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Chapter 23. New Potential Therapies for the Treatment of Atherosclerosis
Chapter 23. New Potential Therapies for the Treatment of Atherosclerosis Scott D. Larsen and Charles H. Spilman The Upjohn Company, Kalamazoo, MI 49001
Introduction- Atherosclerotic cardiovascular diseases are a major cause of morbidity and mortality. Aggresive therapy to decrease low density lipoprotein cholesterol (LDL-C) inhibits the progression of coronary atherosclerotic lesions, promotes regression of existing lesions, and results in a decline in the number of coronary events (1,2).Epidemiologic data consistently support the conclusion that coronary artery disease is also inversely related to high density lipoprotein cholesterol (HDL-C) levels (34. Therefore, the level of total or LDL-C must be carefully evaluated in the face of low HDL-C levels (5). Following a brief overview of current therapies for hypercholesterolemia (HC), this chapter will highlight recent advances in the development of new approaches to the treatment of HC and atherosclerosis. Primary emphasis will be placed on programs which are at or near the stage of clinical development, but a final section will also focus on current basic research that sdggests new ways to confront coronary artery disease. OVERVIEW OF CURRENT THERAPIES Approved treatments for HC have changed little since the last survey of this area in Annual Reports (6) and have been the subject of recent monographs (7,8).This section will deal only briefly with existing therapies to put current research on new approaches in an appropriate context. cid Seauesterants- Patients who prove refractory to dietary intervention are usually treated initially with bile acid sequesterants. New developments in this area related to improvements in potency and palatibility have been reviewed (9). Highlights include tablet formulation (1 0), resins containing amide functionality to augment the binding capacity of the ammonium groups (1l),and water-soluble polymers of reduced molecular weight (12).An alternative approach is inhibition of bile acid transporters with novel tethered cholic acid derivatives (13).
.
.
Nlcotlnates - Nicotinic acid was first prescribed for the treatment of HC over 25 years ago and
continues to be utilized despite its well-known side effects. Efforts to dissect the untoward side effects have involved both prodrugs and timed-release formulations (7,14).The only new analog with significant advantages is acipimox (5-methylpyrazinecarboxylic acid-4-oxide)which is effective at lower doses than nicotinic acid and encumbered with fewer side effects (15).
ProbucQl - Although originally developed as an antihypercholesterolemic (16). probucol's effects on serum C are modest and it is of interest today primarily as a result of its antioxidant properties. Evidence implicates oxidatively-modified LDL as a factor in the generation of the aortic fatty streak, from which atherosclerotic lesions develop (vide infra). Probucol has been demonstrated to retard the progression of atherosclerosis in Watanabe rabbits (17), and clinical studies have indicatedthat it protects human LDL from ex vivo peroxidation and reduces plasma levels of lipid peroxides (1820). Fjbrates - The fibric acids are employed primarily for the treatment of combined hypertriglyceridemia and HC (7). Although no clear structural definition of this class of compounds appears to exist, the most potent possess an a.a-dimethylacetic acid moiety attached by a 0-7carbon spacer to a
phenoxy moiety which may be further substituted. Gemfibrozil (1) enjoys a market edge over other fibrates and maintains nearly 30% of the total US hypolipidemic market. More active fibrates have been developed, most notably beclobrate (a). which is about nine times as potent as gemfibrozil
(21).
Copyright 0 1993 by Academic Press, Inc. ANNUAL R E P O R l S IN MEDI<.INAL
< HEMIVRY-28
L7
All rights o f reproduction
in
any form reserved.
Section IV-Immunology, Endocrinology and Metabolic Diseases
Hagmum. Ed
CI
2
1
..
HMG-CoA R d ~sebfHR) Inhibitors - The standard by which all new therapies for primary HC are now judged is represented by the inhibitors of the rate-limiting step in endogenous C biosynthesis. Virtually all of the members of this class of compounds are 3,5-dihydroxypentanoic acids (or the corresponding 6-lactones) further substituted with a lipophilic anchor that enhances binding to the enzyme. The number of analogs of the original lead in this area, compactin, has grown explosively in the last five years, and their development and SAR have been exhaustively reviewed (22,23). Clinical efficacy of the currently marketed inhibitors (lovastatin, simvastatin, pravastatin) is impressive: average reductions of serum LDL-C levels in patients with heterozygous familial HC range from 32 - 41% at doses of only 40 mg/day (24). Although chronic therapy has yet to reveal the serious side effects which were anticipated to arise from inhibition of C biosynthesis in the periphery (25, 26), current research is strongly focused on developing inhibitors that are selective for the liver, the primary source of endogenous C production (27). New entities of interest with regard to increased potency or hepatic selectivity relative to lovastatin are: CP-83101 (a) (28), CI981 (4) (29) and Bay-w-6228 (5) (30). SQ-33600(6) is particularly noteworthy in that the 5hydroxy moiety of the pentanoic acid fragment has been successfully replaced by a phosphinyl oxygen (31). is reported to inhibit HR with an C The macrocyclic factone I , of 0.1 pM in HepGP cells, is roughly equiptent with lovastatin at reducing HC in rats and represents an unusual departure from the standard mevalonate-mimic structure (32). The observation that an HMG-CoA thioether sulfoxide binds with areater affinitv to HR than the Dhvsioloaicalsubstrate thoester is intriguing and could be of importance in the design of future analogs (33)-
I
HoT30 OH
L
'
.
