reduced lesion size (63%). The change in plasma total cholesterol was due to decreases in the absolute concentrations of VLDL-, LDL- and HDLcholesterol. Triglyceride levels were also decreased [93]. The anti-atherosclerotic activity of tamoxifen was largely ascribed to the marked reduction in plasma lipids, although an increase in TGF-b levels in the aorta may have contributed. These two studies in apo E-deficient mice seem to illustrate that the route of drug administration may have a large impact on the results, and suggest that plasma drug levels should be compared to those in human trials to aid in the interpretation of the data. The age of the animals and the nutritional state at sacrifice (fed vs. fasted) were also different between studies and could have contributed to the differences in drug responses. The difficulty in separating direct effects on lesions (elevation of TGFb) from indirect effects (reduction in plasma cholesterol) is also clear from these data. Interestingly, Grainger and colleagues [93] state that tamoxifen is far more effective than simvastatin at lowering plasma cholesterol in apo E-deficient mice, yet both are thought to act by inhibiting lipoprotein production. Possibly in mice, simvastatin requires an apo E-dependent clearance mechanism. Another mouse model with defective clearance of apo E-containing lipoproteins is caused by the introduction of the dominant human mutant apo E3-Leiden gene [94,95]. In these mice plasma lipid levels depend strongly on the input of lipids into the circulation from the intestine (chylomicrons) and the liver (VLDL). Plasma cholesterol increases from about 3 mmol/l (chow diet) to 40 mmol/l after a high-fat/high-choles-

B.R. Krause, H.M.G. Princen / Atherosclerosis 140 (1998) 15–24

terol diet [94]. This represents a more moderate hypercholesterolemia compared to the apo E-deficient mice, which increase from about 20 mmol/l to 100 mmol/l [84 – 86], and thus is more similar to the human situation. In contrast to nontransgenic mice, apo E3-Leiden mice have lipoprotein particles in the VLDL/LDL size range. By feeding the animals low amounts of cholesterol or a lipogenic (sucrose-containing) diet, plasma cholesterol and triglycerides can be titrated to desired levels [94–96]. Development of atherosclerosis in apo E3-Leiden mice is strongly correlated to plasma cholesterol levels and time of exposure [94,96]. Thus far, nutritional factors and drugs known to decrease the production of chylomicrons and/or VLDL all lower plasma lipids more markedly compared with nontransgenic littermates used as controls (Table 2). For example, dietary fish oil, known to lower serum triglycerides and VLDL levels in humans by inhibition of VLDLtriglyceride production, showed a dose-dependent decrease in plasma cholesterol and triglyceride levels in apo E3-Leiden mice, mainly due to a reduction in VLDL and LDL lipoproteins [97]. LDL and HDLcholesterol levels were unaffected. Kinetic studies indicated that the effects were due to a decreased hepatic VLDL-triglyceride production rate and an increase in VLDL-apo B fractional catabolic rate. Likewise, cholestyramine reduced plasma cholesterol levels more strongly in apo E3-Leiden mice as compared to nontransgenic controls (Havekes and Princen, unpublished data). In contrast to the absence of effects in nontransgenic mice or in rats, lovastatin (decrease in plasma cholesterol) and the ATP-citrate lyase inhibitor SB 204990 (decrease in plasma triglycerides) showed a clear lipid-lowering effect in apo E3-Leiden transgenic mice [98]. Gemfibrozil also decreased plasma triglycerides more strongly in apo E3-Leiden mice compared to nontransgenic controls [98]. Based upon the defect in apo E3-Leiden mice, these results would imply that lovastatin and gemfibrozil would decrease VLDLtriglyceride production in humans, and this indeed has been reported for both drugs [99,100]. Moreover, the above proposed reduction in intrahepatic availability of cholesterol and cholesteryl esters as the primary mechanism of action of statins in rodents, resulting in a decreased VLDL production, can be more reliably monitored in apo E3-Leiden mice which represent a sensitive ‘production’ model. One might predict that SB 204990 would significantly lower lipids in humans based upon these data. Besides the mouse models above, we have found that atorvastatin, and to a lesser extent simvastatin, can decrease plasma cholesterol in the absence of the LDL receptor by demonstrating efficacy in LDL receptor knockout mice [101]. Such data would also appear to support clinical findings of decreased lipoprotein production as the mechanism for LDL reduction by statins

