CA2115675A1 - Method for lowering blood lipid levels - Google Patents

Abstract

Method for treating an animal having an elevated serum lipid level. The method includes the steps of identifying an animal having such an elevated serum lipid level, and introducing into that animal a lipid-lowering amount of an AMP mimetic which stimulates AMP-activated protein kinase.

Description

211~67~
W093/03734 PCT/US92~0~8 ~

DESCRIPTION ~.. "
,~ .. ~ -, -.-.

This application is a continuation;in-part of U.S~
Serial ~o. 07/748,944, filed Au~ust ~3, ls9l~ which is a continuation-in-part of U.S. Serial No. 07/446t979,~iled `:.
5 Ja~uary 1~, 1990~ which i~ a continuation-in~part of U~SO -~
Serial No. 301!453, ~iled Ja~uary 24, 198~, and of U.S. -~
Serial No. 40~,107, fil~d September 15, ~89, which is a co~tinuation-in-part of UOS. Serial 301,2~2, ~:filed ;~-January 24, 1989. The co ~ ~nt of ~hese applications, including their drawings, are hereby incorporat~d by refer~nae.

~ This invention rela~Qs t~ methods for tr~ating ~ :~
;:~ animals having elevat~d serum :Lipid lev~ls, ~ 9~, animals :~
suXfering from hypertrigly~eridemia, hyper~holesterolemia, atherosclerosis or ob~sity.
~ , Ba~karound of the Xnvention Mild hyp~r~riglyceridemia, without much eleva~ion of~
cholesterc~l (~g, Type IV h~erï poproteinemia of 2 0 Fredrickson), i~ quite ~m~on in ~everal disorders , inclu~ling uncon~rolled diabetes mellitus~, rena} ~failure, systemic lupus erythematosus, alcoholism, obesity (Schaef~er & Levy, 312 ~L ~L~ 1300, l985~, and as recently repor~ed, AIDS (Grunfeld et al., 90 ~5 ~ 154, l99l~. Mild hypertri~lyc::eridemia can b~
aggravated by stress and ~arious medic:ati~sns, surh as estr~g~n, oral contrac~ptives, beta-b~;ocker~ arld thiazides~ Mi~tl hypertriglyc:eridemia~ is gerlerally du~ to an increa!;e in very low density lipoproteins ~VLDLs 3, 30 caused by ar~ increase in lipid synthesis, as well as a dee:rease in catabolism. The association of mild ,.:~..
SUBSTITUTE SHEET

W093/03734 PCT/~S92J 28 ~
211~675 hypertriglyceridemia with atherosclerosis is less well established than that of hypercholesterolemia with atherosclero~is (Schwandt, ll ~L__a ~ ~a_~ Y:o~._ 38~
1990). One rea~on for this may be the very high intraindividualvariabilityoffastingtriglyceridelevel~
(Brennex & Heiss, ll nhC__a~ o~ 1054, 1990).
Nevertheless, mo~t physician~ agr~e that mild hypertriglyceridemia should be tre~ted, particularly in diabetics (L~wis et al,, 72 ~ 3 ~3~, l99~
Mark~d to severe hypertriglycQridemi~ ic found in association with hypercholesterolamia in hyperlipoproteinemias type I, III, and V, and is most often due to an increase in both VLDLs and chylomicrons.
The associatiGn of an ~levation of total ~erum cholest~rol, particularly in combination with increased low density lipoproteins (LDL~ and d~crea~ed high den~ity lipoproteins (HD~), wi~h a higher incidence of atherosclerosis and coronary heart dis~ase i~ w ll establ.~hed. In recent years, increasing e~id~nce has also b2en present~d that corr~ction o~
hyperacholesterolemia decreases the subseguent incidence of coronary axtery disea~e (Ha~el, 81 3. Clin._Inv~est.
1653, ~988~.
Obesi~y is de~ined as ~ condition cau~ed by an exc~ssiYe amount o~ adipose ~issue. Present evidence ind~cat~s that obesity c~n be caused not only by an exces~ive intake of ~ood, but also by impairment of the mechanisms that control the normal pr~portion o~ adipos~
ti~sue (abuut 10~ for men and 25% for women). In accordance with the central position of the liver in the build-up o~ triglycerides, obesity is commonly accompanied with moderate to severe h~pertri~lyceridemia~
Current th~rapy of hypertriglyceri~emia and hyper~hol~sterolemia includes specific diets and four main cla~ses of drugs (Lopes-Virella & Colwell, 3 SUBSTITUTE SHEET