-
0
4
NEW THFRAPlES UNDFR DEVFI OPMFNT The undisputed success of current therapy for HC, particularly the HR inhibitors, has not deterred interest in discovering novel methods to reduce serum C or retard the progression of atherosclerosis. This section will detail those approaches that have identified discreet chemical leads possessing well-established biological activity. The majority of work has focused on inhibiting C biosynthesis at points other than HR and on altering the absorption of dietary C.
..
se IHS) Inhibitors - HS catalyzes the C biosynthetic step just prior to the reduction of HMG-CoA. SAR work on the original lead L-659.699 (k) established that manipulationof the lipophilic side chain (34)is detrimentalto activity, but that the p-lactone can be
Chap. 23
Atherosclerosis
Larsen. Spilman
substituted with an N-p-tosyl p-lactam (9,35). Subsequent analog work identified (in)as one of the most potent of these tosyi lactams with an IC, of 2.1 nM (36). A second class of inhibitors was recently isolated from fermentation broths. The most active component (JjJ had a modest ICWof 0.18 pM (37).
RO
s x P X
..
- The first three steps in the C biosynthetic cascade that are committed solely to sterol synthesis involve the conversion of farnesyl pyrophosphate (FPP) to lanosterol. The enzymes responsible are squalene synthase (SS).squalene epoxidase (SE). and 2,3-oxidosqualene-lanosterol cyclase (SLC). Inhibition of these enzymes is considered potentially advantageous over HR inhibition in that the biosyntheses of other isoprenoids essential for cell growth (e.9. ubiquinone) are not affected. SAR studies within a series of stable FPP analogs resulted in the potent competitive SS inhibitor (12) (IC, = 0.05pM), which proved to be two orders of magnitude more active than the closely-related compound illustrating the importance of the ether oxygen for efficient binding to the enzyme (38). Azabicyclo[2.2.2)octane inhibits rat liver microsomal SS with an IC, of 11 nM in the presence of pyrophosphate (39), but it is part of a series of compounds also reported to be 5-HT3antagonists, casting doubt on its clinical potential. A group of fungal metabolites was recently isolated that exhibits extremely potent inhibition of SS (40). Due to their virtually simultaneous discovery by two different groups, they are known collectively as either zaragozic acids or squalestatins. Zaragozic acid C (j.5J has an IC, of 9 nM against rat liver SS (41). Administration of the closely related analog squalestatin 1 subcutaneously to mice results in inhibition of hepatic C synthesis with an ED, of 0.2 rng/kg (42). Furthermore, is orally active in marmosets at reducing serum C (43). Cycloheptylaminomethylene-1,l-biphosphonic acid (YM 175) displays significant noncompetitive inhibition of rat liver SS and reduces hepatic C biosynthesis when administered subcutaneously to rats (44).
w,
a)
(m
CO,H
lene Fmxidase Inhibitors - Inhibitors of mammalian SE fall into two general classes: analogs of the physiological substrate squalene and substituted ally1 amines. Trinorsqualene alcohol (TNSA, 1z)was one of the first squalene mimics to effectively inhibit SE (IC, = 4 pM)(45). The 1.1 4ifluorosqualene is orally active in mice, as indicated by dose-dependent reductions in hepatic C synthesis (46). Roughly equivalent in vitro potency is seen with cyclopropylamine @) against rat hepatic SE (47). SAR studies with TNSA analogs confirm that the length and degree of unsaturation of the polyene is important for activity (48).
Hagmann, Ed.
Section N-Immunology. Endocrinology and Metabolic Diseases
1z R
R=CH2OH R =CH=CF2
R = C H , - N ~
H
Significantly more robust inhibitors evolved from SAR work on the potent fungal SE inhibitor terbinafine 0, which itself is only a weak inhibitor of mammalian SE (49). A dramatic reversal of this selectivity was achieved with NB-598 0, possessing an IC, of 0.75 nM against HepG2 SE (50) with no accompanying antifungal activity. Furthermore, NB-598 effects reductions in serum C and increases in serum squalene in dogs following oral administration (51). Analogs with improved in vifro potency have been reported (52).
Dad
2Q R’ = CHI, R2 = 1-naphthyl R’
= Et,
R2 3:
S
.. Cvcfase Inhibit- - The physiological substrate for SLC, 2,3-oxidosqualene (OS),
Didohas served as the template for a number of synthetic inhibitors. 2,3:18,19-Dioxidosqualene inhibits rat liver SLC non-competitively with an IC, of 0.1 1 pM (53). The location of the second epoxide is important, as indicated by the approximately 1000-fold lower potency of 2,3:22,23dioxidosqualene in the same assay. Simple addition of a methylidene substituent at the 29position of 2,3-oxidosqualene affords a potent (ICso= 0.5 pM) inhibitor of pig liver SLC (54). Of particular mechanistic interest was the observation that this inhibition was irreversible, providing evidence that carbocationic character at C-19 was developing during interaction with the SLC and subsequently being trapped by a biological nucleophile. The hypothesis that 0s actually cyclizes via discreet intermediates with carbocationic character (55) has inspired the synthesis of amino analogs of both squalene and partially cyclized squalene intermediates with the expectation that the charged ammonium group is capable of functioning as a stable bioisotere for a carbocation. A recent acyclic example is N-methyl-10-aza-10 , l l -dihydro-2,3-oxidosqualene with an IC, against hepatic SLC of 5 pM (56). Roughly equivalent activity is observed with the monocyclic derivative 0 , the most potent of a series of cyclic analogs (55). Saturation of the side chain of (22) totally attenuates its inhibitory activity. Evidently even an amide can suffice as a carbocationic mimic; ezadecalin inhibits hepatic SLC with an IC, of 0.7 IM,comparable to the older bicyclb amine (24) (55,57). The piperidine sulfone maintains significant hepatic SLC inhibition (IC, = 5 pM) within a structure markedly simpler than the squalene analogs (58).