21

in hyperlipidemic subjects [100,102,103], and would predict the efficacy observed in homozygous familial hypercholesterolemia with atorvastastin [104]. Thus, this model may prove useful for identifying compounds which more potently inhibit LDL production or enhance clearance by mechanisms not involving the LDL receptor. Another application of the knockout technology to preclinical drug evaluation has been the testing of the ACAT inhibitor CI-976 in the ACAT knockout mouse model recently described by Farese and colleagues [105]. It is clear that the esterification of cholesterol occurring in the liver of these animals is inhibited by CI-976, even though it is not knocked out by the disruption of the gene originally cloned from macrophages [106]. This confirms previous evidence that the compound inhibits esterification in the liver [107,108], and points to the possibility of tissue-specific ‘ACAT-type’ enzymes. Such animal models thus aid in confirming sites of drug action, and illustrate the need for drugs specific for the liver and/or intestinal esterification enzymes for potentially modifying lipoprotein production. In conclusion, the small, genetically-defined murine models, which can be constructed to fit specific needs by transgenesis and knock-out technology, may have considerable advantages as compared with conventional animal models for testing of hypolipidemic drugs. At the very least they could be used in conjunction with conventional models to confirm suspected mechanisms and improve overall predictability. For example, if an experimental drug lowers plasma cholesterol by enhancing apo E-mediated clearance of lipoproteins, it may be inactive in the apo E-deficient mouse yet active in other models. However, one must also keep in mind that mice do not have cholesteryl ester transfer protein (CETP), and that this gene may be required (‘knocked-in’, or use transgenic rabbits) for the lipid-lowering (or anti-atherosclerotic) effect for a given compound. These and other mouse models may exemplify the type of refinement and specificity required to improve our ability to predict the human response, which at the moment, is mainly guesswork using the traditional chow- and cholesterol-fed animal models.

References [1] Steinberg D. Chemotherapeutic approaches to the problem of hyperlipidemia. Adv Pharmacol 1962;1:59. [2] Bocan TMA. Animal models of atherosclerosis and interpretation of drug intervention studies. Curr Pharm Des 1998;4:39. [3] Krause BR, Newton RS. Animal models for the evaluation of inhibitors of HMG-CoA reductase. Adv Lipids Res 1991;1:57. [4] Endo A, Tsujita Y, Kuroda M, Tanzawa K. Effects of ML236B on cholesterol metabolism in mice and rats: lack of hypocholesterolemic activity in normal animals. Biochim Biophys Acta 1979;575:266.