2115~
WV 93/03734 PCr/US92/06 cy/ 691, ~987; and Grundy, 319 New Enaland J. Medi ine 24; 1988).
For example, bile-acid seque~trants ( chole:;tyramine, colestipol) increase the fecal ex~retion 5 of bile acids ~ and lead to an increase in hepatic cholesterol catal~olism. Th~y are useful for treatment of isolated hyperchole-~;t~rolemia with increas~d LDL. One of theîr side ef ~Eec:ts is an inGr~ase in triglycerid~s .
Stimulators of lipoprotein lipase ( .gL., ~::lofibrate) 1~ act by incr~a~;ing the activity o:E~ lipoprol~in lipase, and thereby decrease the plasma VLD~ and tris~lyceride le~rel~
They are useful in tr~atment of hypertri~lyceridS~mia ~
although not all patients respond. In addition, they may induc:e an increase in total cholestProl ~ and in LDI, 15 cholesterol.
Inhibitors of triglyceride arld VL~L synthesis ~, niacin a~d s~mfi~rozil) act by decrea.Ring lipolysis in adipose tissue, and consequently th~ supply of fatty acids available ~or esterification into trigly~erides in th~
20 liver. Niacin (ni~otinic acid) d~cr2ases both serum trig~ycerides and LDL cholesterol, and may incr~a~e HDL
cholesterol. A major problem with niacin is intense flushing and pruritus due to prostaglandin release. It al so has an hyperglycemic effect which renders insulin adjustment in diabetics necessary. ~emfibrozil (a deri~ative of pentènoic acid) stimulates lipoprotein lipase and the synthesis of HDL. It is markedly efficient in lowering triglycerides and in incr~asi ng ~DL, but its ef f ect on LDL is ~ariable.
~nhibit~rs of HM~-CoA reductase (e.~., mevastatin, lovastatin) reduce cholesterol in plasma by inhibiting the rate-limiting enzyme (i~, HMG CoA reduct se) of cholest~rol synthesis. In addition, they r~du~e LDL, apparently by inrreasing the expression of LDL receptors on the surface of the liver cells. They al50 raise HDL in some patient~. . The efficacy of HMG-CoA reductase inhibitors in hypertriglyceridemia is ~nclear. HMG-CoA

SUBSTITUTE SI~EET

WO 93~037~d, PCrf US~2~ ~8 211~67~i ;

reductase inhibitors can produce muscle abnormalities in humans and corneal opat::ities in ~xperimental animals, but have not been found to produce ~erious side effects (Grundy, 319 . New Enql~and J. Medic:ine 24, 1g88) .

5 ~h~
This in~rention r~late~; to a novel means f or decrea~;ing the level of triglycerides, c::holesterol and other related lipids in human sr o~her animal plasma. The method is ~a~;ed upon the f ind ng that AI~ibosid~
10 monophosphate ( Z~P), and related analogs (which are structural mimetics o~ ~), are effective in rQducing the amount of synthesi~; of thes~ lipids. These compounds have their ef f ect by stiDIulating a protein kinase (~qP-activa~ed pro~ein ~kina~;e) which regulates the ac~ rity o~
î5 enzym~s that control the synthesis ~f fatty acids cholesterol, and a lip se which may in turn effec:t the action o~ lipolytic hormones. For exaIaple, th~ compounds may block the action of lipolytic hor~aones on aldipe~s~
ti~isue hormone sensiti~e lipa~e, and thereby preve~t ~Please o~ fa~ty a~ids ~or export to liver ~or re-es~erification into triglyceridQs. T~us, administration of these compounds ~o an animal having ele~ated serum lipid levels i~ ~ffective to low~r such lipid lev~ls, and thus is a treatment for hyperkriglyceridemia, hyp~rcholesterolemia and obesity.
Thus, in a fir~t a~p~ct, the inven~ion features a method for treating an animal, e a., one having an elevated serum lipid lev~l. The method may includ~ the step of identifying an animal having such an elevated serum lipid level. The method includes introduci~g into that animal a lipid-loweriny amount of an ~MP mimetic, or pro-drug (a comp~und which can be administered (e.g.;
orally) to generate an A~P mim~tic in vivo, ~ g , it includes compounds which upon administra~ion are ~cti~ated to produce the AMP mi~etic, e.q., esters which can bQ
cleaved, or ~ucleosides which can be phospoxylated or SUBSTITUTE SHET