..
sterol 14a-Demgtbvlase f L D m - Inhibition of the P-450 enzyme LDM has the potential to attenuate cholesterol biosynthesis by a dual mechanism: direct blockage of the conversion of lanosterol to C and indirect inhibition via a putative feedback mechanism whereby the accumulation of oxylanosterol intermediates (2s. and 21) regulates the expression of HR is reported to function as an ineversible inhibitor of rat liver LDM (59). The ethynyl lanosterol (60) while epoxide (?s) is a competitive inhibitor with a Ki of 0.6 pM (61). Lanosterols bearing
(a)
Chap. 23
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Larsen. Spllman &l
substitution at the 15-carbon have been targeted due to their expected resistance to elimination of the 14-methyl group as formic acid, the final transformation induced by LDM. Oxime (& is a lJ modest inhibitor of LDM (IC, = 55 pM), but is an effective oral hypocholesterolemic in hamsters (62). Reports of clinical hypocholesterolemic activity (63) with ketoconazole, an imidazole LDM inhibitor marketed as an antifungal, have fostered continued interest in the development of more potent analogs. A recent example is imidazole which has an ICx, against hepatic LDM that is less than one-tenth that of ketoconazole and is at least three times as potent as ketoconazole at reducing serum C in hamsters (64).
2.6 R = CHzOH,
R = CHO, 28 R = CH-CWCH,
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X=Hz X=Hz X = Hz
,
A-7,
X=H2
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&vlCoA:Chdesterol A c v W r a s e M ATUohibRors - It is now widely accepted that ACAT. an enzyme responsible for esterfying C with fatty acids, plays a key role in both the absorption of dietary C and in the accumulation of C within arterial tissue, two processes that are intimately involved in the atherogenic process (65). Research aimed at developing regulators of ACAT has been intense despite their disappointing clinical performance to date (66). The discovery and development of recent leads through 1992 have been comprehensively reviewed (67). A simple description of an ACAT pharmacophore is precluded by the diversity of reported inhibitors, but the majority to date have been lipophilic derivatives of amides, ureas. and imaazoles. Some of the most effective members of these classes which are currently in clinical development are CI976 (68), DuP-128 @) (69), RP-70676 cas) (70), YM17E (72). (71), and 447C88
&'p(CH&cH3
m)
OCH,
phIN%S-(CHJ-F
CH30
OCH,
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N H
F CH3
CH3
Attenuation of dietary C absorption has been readily achieved with a variety of ACAT inhibitors, regardless of their systemic bioavailability, as a result of inhibition of intestinal ACAT. Current research is strongly focused on developing inhibitors that act directly at the artery to determine if lesion development can be retarded. Although reports of arterial ACAT inhibition have appeared, the experimental results are often clouded by a concomitant reduction in serum C, which alone is sufficient to alter arterial ACAT activity and lesion progression. Cyclandelate (3,3,5-trimethyl
m
Section N-Immunology, Endocrinology and Metabolic Diseases
Hagmann. Ed
cyclohexyl hydroxyphenylacetate) was the first ACAT inhibitor to reduce aortic C without significant changes in serum C (73). Similarly, (3-976 decreases foam cell formation and C ester content in mechanically-inducedarterial lesions in micropigs at a dose which does not alter plasma LDL-C or HDL-C levels (74). Treatment of rabbits with pre-established HC with €5324 reduced aortic arch ACAT activity by 50% without decreasing plasma C levels below controls (75). KF-17828 is reported to accelerate the regression of HC in previously C-fed hamsters, implying a systemic effect more profound than simple withdrawal of dietary C (76).
(m
&z
Et
Several new compounds are of interest for their structural novelty. 2,3,5-Trimethyl-4hydroxyphenyl oleamide is a modest ACAT inhibitor (IC, = 0.25 pM), but is effective at reducing serum C in hypercholesterolemic hamsters and protects LDL from oxidation (77). Pantothenic acid derivative @), clearly designed to mimic the preferred ACAT substrate oleoyl-CoA, is a modest ACAT inhibitor (ICm = 1.2 pM) (78). Replacement of the oleyl portion with a urea affords F-1394 with dramatically improved potency (IC, = 12 nM) against rat liver ACAT (79). A recently isolated fungal metabolite, the cyclic hexapeptide beauvericin (I& z 3 pM), is one of the most potent ACAT inhibitors of microbial origin (80).
%'q
0 x 0 &@
'4N
0
..