22

B.R. Krause, H.M.G. Princen / Atherosclerosis 140 (1998) 15–24

[5] Tsujita Y, Kuroda M, Shimada Y, Tanzawa K, Arai M, Kaneko I, Tanaka M, Masuda H, Tarumi C, Watanabe Y. CS-514, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase: tissue-selective inhibition of sterol synthesis and hypolipidemic effect on various animal species. Biochim Biophys Acta 1986;877:50. [6] Fujioka T, Nara F, Tsujita Y, Fukushige J, Fukami M, Kuroda M. The mechanism of lack of hypocholesterolemic effects of pravastatin sodium, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, in rats. Biochim Biophys Acta Lipids Lipid Metab 1995;1254:7. [7] Krause BR, Bousley R, Kieft K, Robertson D, Stanfield R, Urda E, Newton RS. Comparison of lifibrol to other lipid-regulating agents in experimental animals. Pharmacol Res 1994;29:345. [8] Alberts AW, Chen J, Kuron G, Hunt V, Huff J, Hoffman C, Rothrock J, Lopez M, Joshua H, Harris E, Patchett A, Monaghan R, Currie S, Stapley E, Albers Schonberg G, Hensens O, Hirshfield J, Hoogsteen K, Liesch J, Springer J. Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutarylcoenzyme A reductase and a cholesterol-lowering agent. Proc Natl Acad Sci USA 1980;77:3957. [9] Huff MW, Burnett JR. 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors and hepatic apo lipoprotein B secretion. Curr Opin Lipidol 1997;8:138. [10] Krause BR, Newton RS. Lipid-lowering activity of atorvastatin and lovastatin in rodent species: Triglyceride-lowering in rats correlates with efficacy in LDL animal models. Atherosclerosis 1995;117:237. [11] Humble E, AL-Shurbaji A, Lund E, Berglund L. Studies on rat liver phosphatidate phosphohydrolase and plasma lipids: effect of HMG-CoA reductase inhibitors. Biochim Biophys Acta Lipids Lipid Metab 1994;1214:32. [12] Kritchevsky D, Tepper SA, Klurfeld DM. Influence of mevinolin on experimental atherosclerosis in rabbits. Pharmacol Res Commun 1981;13:921. [13] Kroon PA, Hand KM, Huff JW, Alberts AW. The effects of mevinolin on serum cholesterol levels of rabbits with endogenous hypercholesterolemia. Atherosclerosis 1982;44:41. [14] Chao YS, Yamin TT, Alberts AW. Catabolism of low density lipoproteins by perfused rabbit livers: cholestyramine promotes receptor-dependent hepatic catabolism of low density lipoproteins. Proc Natl Acad Sci USA 1982;79:3983. [15] Khosla P, Samman S, Carroll KK, Huff MW. Turnover of 125I-VLDL and 131I-LDL apolipoprotein B in rabbits fed diets containing casein or soy protein. Biochim Biophys Acta 1989;1002:157. [16] Chao YS, Yamin TT, Seidenberg J, Kroon PA. Secretion of cholesteryl-ester-rich lipoproteins by the perfused livers of rabbits fed a wheat-starch-casein diet. Biochim Biophys Acta 1986;876:392. [17] Krause BR, Pape ME, Kieft K, Auerbach B, Bisgaier CL, Homan R, Newton RS. ACAT inhibition decreases LDL cholesterol in rabbits fed a cholesterol-free diet: Marked changes in LDL cholesterol without changes in LDL receptor mRNA abundance. Arterioscler Thromb 1994;14:598. [18] Huff MW, Carroll KK. Effects of dietary protein on turnover, oxidation and absorption of cholesterol, and on steroid excretion in rabbits. J Lipid Res 1980;21:546. [19] Auerbach BJ, Krause BR, Bisgaier CL, Newton RS. Comparative effects of HMG-CoA reductase inhibitors on apo B production in the casein-fed rabbit: Atorvastatin versus lovastatin. Atherosclerosis 1995;115:173. [20] Bocan TMA, Mazur MJ, Mueller SB, Brown EQ, Sliskovic DR, O’Brien PM, Creswell MW, Lee H, Uhlendorf PD, Roth BD, Newton RS. Antiatherosclerotic activity of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase in cholesterol-fed rabbits: A biochemical and morphological evaluation. Atherosclerosis 1994;111:127.