WO 93/03734 2 1 1 ~ 6 7 ~ P~/US92/0682$

bases whi ::h can be phosphoribosylated to form the AMP~
mimetiC) of an AMP mimetic, which sti~ulates AMP-activated protein kinase.
The t rm "alevated" is meant to ~nc:ompass a level of 5 lipid which is abo~re an accepted normal range for that lipid in the animal, or which is krlown to be associated with a pathologi~ proce~s. 5uch level~; can be measured by any standard means, ~or ~xample ~ they can be measur2d chemically, bioc:hemically, or everl by s~udy of tha 10 sy~ptoms of an animal which may ref lect an eleYate~ lipid level . Suc:h symptoms may incIude di~;orders whic:h are commonly associated with elevated lipid levels~ such as diabekes mellitus, renal failure, atherosclerosis, heart dis~ase , str~ke , etc ., as disclassed above . Thus , to the 15 extent that an animal may nct be :specifically dia~nosed as ha~ring an elevated lipid level, it is appropriate in ~his inventiorl to t reat animals which have a ~igni~icant potential of ha~ing such elevated lipid ~levels. Thus, th~
: term "~i~entifying'~ includes i.dentifying th~se animals 20 which have su::h a si~7nif~c:arlt po~ent:ial. Those skilled in the art will recognize that the phra~;e "signii~icant poten~ial" includes those disorders which are co~ only : recogniz d by those skilled in the art as being a~sociated with el~vated lipid levels. For example, it can bc conc~uded that an ele~ated serum lipid leveI is pres~nt in ob~se persons (i.e.~ those having ~xcess ad~pose tissu~
or those with the effects of elavated lipi~ , those having atherosclerosis or atherosclero~i5~related complications, such as transient ischemic attacks, strokes, hear~ attacks, angin~, and peripheral vascular disease~
By ~'lipid" is meant to include any of a large number of lipids pr~sent in th~ serum of an animal, including ~as discussed above~ but not limit~d to chole~terol, triglycerides, lipoprotei~s, low density lipoproteins, very low density lipoproteins, and chylomicrons.

SUBSTITllTE SHEFI

W093/03734 PCT/US92/L !8 2115 67~

The method for treating includes introducing the desirsd AMP mim~tic or pro-drug by any standard me~hodology, including transdermal, injectio~ i~to muscle tissue, or blood stream, or by oral or other parenteral administration.
A "lipid-lowering amount1' includes an amount which is effective over a period of several hours or day~ to lower the sQrum lipid l~vel in a way which can be detected either chemically, biochemically, or by a change in the appearance or symptoms of the patient~ That is~ ~'lipid lowering" means a lowering of the le~rel of lipid in a clinically ~ignificant ~anner, well known to tho of ordinary skill in the art~
AMP mimetics or pro-drugs are well known ~o those in the art a~d include, ~, AIC~riboside (5~-amino-4-imidazolecarboxamide riboside), AICAribotide (ZKP), and analogs thereof, and any pro-drugs which can b~ used to produce such AIC~riboside, Z~?, and analags thereof, within an animal body, or tubercidin (4-zmino~ D~
ribofuranosylpyrrolo~2,3-d]pyrimidine), tubercidin bas~, tubercidin monophosphate, and prodrugs thereo~, whi~h can produce AMP mimetic~ within a body of the animal being treated. For example, tho~e pro-drugs and related analogs described in PCT Application WO 90jO9163, published August 23, 1s~o, are potentially useful in this invention~
Such mimetics can be synthe~ized by the methods described in this publication. By "bas~" is meant a compou~d which when phosphoribosylated i~ a nucleotide and s~rves as an AMP mimetic.
AMP-mimetics or pro-drugs whi~h are suitable for lipid-lowering within an animal can b~ re.adily identified by any standard biochemical test, ~g~, drug phaxmacokinetics, measurement of lipid level5 in biological fluids, or lipid metabolism, either in YiVo or ~E_~ZLE}~ gxamples of such te~ts ar~ described below.
Potentially useful AMP mimetics or pro-drugs are used in such tests, and those useful in this invention pro~ide SUBSTITUTE SHEET