0
H
0
H
(CH2)7CH=CH(CH&CH,
. . . - Beyond intestinal ACAT inhibition, the level of effort
directed towards inhibiting C or fat absorption has been relatively minor. A recent patent reports significant reductions in both TC and LDL-C in human subjects given high-viscosity water-soluble cellulose ethers, presumably as a result of inhibition of C absorption (81). P-Tigogenin cellobioside (CP-88818) lowers plasma LDWLDL C in a variety of both C-fed and chow-fed animals (82). The mechanism of action is reported to involve sequestration of both dietary and endogenous biliary C. Phosphorylcholine derivatives of C (e.g. effect reductions in dietary C absorption in mice of 60-80% by an undetermined mechanism (83). Cholesterol ester hydrolase (CEH) inhibition remains of some interest, despite controversy over its overall impact on C absorption (65). WAY-121898 (41) reportedly inhibits CEH with an C I , of 0.2 pM and reduces absorption of a single dose of C in normal-fed rats with an EDmof 10 mgkg (84). The pancreatic lipase inhibitor tetrahydrolipstatin (Ro-18-0647) is an inhibitor of C oleate absorption, but not free C, in rats (S), and continues to be the subject of analog work (86).
m)
. .
Antloxldants - It is now widely accepted that oxidative modification of LDL accentuates its atherogenicity by a variety of mechanisms and that agents acting to inhibit this process may retard the atherosclerotic process (87,88). This view is supported by studies with the antioxidants vitamin E (89) and probucol (vide supra) which clearly demonstrate a variety of antiatherogenic
Chap 23
Atherosclerosis
Larsen. Spilman 288
effects, and by epidemiological data linking coronary heart disease with susceptibility of LDL to oxidation (90). The search for more potent antioxidants has primarily uncovered compounds similar to probucol or vitamin E, i.e. lipophilic hindered phenols. Noteworthy are the spirocycle 0,which was 10-100 times as potent as probucol against LDL oxidation by Cu" or endothelial cells (91) and tocopherol analog (&) which reduced serum C in a dose-dependent manner in mice (92). The desmethyl probucol analog MDL-29311 is slightly more effective than probucol at inhibiting LDL oxidation ex vivo and retarding lesion progression in Watanabe rabbits, perhaps due to superior bioavailability (93). I t-Bu
Non-phenolic antioxidants have also been reported. The hydroxyurea (44) has an ICmof 0.5
pM for inhibition of endothelial cell-induced LDL oxidation (94). A 71% reduction in arterial lesion
(m,
area in C-fed rabbits was observed upon administration of N,N-diphenyl-phenylenediamine accompanied by significant protection of the LDL from oxidation (95). Phenothiazine & (J was approximately ten times more potent than probucol at blocking Cu+'-catalyzed oxidation of LDL (96).
POTFNTIAL NEW THERAPEUTIC APPROACHFS Several genes that are important in lipoprotein metabolism provide the opportunity for new therapeutic approaches. For example, a great deal is already known about the molecular mechanisms that regulate HR transcription (97) and degradation (98-100). It is now quite easy to develop systems to discover drugs that specifically regulate gene transcription. Such an assay has been developed for the HR gene (101). and assays could be developed for any gene of interest in the C biosynthetic pathway. Posttranscriptional down-regulation of HR has been reported with 15a-fluoro-lanost-7-en-3~-ol(102), 24(S). 25-oxidolanosterol (103). and ytocotriend (104). Investigations to study the regulation of expression of HS (105), SS (106). and SE (107,108) are also underway. LDL receptors play a crital role in the catabolism of C, and a great deal is known about the molecular regulation of LDL receptor expression (109-111). Therapeutic agents that stimulate the production of LDL receptors, and thus lower serum LDL-C, could be discovered in an assay employing the transcriptional regulatory elements from the LDL receptor gene (112). Transgenic mice that overexpress the LDL receptor have lower plasma C concentrations than normal mice (113), and do not develop HC when fed a high-fat diet (114). One caveat to this approach is the suggestion that up-regulation of LDL receptors in peripheral tissues could lead to foam cell formation and contribute to atherogenesis (115). Apolipoprotein E (apo E) is a ligand for the LDL receptor and an important component of several lipoproteins (116,117). Intravenous administration of apo E to rabbits causes a reduction in plasma C (118). Severe HC and atherosclerosis develop rapidly in mice that lack apo E (119,120). Transgenic mice that overexpress apo E have low lipoprotein C levels and are protected from developing HC when fed a high-C diet (121). These experimental observations suggest that increasing the expression of apo E could provide protection against atherosclerosis. The first step in the conversion of C to bile acids is catalyzed by cholesterol 7a-hydroxylase (C7H)(122). Drugs that stimulate C7H activity would be expected to increase C catabolism. Stimulating expression of the C7H gene might lead to a disproportionate increase in the expression of the LDL receptor (123). and thus a large decrease in LDL-C. Drugs that lower lipoprotein(a), a lipoprotein particle that has been shown to have a positive association with atherosclerosis (124-126), could be important for the prevention of atherosclerosis in those patients who have elevated levels. Since the incidence of coronary heart disease is inversely correlated with HDL-C levels, drugs
Section W-Immunology. Endocrinology and Metabolic Diseases
Hagmann. Ed
that increase H D L could b e very important agents for t h e prevention of atherosclerosis. Transgenic mice that express high levels of human apo A-I, the major protein component of HDL, have elevated HDL-C and do not develop fatty streak lesions when fed a high-fat diet (127). Another potential mechanism to increase H D L is to inhibit the activity of cholestetyl ester transfer protein (CETP), a plasma protein responsible for the transfer of cholesteryl ester from HDL to other lipoproteins (128). Humans who have a CETP deficiency have very high HDL levels (129). Non-insulin-dependent diabetes mellitus (NIDDM) is associated with hypertriglyceridemia, decreased HDL-C and atherosclerosis (130.131). Patients with NIDDM are insulin resistant; thus, drugs such a s pioglitazone that improve the sensitivity to insulin (132) may have a favorable impact on HDL-C levels.