[21] Quig DW, Zilversmit DB. Dissociation between cholesterol secretion and plasma lipid transfer activity in rabbits. FASEB J 1988;2:2712. [22] La Ville A, Moshy R, Turner PR, Miller NE, Lewis B. Inhibition of cholesterol synthesis reduces low-density-lipoprotein apoprotein B production without decreasing very-low-density-lipoprotein apoprotein B synthesis in rabbits. Biochem J 1984;219:321. [23] Ishida F, Sato A, Iizuka Y, Sawasaki Y, Aizawa A, Kamei T. Effects of MK-733, an inhibitor of 3-hydroxy-3-methylglutarylcoenzyme A reductase, on absorption and excretion of [3H]cholesterol in rabbits. Biochim Biophys Acta 1988;963:35. [24] Ma PT, Gil G, Sudhof TC, Bilheimer DW, Goldstein JL, Brown MS. Mevinolin, an inhibitor of cholesterol synthesis, induces mRNA for low density lipoprotein receptor in livers of hamsters and rabbits. Proc Natl Acad Sci USA 1986;83:8370. [25] Otto J, Ordovas JM, Smith D, Van Dongen D, Nicolosi RJ, Schaefer EJ. Lovastatin inhibits diet induced atherosclerosis in F1B Golden Syrian hamsters. Atherosclerosis 1995;114:19. [26] Amin D, Gustafson S, Perrone MH. Lovastatin is hypertriglyceridemic in Syrian golden hamsters. Biochem Biophys Res Commun 1988;157:530. [27] Morand OH, Aebi JD, Dehmlow H, Ji YH, Gains N, Lengsfeld H, Himber J. Ro 48-8.071, a new 2,3-oxidosqualene:lanosterol cyclase inhibitor lowering plasma cholesterol in hamsters, squirrel monkeys, and minipigs: comparison to simvastatin. J Lipid Res 1997;38:373. [28] Spady DK, Dietschy JM. Interaction of dietary cholesterol and triglycerides in the regulation of hepatic low density lipoprotein transport in the hamster. J Clin Invest 1988;81:300. [29] Dvornik D, Cayen MN. Drugs affecting lipoprotein disposition in laboratory animals. In: Fumagalli R, Kritchevsky D, Paoletti R, editors. Drugs Affecting Lipid Metabolism. Amsterdam: Elsevier/North-Holland Biomedical Press, 1980:263. [30] Rodney G, Uhlendorf P, Maxwell RE. The hypolipidemic effect of gemfibrozil (CI-719) in laboratory animals. Proc roy Soc Med 1976;69:6. [31] Stahlberg D, Angelin B, Einarsson K. Effects of treatment with clofibrate, bezafibrate and ciprofibrate on the metabolism of cholesterol in rat liver microsomes. J Lipid Res 1989;30:953. [32] Krause BR, Barnett BC, Essenburg AD, Kieft KA, Auerbach BJ, Bousley R, Stanfield R, Newton RS, Bisgaier CL. Opposite effects of bezafibrate and gemfibrozil in both normal and hypertriglyceridemic rats. Atherosclerosis 1996;127:91. [33] Haubenwallner S, Essenburg AD, Barnett BC, Pape ME, DeMattos RB, Krause BR, Minton LL, Auerbach BJ, Newton RS, Leff T, Bisgaier CL. Hypolipidemic activity of select fibrates correlates to changes in hepatic apolipoprotein C-III expression: A potential physiologic basis for their mode of action. J Lipid Res 1995;36:2541. [34] Newton RS, Krause BR. Alterations in plasma high density lipoprotein levels with gemfibrozil treatment. In: Miller NE, Tall AR, editors. High Density Lipoproteins and Atherosclerosis III. Amsterdam: Elsevier Scientific, 1992:259. [35] Staels B, Van Tol A, Andreu T, Auwerx J. Fibrates influence the expression of genes involved in lipoprotein metabolism in a tissue-selective manner in the rat. Arterioscler Thromb 1992;12:286. [36] Pfeiffer M, Thiery J, Walli AK, Seidel D. Gemfibrozil decreases cholesterol in the atherosclerotic aorta of rabbits (Abstract). Drugs Affecting Lipid Metabolism X 1989;127. [37] Matsuoka N, Jingami H, Masuzaki H, Mizuno M, Nakaishi S, Suga J, Tanaka T, Yamamoto T, Nakao K. Effects of gemfibrozil administration on very low density lipoprotein receptor mRNA levels in rabbits. Atherosclerosis 1996;126:221. [38] Plancke MO, Olivier P, Clavey V, Marzin D, Fruchart JC. Aspects of cholesterol metabolism in normal and hypercholesterolemic Syrian hamsters. Influence of fenofibrate. Methods Find Exp Clin Pharmacol 1988;10:575.