W093/0373~ 2 ~ 7 ~ pcT/us92/o6x28 results similar to or be~ter than AICAriboside. Other examples of such tests are prvvided by Davie~ e~ al~, 186 Eur. J. Biochem. 123, 1989 and Carling et al., 186 Biochem. 129, 1~89. Generally, in in vitro tests it is determined whether the AMP mimetic or pro-drug enhance~
enzymatic activity of an AMP-activat~d protein kinase.
Thus, the pro-drugs and related drugs, such as th~se described in PCT 90l09163, can be readily sereened to determine whather th~y are useful in the method o~ this invention .
AMP-activated protein kina~e~ ~re well known to those in the ar~. For example, one embcdiment is desGrib~d in nume~ous pub:Lications by ~r. Hardie and his colleagues, see, ~lardie et al., 14 ~ 20, 1989, Munday et al., 175 ~y~ Lc~ 31, 1988; Davi s ~t al., 187 Eur._J. Biochem~ 183, 1990; Sim and Hardie, 233 r~ s~ 294,:1988; ~unday et al., 235 ~Y~ ~
144, 19$8; Carling et ~ 186 EurO J. Biohem. 12g, 1989;
and Davies et al., 186 Eur. J- E~5E~L 123, ~989 This inv~ntion provides a simple way in which several lipid synthesis pathways can be simultaneously inhibited.
This inhibition ca~ be produced by administration of a single drugl as discussed aboYe, and thus ha. significant advantages over prior art me-thods of lowering lipid synthesis which reguire administration of several drugs.
In addition, the inhibition by a drug oP this invention al~o causes inhibition of hepatic synthesis of triglycerides, which had not to date been possible.
The method of this invention is advantageous since it specifically inhibits activity of liver-specific pr~teins, and has little or no effects in certain other tissues, such as cornea and muscle tissue~. The drugs are also ad~antageous since they control both lipid and chol~sterol biosynthesis, and are potenti~lly far more potent than existing drugs.

SUBSTITUTE SHEET

W093/03734 P~/US92/l !8 21i~6~

Other features and advantages of the invention will be apparent from ~he following description of the pref erxed embodiments thereof, and f rom the claims .

erred Em~
The drawings will f ix~;t b:rief ly b~ de~;cribed .

Drawinqs Fig. 1 is a . chematic representation of two lipid synth~sis pathways in animals;
Fig . 2 is a graph showing the ef ~ect of AICAri~oside 10 on synthesis of fatty acids ~rs:~m endogenous sub~trates;
Fig. 3 is a graph showing the dose effect of AICAriboside s:n synthesis of fatty a::ids from glucose;
Fi~ . 4 is a graph showing ~h~ dose ef ~e t on AXCP.riboside on synthesis of fatty acids from lactate;
Fig . S is a graph showing the~ ef f ect of ~ Ariboside on synthesis o:E fatty acids from leu ine;
Fig. 6 is a graph showing th~ effect of AICAril:~oside on synthesis of fatty acids from 2 ketoisoc:aproate;
Fig. 7 iB a ~raph showin~ th~ effect of AIC~riboside on acetyl-CoA carboxylase activity in isolated hepatocytes;
Fig. 8 is a graph showing the ~ffect of AICAriboside on incorporation of tritiated water into lipids; and Fig. 9 is a graph showing the effect of AICAriboside on the non-saponifiable lipid fraction.
Fig. lQ is a graph showing the effect of IP injection of AIC~riboside (500 mg/kg) vn rat liver HM~CoA reductase activity.
Figs. 11 and 12 are graphs sh~wing AMP-stimulated protein kinas~ activi~y relative to Z~P and ~MP
concentrationi~
Fig. 13 is a hi~tog~am showing ~P stimulated protein kinase activity by AMP and other reagents.