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aaS
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w,
m,
m,
a, a,
a,
a
a.
Introduction- Atherosclerotic cardiovascular diseases are a major cause of morbidity and mortality. Aggresive therapy to decrease low density lipoprotein cholesterol (LDL-C) inhibits the progression of coronary atherosclerotic lesions, promotes regression of existing lesions, and results in a decline in the number of coronary events (1,2).Epidemiologic data consistently support the conclusion that coronary artery disease is also inversely related to high density lipoprotein cholesterol (HDL-C) levels (34. Therefore, the level of total or LDL-C must be carefully evaluated in the face of low HDL-C levels (5). Following a brief overview of current therapies for hypercholesterolemia (HC), this chapter will highlight recent advances in the development of new approaches to the treatment of HC and atherosclerosis. Primary emphasis will be placed on programs which are at or near the stage of clinical development, but a final section will also focus on current basic research that sdggests new ways to confront coronary artery disease. OVERVIEW OF CURRENT THERAPIES Approved treatments for HC have changed little since the last survey of this area in Annual Reports (6) and have been the subject of recent monographs (7,8).This section will deal only briefly with existing therapies to put current research on new approaches in an appropriate context. cid Seauesterants- Patients who prove refractory to dietary intervention are usually treated initially with bile acid sequesterants. New developments in this area related to improvements in potency and palatibility have been reviewed (9). Highlights include tablet formulation (1 0), resins containing amide functionality to augment the binding capacity of the ammonium groups (1l),and water-soluble polymers of reduced molecular weight (12).An alternative approach is inhibition of bile acid transporters with novel tethered cholic acid derivatives (13).
.
.
Nlcotlnates - Nicotinic acid was first prescribed for the treatment of HC over 25 years ago and
continues to be utilized despite its well-known side effects. Efforts to dissect the untoward side effects have involved both prodrugs and timed-release formulations (7,14).The only new analog with significant advantages is acipimox (5-methylpyrazinecarboxylic acid-4-oxide)which is effective at lower doses than nicotinic acid and encumbered with fewer side effects (15).
ProbucQl - Although originally developed as an antihypercholesterolemic (16). probucol's effects on serum C are modest and it is of interest today primarily as a result of its antioxidant properties. Evidence implicates oxidatively-modified LDL as a factor in the generation of the aortic fatty streak, from which atherosclerotic lesions develop (vide infra). Probucol has been demonstrated to retard the progression of atherosclerosis in Watanabe rabbits (17), and clinical studies have indicatedthat it protects human LDL from ex vivo peroxidation and reduces plasma levels of lipid peroxides (1820). Fjbrates - The fibric acids are employed primarily for the treatment of combined hypertriglyceridemia and HC (7). Although no clear structural definition of this class of compounds appears to exist, the most potent possess an a.a-dimethylacetic acid moiety attached by a 0-7carbon spacer to a
phenoxy moiety which may be further substituted. Gemfibrozil (1) enjoys a market edge over other fibrates and maintains nearly 30% of the total US hypolipidemic market. More active fibrates have been developed, most notably beclobrate (a). which is about nine times as potent as gemfibrozil
(21).
Copyright 0 1993 by Academic Press, Inc. ANNUAL R E P O R l S IN MEDI<.INAL
< HEMIVRY-28
L7
All rights o f reproduction
in
any form reserved.
Section IV-Immunology, Endocrinology and Metabolic Diseases
Hagmum. Ed
CI
2
1
..
HMG-CoA R d ~sebfHR) Inhibitors - The standard by which all new therapies for primary HC are now judged is represented by the inhibitors of the rate-limiting step in endogenous C biosynthesis. Virtually all of the members of this class of compounds are 3,5-dihydroxypentanoic acids (or the corresponding 6-lactones) further substituted with a lipophilic anchor that enhances binding to the enzyme. The number of analogs of the original lead in this area, compactin, has grown explosively in the last five years, and their development and SAR have been exhaustively reviewed (22,23). Clinical efficacy of the currently marketed inhibitors (lovastatin, simvastatin, pravastatin) is impressive: average reductions of serum LDL-C levels in patients with heterozygous familial HC range from 32 - 41% at doses of only 40 mg/day (24). Although chronic therapy has yet to reveal the serious side effects which were anticipated to arise from inhibition of C biosynthesis in the periphery (25, 26), current research is strongly focused on developing inhibitors that are selective for the liver, the primary source of endogenous C production (27). New entities of interest with regard to increased potency or hepatic selectivity relative to lovastatin are: CP-83101 (a) (28), CI981 (4) (29) and Bay-w-6228 (5) (30). SQ-33600(6) is particularly noteworthy in that the 5hydroxy moiety of the pentanoic acid fragment has been successfully replaced by a phosphinyl oxygen (31). is reported to inhibit HR with an C The macrocyclic factone I , of 0.1 pM in HepGP cells, is roughly equiptent with lovastatin at reducing HC in rats and represents an unusual departure from the standard mevalonate-mimic structure (32). The observation that an HMG-CoA thioether sulfoxide binds with areater affinitv to HR than the Dhvsioloaicalsubstrate thoester is intriguing and could be of importance in the design of future analogs (33)-
I
HoT30 OH
L
'
.