B.R. Krause, H.M.G. Princen / Atherosclerosis 140 (1998) 15–24 [39] Schoonjans K, Staels B, Auwerx J. Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J Lipid Res 1996;37:907. [40] Staels B, Auwerx J. Role of PPAR in the pharmacological regulation of lipoprotein metabolism by fibrates and thiazolidinediones. Current Pharm Design 1997;3:1. [41] Staels B, Vu-Dac N, Kosykh VA, Saladin R, Fruchart J-C, Dallongeville J, Auwerx J. Fibrates downregulate apolipoprotein C-III expression independent of induction of peroxisomal acyl coenzyme A oxidase. A potential mechanism for the hypolipidemic action of fibrates. J Clin Invest 1995;95:705. [42] Berthou L, Duverger N, Emmanuel F, Langouet S, Auwerx J, Guillouzo A, Fruchart J-C, Rubin E, Denefle P, Staels B, Branellec D. Opposite regulation of human versus mouse apolipoprotein A-I by fibrates in human apolipoprotein A-I transgenic mice. J Clin Invest 1996;97:2408. [43] Schoonjans K, Peinado-Onsurbe J, Lefebvre AM, Heyman RA, Briggs M, Deeb S, Staels B, Auwerx J. PPARa and PPARgamma activators direct a distinct tissue-specific transcriptional response via a PPRE in the lipoprotein lipase gene. EMBO J 1996;15:5336. [44] Lefebvre AM, Peinado-Onsurbe J, Leitersdorf I, Briggs MR, Paterniti Jr J, Fruchart J-C, Fievet C, Auwerx J, Staels B. Regulation of lipoprotein metabolism by thiazolidinediones occurs through a distinct but complementary mechanism relative to fibrates. Arterioscler Thromb Vasc Biol 1997;17:1756. [45] Lefebvre AM, Peinado-Onsurbe J, Leitersdorf I, Briggs MR. Thiazolidinediones exert a hypotriglyceridemic effect by a distinct, but complementary mechanism relative to fibrates. Atherosclerosis 1997;134:57. [46] Stafford RS, Blumenthal D, Pasternak RC. Variations in cholesterol management practices of U.S. physicians. J Am Coll Cardiol 1997;29:139. [47] Cayen MN, Kallai Sanfacon MA, Dubuc J, Greselin E, Dvornik D. Evaluation of the lipid-lowering activity of AY25,712 in rats. Atherosclerosis 1982;45:267. [48] Muller KR, Cortesi RG. The value of the normolipidaemic rat as an experimental animal in hypocholesterolemic drug research. Artery 1978;4:564. [49] Ohata I, Sakamoto N, Nitatori R, Maeno H. Aminophenyl ether compound (YM-95831), a new hypolipidemic agent with a high density lipoprotein elevating activity in rats. ArzneimForsch 1983;33:237. [50] Magide AA, Myant NB. Effect of nicotinic acid on cholesterol metabolism in monkeys. Clin Sci Mol Med 1974;46:527. [51] Bentzen C, Guyon Y, Niesor E. Nicotinic acid decreases specifically plasma Lp(a) in cynomolgus monkeys without affecting other lipoproteins. Atherosclerosis 1997;134:137. [52] Merrill JM, Lemley-Stone J. Effects of nicotinic acid on serum and tissue cholesterol in rabbits. Circ Res 1957;5:617. [53] Cighetti G, Bosisio E, Galli G, Kienle MG. The effect of cholestyramine on liver HMG-CoA reductase and cholesterol 7-alpha-hydroxylase in various laboratory animals. Life Sci 1983;33:2483. [54] Maxwell RE, Nawrocki JW, Uhlendorf PD. Some comparative effects of gemfibrozil, clofibrate, bezafibrate, cholestyramine and compactin on sterol metabolism in rats. Atherosclerosis 1983;48:195. [55] Groot PHE, Pearce NJ, Suckling KE, Eisenberg S. Effects of cholestyramine on lipoproteins and metabolism in Syrian hamsters. Biochim Biophys Acta 1992;23:76–88. [56] Benson GM, Alston DR, Bond BC, Gee AN, Glen A, Haynes C, Hickey DMB, Iqbal S, Jackson B, Jaxa-Chamiec AA, Johnson MR, Roberts MG, Slingsby BP, Whittaker CM, Suckling KE. SK&F 97426-A, a more potent bile acid sequestrant and hypocholesterolemic agent than cholestyramine in the hamster. Atherosclerosis 1993;101:51.