SUBSTITUTE SHEET

2 ~1 5 6 1 5 WO 9~fO3734 PCr~U~9~

Lipid Svnthesis Pathways ~Re~eerring to Fig. 1, the following i5 a 3:srief description of the vari~us biochemi ::al pathways, and enzym~s involved therein, which are relat:ed to the utility 5 of this inVention.
In humans, triglycerides are ~ynthesized mainly in the liver , frc:m ~Eatty acids either newly synthesized by the liYer i~self, or coming ~rom lipolysis in adipose tissue. Hepatic synthasis of fatty acids includes the 10 conversion of various substrates into acetyl-CoA, ~, glucose and other monosacc:harides, such as fr~ctose and _. galal:tose, lac:tate, pyruvate, ketogenic: ami~o aeids and l:heir keto deriYatives. Acetyl CoA is c:onverted into malonyl~CoA by acetyl-Co~ car~oxylase. ~alonyl-Co~ is 15 sub~ tly e~ongated into loxlg-chain f~tty at:y~ CoR, which is esterif ied into tri~lycericles W~t}l glyc:ero~

3-phosphate. Theæ~ triglycerid@s ~ are exported oward peripheral tis~;ues, including adipose tissue, in the form of v~ry-lowd~nsity lipoproteins. ~ipoprotein lipa~
~0 l~cated at t~e outer surface of peripheral cells, hydrolyzes the triglycerides to fatty acids and glycerol which can be taken up by the cells.
~ cetyl-CoA carboxylase is the rate-limiting enzyme of h~patic fatty acid synthesis. It~ acti~ity is controlled by citrate, which is stimulatory, and by long-Gh~i~ fatty acyl-CoA, which is inhibitory. ~cetyl-CoA carboxylase i5 moreo~er regulated by reversible phosphorylation/
dephosphorylation. The dephosphorylated form o~ ~cetyl-CoA carboxylas~ is active, and the phosphorylated form inactive. A ~ariety of protein kinases, including cAMP~
dependent protein kina~e and protein kinase C, are able ~o phosphorylate acetyl-CoA car~oxylase in vltrQ. However, strong evidence has been presented tha~, in intact cells, AMP activated prstein kinase phosphorylates and 3 5 inactivates acetyl-CoA carboxylase (see, H~rdie et al., 14 Trends Blochem._Sci, 20, 198g)~

SUBSTITUTE SHEET

~V093/03734 P~T/US9~ 28 211~7~

Cholester~l is synthesized by nearly all tissues in humans, but the liver makes one of the largest contributions to the body ' s choles~erol pool . In the pla~ma, cholesterQl is transported mainly in the fo~m of 5 low-density lipoproteins tLDL), which result from the conversion of ~LDL after th~se have relsa~ed their triglycerides to peripheral tissue~. Re~erse transpo~t of cholesterol, i.e., from ti~sues back to the liver for ca~abolism and ~xcretion, is postula~ed to occur via high~
lo den~ity lipoprotein~. Similar to that of fatty s6ids, synthesis Df cholesterol also proc~eds from acetyl-CoA, which is first converted into acetDace~yl-CoA, and thsreafter into 3-hydroxy-3 methylglutaryl-CoA (HMG-CoA)~
The subsequent reduction of H~G-CoA into mevalonate, 15 ca~alyzed by ~M~-CoA reductase, is tha rat~ limiting step of ch~lesterol synth~sis~ ~ ensuing 9 step s~quence leads from mevalonat~ tQ cholest.ervl. HMG-C~A reductase is located inside the smooth endoplasmic reticulum.
Expressisn of the enzyme is regulated by the cholesterol 20 level: excess cholesterol deereases the synth~si~ of the protein and increases its degradatio~. Similar to acetyl~
CoA carboxylase, ~MG CoA reductase is regulated by phosphorylat.ion~dephosphoryl~tion. Purified HMG~CoA
reductase can be phosphorylated and inactivated in_yitrv 25 by se~eral protein kinases, including protein kinase C, a Ca~/calmodulin-dependent protein kina~e, and AMP-activated protein kinase (~ardie et al., 14 Trends ~iQ~hem. Sci. 20, ~98g). Recently, evidence has been presented that AMP-activated prot~in kinase phospho~ylates HMG-CoA reductase ~ y~y~ (Clarke & Hardie, 9 EMBOJ Qurnal 2439, l99o~.