-
0
4
NEW THFRAPlES UNDFR DEVFI OPMFNT The undisputed success of current therapy for HC, particularly the HR inhibitors, has not deterred interest in discovering novel methods to reduce serum C or retard the progression of atherosclerosis. This section will detail those approaches that have identified discreet chemical leads possessing well-established biological activity. The majority of work has focused on inhibiting C biosynthesis at points other than HR and on altering the absorption of dietary C.
..
se IHS) Inhibitors - HS catalyzes the C biosynthetic step just prior to the reduction of HMG-CoA. SAR work on the original lead L-659.699 (k) established that manipulationof the lipophilic side chain (34)is detrimentalto activity, but that the p-lactone can be
Chap. 23
Atherosclerosis
Larsen. Spilman
substituted with an N-p-tosyl p-lactam (9,35). Subsequent analog work identified (in)as one of the most potent of these tosyi lactams with an IC, of 2.1 nM (36). A second class of inhibitors was recently isolated from fermentation broths. The most active component (JjJ had a modest ICWof 0.18 pM (37).
RO
s x P X
..
- The first three steps in the C biosynthetic cascade that are committed solely to sterol synthesis involve the conversion of farnesyl pyrophosphate (FPP) to lanosterol. The enzymes responsible are squalene synthase (SS).squalene epoxidase (SE). and 2,3-oxidosqualene-lanosterol cyclase (SLC). Inhibition of these enzymes is considered potentially advantageous over HR inhibition in that the biosyntheses of other isoprenoids essential for cell growth (e.9. ubiquinone) are not affected. SAR studies within a series of stable FPP analogs resulted in the potent competitive SS inhibitor (12) (IC, = 0.05pM), which proved to be two orders of magnitude more active than the closely-related compound illustrating the importance of the ether oxygen for efficient binding to the enzyme (38). Azabicyclo[2.2.2)octane inhibits rat liver microsomal SS with an IC, of 11 nM in the presence of pyrophosphate (39), but it is part of a series of compounds also reported to be 5-HT3antagonists, casting doubt on its clinical potential. A group of fungal metabolites was recently isolated that exhibits extremely potent inhibition of SS (40). Due to their virtually simultaneous discovery by two different groups, they are known collectively as either zaragozic acids or squalestatins. Zaragozic acid C (j.5J has an IC, of 9 nM against rat liver SS (41). Administration of the closely related analog squalestatin 1 subcutaneously to mice results in inhibition of hepatic C synthesis with an ED, of 0.2 rng/kg (42). Furthermore, is orally active in marmosets at reducing serum C (43). Cycloheptylaminomethylene-1,l-biphosphonic acid (YM 175) displays significant noncompetitive inhibition of rat liver SS and reduces hepatic C biosynthesis when administered subcutaneously to rats (44).
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a)
(m
CO,H
lene Fmxidase Inhibitors - Inhibitors of mammalian SE fall into two general classes: analogs of the physiological substrate squalene and substituted ally1 amines. Trinorsqualene alcohol (TNSA, 1z)was one of the first squalene mimics to effectively inhibit SE (IC, = 4 pM)(45). The 1.1 4ifluorosqualene is orally active in mice, as indicated by dose-dependent reductions in hepatic C synthesis (46). Roughly equivalent in vitro potency is seen with cyclopropylamine @) against rat hepatic SE (47). SAR studies with TNSA analogs confirm that the length and degree of unsaturation of the polyene is important for activity (48).
Hagmann, Ed.
Section N-Immunology. Endocrinology and Metabolic Diseases
1z R
R=CH2OH R =CH=CF2
R = C H , - N ~
H
Significantly more robust inhibitors evolved from SAR work on the potent fungal SE inhibitor terbinafine 0, which itself is only a weak inhibitor of mammalian SE (49). A dramatic reversal of this selectivity was achieved with NB-598 0, possessing an IC, of 0.75 nM against HepG2 SE (50) with no accompanying antifungal activity. Furthermore, NB-598 effects reductions in serum C and increases in serum squalene in dogs following oral administration (51). Analogs with improved in vifro potency have been reported (52).
Dad
2Q R’ = CHI, R2 = 1-naphthyl R’
= Et,
R2 3:
S
.. Cvcfase Inhibit- - The physiological substrate for SLC, 2,3-oxidosqualene (OS),
Didohas served as the template for a number of synthetic inhibitors. 2,3:18,19-Dioxidosqualene inhibits rat liver SLC non-competitively with an IC, of 0.1 1 pM (53). The location of the second epoxide is important, as indicated by the approximately 1000-fold lower potency of 2,3:22,23dioxidosqualene in the same assay. Simple addition of a methylidene substituent at the 29position of 2,3-oxidosqualene affords a potent (ICso= 0.5 pM) inhibitor of pig liver SLC (54). Of particular mechanistic interest was the observation that this inhibition was irreversible, providing evidence that carbocationic character at C-19 was developing during interaction with the SLC and subsequently being trapped by a biological nucleophile. The hypothesis that 0s actually cyclizes via discreet intermediates with carbocationic character (55) has inspired the synthesis of amino analogs of both squalene and partially cyclized squalene intermediates with the expectation that the charged ammonium group is capable of functioning as a stable bioisotere for a carbocation. A recent acyclic example is N-methyl-10-aza-10 , l l -dihydro-2,3-oxidosqualene with an IC, against hepatic SLC of 5 pM (56). Roughly equivalent activity is observed with the monocyclic derivative 0 , the most potent of a series of cyclic analogs (55). Saturation of the side chain of (22) totally attenuates its inhibitory activity. Evidently even an amide can suffice as a carbocationic mimic; ezadecalin inhibits hepatic SLC with an IC, of 0.7 IM,comparable to the older bicyclb amine (24) (55,57). The piperidine sulfone maintains significant hepatic SLC inhibition (IC, = 5 pM) within a structure markedly simpler than the squalene analogs (58).