23

[57] Kowala MC, Nunnari JJ, Durham SK, Nicolosi RJ. Doxazosin and cholestyramine similarly decrease fatty streak formation in the aortic arch of hyperlipidemic hamsters. Atherosclerosis 1991;91:35. [58] Huff JW, Gilfillan JL, Hunt HJ. Effect of cholestyramine, a bile acid binding polymer, on plasma cholesterol and fecal bile acid excretion in the rat. Proc Soc Exp Biol Med 1963;114:352. [59] Auerbach B, Bousley R, Stanfield R, Bisgaier CL, Homan R, Krause BR. Modulation of apoB kinetics and lipoprotein cholesterol in casein-fed rabbits by CI-1011: in vivo evidence for liver ACAT inhibition. Int Symp Drugs Affecting Lipid Met :Nov 1995;7-10:135. [60] Lee HT, Sliskovic DR, Picard JA, Roth BD, Wierenga W, Hicks JL, Bousley RF, Hamelehle KL, Homan R, Speyer C, Stanfield RL, Krause BR. Inhibitors of acyl-CoA: cholesterol O-acyl transferase (ACAT) as hypocholesterolemic agents. CI1011: an acyl sulfamate with unique cholesterol-lowering activity in animals fed noncholesterol-supplemented diets. J Med Chem 1996;39:5031. [61] Krause BR, Auerbach BJ. ACAT inhibition by CI-1011 lowers plasma triglycerides in rats by enhancing the clearance of VLDL (Abstract). Atherosclerosis 1997;134:128. [62] Ramharack R, Spahr MA, Sekerke C, Stanfield R, Bousley R, Lee H, Krause BR. CI-1011 lowers lipoprotein(a) and plasma cholesterol concentrations in chow-fed cynomlgus monkeys. Atherosclerosis 1997;136:79. [63] Harris WS, Dujovne C, Von Bergmann K, Neal J, Akester J, Windsor MT, Greene D, Look Z. Effects of the ACAT inhibitor CL 277,082 on cholesterol metabolism in humans. Clin Pharmacol Ther 1990;48:189. [64] Vora J, Stern R, Lathia C. Clinical pharmacokinetics of CI1011, an ACAT inhibitor. Pharm Res 1997;14:505. [65] Krause BR, Bocan TMA. ACAT inhibitors: physiologic mechanisms for hypolipidemic and anti-atherosclerotic activities in experimental animals. In: Ruffolo RR, Hollinger MA, editors. Inflammation: Mediators and Pathways. Boca Raton, FL: CRC Press, 1995:173. [66] Krause BR, Sliskovic DR, Bocan T. Emerging therapies in atherosclerosis. Exp Opin Invest Drugs 1995;4:353. [67] Matsuda K. ACAT inhibitors as antiatherosclerotic agents: compounds and mechanisms. Med Res Rev 1994;14:271. [68] Bocan TMA, Rosebury WS, Bak Mueller S, Milad M, Lathia C, Homan R. Might ACAT inhibitors promote development of a stable plaque morphology? Circulation 1997;96:491. [69] Nicolosi RJ, Wilson TA, Krause BR. The ACAT inhibitor, CI-1011, is effective in the prevention and regression of early atherosclerosis in hamsters. Atherosclerosis 1998;137:77. [70] Smith JD, Breslow JL. The emergence of mouse models of atherosclerosis and their relevance to clinical research. J Int Med 1997;242:99. [71] Plump A. Atherosclerosis and the mouse: a decade of experience. Ann Med 1997;29:193. [72] Berthou L, Duverger N, Emmanuel F, Langouet S, Auwerx J, Guillouzo A, Fruchart J-C, Rubin E, Dene`fle P, Staels B, Branellec D. Opposite regulation of human versus mouse apolipoprotein A-I by fibrates in human apolipoprotein A-I transgenic mice. J Clin Invest 1996;97:2408. [73] Olivier P, Plancke MO, Marzin D, Clavey V, Sauzieres J, Fruchart J-C. Effects of fenofibrate, gemfibrozil and nicotinic acid on plasma lipoprotein levels in normal and hyperlipidemic mice. Atherosclerosis 1988;70:107. [74] Hayek T, Chajek-Shaul T, Walsh A, Azrolan N, Breslow J. Probucol decreases apolipoprotein A-I transport rate and increases high density lipoprotien cholesteryl ester fractional catabolic rate in control and human apolipoprotein A-I transgenic mice. Arterio Thromb 1991;11:1295. [75] Shepard J, Packard CJ, Morgan HG, Third JLHC, Stewart JM, Lawrie TDV. The effects of cholestyramine on high density lipoprotein metabolism. Atherosclerosis 1979;33:433.