..
sterol 14a-Demgtbvlase f L D m - Inhibition of the P-450 enzyme LDM has the potential to attenuate cholesterol biosynthesis by a dual mechanism: direct blockage of the conversion of lanosterol to C and indirect inhibition via a putative feedback mechanism whereby the accumulation of oxylanosterol intermediates (2s. and 21) regulates the expression of HR is reported to function as an ineversible inhibitor of rat liver LDM (59). The ethynyl lanosterol (60) while epoxide (?s) is a competitive inhibitor with a Ki of 0.6 pM (61). Lanosterols bearing
(a)
Chap. 23
Atherosclerosis
Larsen. Spllman &l
substitution at the 15-carbon have been targeted due to their expected resistance to elimination of the 14-methyl group as formic acid, the final transformation induced by LDM. Oxime (& is a lJ modest inhibitor of LDM (IC, = 55 pM), but is an effective oral hypocholesterolemic in hamsters (62). Reports of clinical hypocholesterolemic activity (63) with ketoconazole, an imidazole LDM inhibitor marketed as an antifungal, have fostered continued interest in the development of more potent analogs. A recent example is imidazole which has an ICx, against hepatic LDM that is less than one-tenth that of ketoconazole and is at least three times as potent as ketoconazole at reducing serum C in hamsters (64).
2.6 R = CHzOH,
R = CHO, 28 R = CH-CWCH,
A -8, A-8, A 8,
-
X=Hz X=Hz X = Hz
,
A-7,
X=H2
A -8,
X
&Q R = CH3,
HO
NOH
H
31
CI
&vlCoA:Chdesterol A c v W r a s e M ATUohibRors - It is now widely accepted that ACAT. an enzyme responsible for esterfying C with fatty acids, plays a key role in both the absorption of dietary C and in the accumulation of C within arterial tissue, two processes that are intimately involved in the atherogenic process (65). Research aimed at developing regulators of ACAT has been intense despite their disappointing clinical performance to date (66). The discovery and development of recent leads through 1992 have been comprehensively reviewed (67). A simple description of an ACAT pharmacophore is precluded by the diversity of reported inhibitors, but the majority to date have been lipophilic derivatives of amides, ureas. and imaazoles. Some of the most effective members of these classes which are currently in clinical development are CI976 (68), DuP-128 @) (69), RP-70676 cas) (70), YM17E (72). (71), and 447C88
&'p(CH&cH3
m)
OCH,
phIN%S-(CHJ-F
CH30
OCH,
Ph
(CH,),CH,
a R = NI
N H
F CH3
CH3
Attenuation of dietary C absorption has been readily achieved with a variety of ACAT inhibitors, regardless of their systemic bioavailability, as a result of inhibition of intestinal ACAT. Current research is strongly focused on developing inhibitors that act directly at the artery to determine if lesion development can be retarded. Although reports of arterial ACAT inhibition have appeared, the experimental results are often clouded by a concomitant reduction in serum C, which alone is sufficient to alter arterial ACAT activity and lesion progression. Cyclandelate (3,3,5-trimethyl
m
Section N-Immunology, Endocrinology and Metabolic Diseases
Hagmann. Ed
cyclohexyl hydroxyphenylacetate) was the first ACAT inhibitor to reduce aortic C without significant changes in serum C (73). Similarly, (3-976 decreases foam cell formation and C ester content in mechanically-inducedarterial lesions in micropigs at a dose which does not alter plasma LDL-C or HDL-C levels (74). Treatment of rabbits with pre-established HC with €5324 reduced aortic arch ACAT activity by 50% without decreasing plasma C levels below controls (75). KF-17828 is reported to accelerate the regression of HC in previously C-fed hamsters, implying a systemic effect more profound than simple withdrawal of dietary C (76).
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Several new compounds are of interest for their structural novelty. 2,3,5-Trimethyl-4hydroxyphenyl oleamide is a modest ACAT inhibitor (IC, = 0.25 pM), but is effective at reducing serum C in hypercholesterolemic hamsters and protects LDL from oxidation (77). Pantothenic acid derivative @), clearly designed to mimic the preferred ACAT substrate oleoyl-CoA, is a modest ACAT inhibitor (ICm = 1.2 pM) (78). Replacement of the oleyl portion with a urea affords F-1394 with dramatically improved potency (IC, = 12 nM) against rat liver ACAT (79). A recently isolated fungal metabolite, the cyclic hexapeptide beauvericin (I& z 3 pM), is one of the most potent ACAT inhibitors of microbial origin (80).