24

B.R. Krause, H.M.G. Princen / Atherosclerosis 140 (1998) 15–24

[76] Frazer KA, Narla G, Zhang JL, Rubin EM. The apolipoprotein(a) gene is regulated by sex hormones and acute-phase inducers in YAC transgenic mice. Nat Genet 1995;9:424. [77] Linton MF, Farese Jr RV, Chiesa G, Grass DS, Chin P, Hammer RE, Hobbs HH, Young SG. Transgenic mice expressing high plasma concentrations of human apolipoprotein B100 and lipoprotein(a). J Clin Invest 1993;92:3029. [78] Callow MJ, Stoltzfus LJ, Lawn RM, Rubin EM. Expression of human apolipoprotein B and assembly of lipoprotein(a) in transgenic mice. Proc Natl Acad Sci USA 1994;91:2130. [79] Zysow BR, Kauser K, Lawn RM, Rubanyi GM. Effects of estrus cycle, ovariectomy, and treatment with estrogen, tamoxifen and progesterone on apolipoprotein(a) gene expression in transgenic mice. Arterio Thromb Vasc Biol 1997;17:1741. [80] Shewmon DA, Stock JL, Rosen CJ, Heiniluoma KM, Hogue MM, Morrison A, Doyle EM, Ukena T, Weale V, Baker S. Tamoxifen and estrogen lower circulating lipoprotein(a) concentrations in healthy postmenopausal women. Arterioscler Thromb 1994;14:1586. [81] Ramires JAF, Mansur AP, Solimene MC, Maranhao R, Chamone D, da Luz P, Pileggi F. Effect of gemfibrozil versus lovastatin on increased serum lipoprotein(a) levels of patients with hypercholesterolemia. Int J Cardiol 1995;48:115. [82] Gurakar A, Hoeg JM, Kostner GM, Papadopoulos NM, Brewer HB. Levels of lipoprotein(a) decline with neomycin and niacin treatment. Atherosclerosis 1985;57:293. [83] Ramharack R, Spahr MA, Hicks GW, Kieft KA, Brammer DW, Minton LL, Newton RS. Gemfibrozil significantly lowers cynomolgus monkey plasma lipoprotein[a]-protein and liver apolipoprotein[a] mRNA levels. J Lipid Res 1995;36:1294. [84] Plump AS, Smith JD, Hayek T, Aalto Setala K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell 1992;71:343. [85] Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science 1992;258:468. [86] Van Ree JH, Van den Broek WJAA, Dahlmans VEH, Groot PHE, Vidgeon-Hart M, Frants RR, Wieringa B, Havekes LM, Hofker MH. Diet-induced hypercholesterolemia and atherosclerosis in heterozygous apolipoprotein E-deficient mice. Atherosclerosis 1994;111:25. [87] Zhang SH, Reddick RL, Avdievich E, Suries LK, Jones RG, Reynolds JB, Quarfordt SH, Maeda N. Paradoxical enhancement of atherosclerosis by probucol treatment in apolipoprotein E-deficient mice. J Clin Invest 1997;99:2858. [88] Tangirala RK, Casanada F, Miller E, Witztum JL, Steinberg D, Palinski W. Effect of the antioxidant N,N’-diphenyl 1,4phenylenediamine (DPPD) on atherosclerosis in apo E-deficient mice. Arterioscler Thromb Vasc Biol 1995;15:1625. [89] Yamamoto A, Hara H, Takaichi S, Wakasugi J, Tamikawa M. Effect of probucol on macrophages, leading to regression of xanthomas and atheromatous vascular lesions. Am J Cardiol 1988;62:31B. [90] Moghadasian MH, McManus BM, Pritchard H, Frohlich J. ‘Tall oil’-derived phytosterols reduce atherosclerosis in apo E-deficient mice. Arterioscler Thromb Vasc Biol 1997;17:119. [91] Homan R, Krause BR. Established and emerging strategies for inhibition of cholesterol absorption. Curr Pharm Design 1997;3:29. [92] Bourassa PAK, Milos PM, Gaynor BJ, Breslow JL, Aiello RJ. Estrogen reduces atherosclerotic lesion development in apolipoprotein E-deficient mice. Proc Natl Acad Sci USA 1996;93:10022. [93] Reckless J, Metcalfe JC, Grainger DJ. Tamoxifen decreases cholesterol sevenfold and abolishes lipid lesion development in apolipoprotein E knockout mice. Circulation 1997;95:1542.