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. . . - Beyond intestinal ACAT inhibition, the level of effort
directed towards inhibiting C or fat absorption has been relatively minor. A recent patent reports significant reductions in both TC and LDL-C in human subjects given high-viscosity water-soluble cellulose ethers, presumably as a result of inhibition of C absorption (81). P-Tigogenin cellobioside (CP-88818) lowers plasma LDWLDL C in a variety of both C-fed and chow-fed animals (82). The mechanism of action is reported to involve sequestration of both dietary and endogenous biliary C. Phosphorylcholine derivatives of C (e.g. effect reductions in dietary C absorption in mice of 60-80% by an undetermined mechanism (83). Cholesterol ester hydrolase (CEH) inhibition remains of some interest, despite controversy over its overall impact on C absorption (65). WAY-121898 (41) reportedly inhibits CEH with an C I , of 0.2 pM and reduces absorption of a single dose of C in normal-fed rats with an EDmof 10 mgkg (84). The pancreatic lipase inhibitor tetrahydrolipstatin (Ro-18-0647) is an inhibitor of C oleate absorption, but not free C, in rats (S), and continues to be the subject of analog work (86).
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Antloxldants - It is now widely accepted that oxidative modification of LDL accentuates its atherogenicity by a variety of mechanisms and that agents acting to inhibit this process may retard the atherosclerotic process (87,88). This view is supported by studies with the antioxidants vitamin E (89) and probucol (vide supra) which clearly demonstrate a variety of antiatherogenic
Chap 23
Atherosclerosis
Larsen. Spilman 288
effects, and by epidemiological data linking coronary heart disease with susceptibility of LDL to oxidation (90). The search for more potent antioxidants has primarily uncovered compounds similar to probucol or vitamin E, i.e. lipophilic hindered phenols. Noteworthy are the spirocycle 0,which was 10-100 times as potent as probucol against LDL oxidation by Cu" or endothelial cells (91) and tocopherol analog (&) which reduced serum C in a dose-dependent manner in mice (92). The desmethyl probucol analog MDL-29311 is slightly more effective than probucol at inhibiting LDL oxidation ex vivo and retarding lesion progression in Watanabe rabbits, perhaps due to superior bioavailability (93). I t-Bu
Non-phenolic antioxidants have also been reported. The hydroxyurea (44) has an ICmof 0.5
pM for inhibition of endothelial cell-induced LDL oxidation (94). A 71% reduction in arterial lesion
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area in C-fed rabbits was observed upon administration of N,N-diphenyl-phenylenediamine accompanied by significant protection of the LDL from oxidation (95). Phenothiazine & (J was approximately ten times more potent than probucol at blocking Cu+'-catalyzed oxidation of LDL (96).
POTFNTIAL NEW THERAPEUTIC APPROACHFS Several genes that are important in lipoprotein metabolism provide the opportunity for new therapeutic approaches. For example, a great deal is already known about the molecular mechanisms that regulate HR transcription (97) and degradation (98-100). It is now quite easy to develop systems to discover drugs that specifically regulate gene transcription. Such an assay has been developed for the HR gene (101). and assays could be developed for any gene of interest in the C biosynthetic pathway. Posttranscriptional down-regulation of HR has been reported with 15a-fluoro-lanost-7-en-3~-ol(102), 24(S). 25-oxidolanosterol (103). and ytocotriend (104). Investigations to study the regulation of expression of HS (105), SS (106). and SE (107,108) are also underway. LDL receptors play a crital role in the catabolism of C, and a great deal is known about the molecular regulation of LDL receptor expression (109-111). Therapeutic agents that stimulate the production of LDL receptors, and thus lower serum LDL-C, could be discovered in an assay employing the transcriptional regulatory elements from the LDL receptor gene (112). Transgenic mice that overexpress the LDL receptor have lower plasma C concentrations than normal mice (113), and do not develop HC when fed a high-fat diet (114). One caveat to this approach is the suggestion that up-regulation of LDL receptors in peripheral tissues could lead to foam cell formation and contribute to atherogenesis (115). Apolipoprotein E (apo E) is a ligand for the LDL receptor and an important component of several lipoproteins (116,117). Intravenous administration of apo E to rabbits causes a reduction in plasma C (118). Severe HC and atherosclerosis develop rapidly in mice that lack apo E (119,120). Transgenic mice that overexpress apo E have low lipoprotein C levels and are protected from developing HC when fed a high-C diet (121). These experimental observations suggest that increasing the expression of apo E could provide protection against atherosclerosis. The first step in the conversion of C to bile acids is catalyzed by cholesterol 7a-hydroxylase (C7H)(122). Drugs that stimulate C7H activity would be expected to increase C catabolism. Stimulating expression of the C7H gene might lead to a disproportionate increase in the expression of the LDL receptor (123). and thus a large decrease in LDL-C. Drugs that lower lipoprotein(a), a lipoprotein particle that has been shown to have a positive association with atherosclerosis (124-126), could be important for the prevention of atherosclerosis in those patients who have elevated levels. Since the incidence of coronary heart disease is inversely correlated with HDL-C levels, drugs
Section W-Immunology. Endocrinology and Metabolic Diseases
Hagmann. Ed
that increase H D L could b e very important agents for t h e prevention of atherosclerosis. Transgenic mice that express high levels of human apo A-I, the major protein component of HDL, have elevated HDL-C and do not develop fatty streak lesions when fed a high-fat diet (127). Another potential mechanism to increase H D L is to inhibit the activity of cholestetyl ester transfer protein (CETP), a plasma protein responsible for the transfer of cholesteryl ester from HDL to other lipoproteins (128). Humans who have a CETP deficiency have very high HDL levels (129). Non-insulin-dependent diabetes mellitus (NIDDM) is associated with hypertriglyceridemia, decreased HDL-C and atherosclerosis (130.131). Patients with NIDDM are insulin resistant; thus, drugs such a s pioglitazone that improve the sensitivity to insulin (132) may have a favorable impact on HDL-C levels.
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