[94] Van Vlijmen BJM, Van den Maagdenberg AMJM, Gijbels MJJ, Van der Boom H, HogenEsch H, Frants RR, Hofker MH, Havekes LM. Diet-induced hyperlipoproteinemia and atherosclerosis in apolipoprotein E3-Leiden transgenic mice. J Clin Invest 1994;93:1403. [95] Van Vlijmen BJM, Van’t Hof HB, Mol MJTM, Van der Boom H, Van der Zee A, Frants RR, Hofker MH, Havekes LM. Modulation of very low density lipoprotein production and clearance contributes to age- and gender-dependent hyperlipoproteinemia in apolipoprotein E3-Leiden transgenic mice. J Clin Invest 1996;97:1184. [96] Groot PHE, Van Vlijmen BJM, Benson GM, Hofker MH, Schiffelers R, Vidgeon-Hart M, Havekes LM. Quantitative assessment of aortic atherosclerosis in APO E*3 Leiden transgenic mice and its relationship to serum cholesterol exposure. Arterioscler Thromb Vasc Biol 1996;16:926. [97] Van Vlijmen BJM, Mensink RP, Van’t Hof HB, et al. Effects of dietary fish oil on serum lipids and VLDL kinetics in hyperlipidemic apolipoprotein E*3-Leiden transgenic mice. J Lipid Res 1998;39:1181 – 8. [98] Van Vlijmen BJM, Pearce NJ, Bergo M, Staels B, Yates JW, Gribble AD, Bond BC, Hofker MH, Havekes L, Groot PHE. Apolipoprotein E3-Leiden transgenic mice as test model for hypolipidaemic drugs. Arzneimittel 1998;48:396. [99] Kesaniemi YA, Grundy SM. Influence of gemfibrozil and clofibrate on metabolism of cholesterol and plasma triglycerides in man. J Am Med Assoc 1984;251:2241. [100] Arad Y, Ramakrishnan R, Ginsberg HN. Effects of lovastatin therapy on very-low-density lipoprotein triglyceride metabolism in subjects with combined hyperlipidemia: evidence for reduced assembly and secretion of triglyceride-rich lipoproteins. Metabolism 1992;41:487. [101] Bisgaier CL, Essenburg AD, Auerbach B, Pape M, Sekerke C, Gee A, Wolle S, Newton RS. Attenuation of plasma low density lipoprotein cholesterol by select 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in mice devoid of low density lipoprotein receptors. J Lipid Res 1997;38:2502. [102] Arad Y, Ramakrishnan R, Ginsberg HN. Lovastatin therapy reduces low density lipoprotein apo B levels in subjects with combined hyperlipidemia by reducing the production of apo B-containing lipoproteins: implications for the pathophysiology of apo B production. J Lipid Res 1990;31:567. [103] Grundy SM, Vega GL. Influence of mevinolin on metabolism of low density lipoproteins in primary moderate hypercholesterolemia. J Lipid Res 1985;26:1464. [104] Marais AD, Firth JC, Bateman ME, Byrnes P, Martens C, Mountney J. Atorvastatin: an effective lipid-modifying agent in familial hypercholesterolemia. Arterioscler Thromb Vasc Biol 1997;17:1527. [105] Meiner VL, Cases S, Myers HM, Sande ER, Bellosta S, Schambelan M, Pitas RE, McGuire J, Herz J, Farese Jr RV. Disruption of the acyl-CoA:cholesterol acyltransferase gene in mice: Evidence suggesting multiple cholesterol esterification enzymes in mammals. Proc Natl Acad Sci USA 1996;93:14041. [106] Chang CCY, Huh HY, Cadigan KM, Chang TY. J Biol Chem 1993;268:20747. [107] Krause BR, Anderson M, Bisgaier CL, Bocan T, Bousley R, DeHart P, Essenburg A, Hamelehle K, Homan R, Kieft K. In vivo evidence that the lipid-regulating activity of the ACAT inhibitor CI-976 in rats is due to inhibition of both intestinal and liver ACAT. J Lipid Res 1993;34:279. [108] Carr TP, Hamilton Jr RL, Rudel LL. ACAT inhibitors decrease secretion of cholesteryl esters and apolipoprotein B by perfused livers of African green monkeys ACAT inhibitors decrease secretion of cholesteryl esters and apolipoprotein B by perfused livers of African green monkeys. J Lipid Res 1995;36:25.