AMP~a~ _Protein Kinase A~P-activated protein kinase (A~P-PK) belong~ to a group of enzymes which~ u~ing ATP, phosphorylate proteins at sexine or oe~sionally threcnine residues. Best known in this ~roup are cyclic AMP-dependent protein kinase (cA-PK~, Ca~/calmodulin-dependent protein ~inases, and protei~

SUBSTITUTE SHEET

WO 93~03734 211 5 ~ ~ 5 PCI/US92/06828 11 , kinas~ C. Protein kinases are componerlts of the trarlsduc:tion mechanism~; whereby hormones and other fac:tors regulate physiologic:al functlons. Their ac:tion elicit:s conf ormational change~; ~ha~ modify either the c:atalytic 5 activity of enzyme~; or the flmc~ion of other regulatory proteins . T~e conf ormational ~hanges indu~ed by phosphorylation can b~ rP~rersed by pro~ein phs~sphatases, of which several type~ have been c:haracterlzed in r~cent year~
AMP-PK phosphorylates three enzyme~, each altalyzing a key regulatory st~p of lipid metabolism: ~13 aeetyl-CoA
carb~xylas2, the rate-limiting s~ep of f atty acids : :
synthesis, which is IllD~t ac:t~ ve in liver; ~ 2 ] HMG-Co~
-reductase, the f irst cc)lamitted s ep of cholesterol 15 synth~sis, also predominarlt1y 1ocated in li~r; and t 3 ]
hormone-sen~;itive 1ipase, the enzyme that contro1s the r~lease of f atty a ::ids f rora 1:rig1ycerides in adipose tis~ue (Hardie et al. ~ 14 ~E~b~i 20~ 1989) ~
In accordance with its role in lipid metaboli~;m, the 20 highest acti~ities o~ PK are found in 1iv~:r and lactating mammary g1and, which are very active in both fatty acid and cholesterol synthesis ~Davies et al. ~ 186 _~o~ 123, 1989). Lower activities exi~t in tissues whi ::h hav~ an active fatty acid metabolism, 25 name1y~ adipose ti~ue, adrena1 cc~r~x, 1ung, mae~roph~ge~
and heart. Th~ tissues with the 1Owest ac:tivity of AMP PK
are brain and rnuscle, two tissues in which rates of lipid synthe~ ; are very low, at least in adults.
AMP-PK ha~ been purified 4800-fold from rat liver, to 30 a spec:ific acti~ity of 1.25 ,umol/min/mg (C rlirlg et al., 186 Eur. J. Biochem. 129, 1989). Although the preparation did no~ disp1ay a sing1e band on an electrophoretic gel, the f act that its specif ic activi~y was compara~1e with that of other protein kinases s~aggests th~t it was 3 5 approac:hing homogeneity . Mo1ecu1ar mass of subunits is es~imated at 63 kDa, and of ~he ho1Oenzyme at 100 ~ 30 kDa, indis::ating that native AMP-PK might be a dimer~

SUBSTITUTE SHEET

WO 93J03734 P~/~JS92/~
211~7 ~

Most inYestigations of the catalytic properties of AMP-Pg ha~e b~en perf ormed with ac:etyl-Co~ carboxylase as substrate. In the absence of ~ PK has a Km of 86 ~M for ATP and of 1., 9 ,uM fvr acetyl-CoA carboxylase. AMP
5 increases Vmax 3 - tc) 6-f old, without signif ic:antly modifying ~n f or either ATP or acetyl-CoP~ ::arboxylase .
The sensitivity to ~iP depends on he concentration o ATP: at O ~ 2 mM ATP, half maximal stimulation by AMP is observed at 1.4 ,uM; at the near physiologica? ATP
10 c~nc:en~ratioll of 2 mM, hal~-maxiDIal ~3ti~ulation requires 14 ~ Some AMP analogu~s are reportedl ineffectiv~ as _~ substituting for AMP ~Carlin-~ et al., 1~6 Eur._J._Biochem.
1~9, 1989)~ ~oweverr 8-bromoadeno~ine 5-monophospha~e is a weak stimulator at low concentrations~ but it is an inhibi~or at high con~entra1:ions. The regulatory co~Gentrations of AMP are an order of magnitude lower than those measured in acid ex~raGt~; of liver. However, the latter have o~ten b~en claimed to be artefactual;
resulting from degradation of ATP. Calculations based on equilibrium constants and nuclear magnetic resonance studies have led to estimates that the free concentration of liver AMP may be around 1 ~M. Any incx~ase in AMP
within this range would thus po~ently stimulate A~P-PK, ~ MP-PK has been shown ~o inactivate acetyl-CoA
car~oxylas2 in a cell-~ree sy ~em by phosphorylating Ser-79 of the prot~in (Hardi~ e~ al., 14 Trends Biochem. ~SGi 20, 1989~. Addition of glucagon to intact cell.~, namely isolated rat hepatocytes and adipocyte~, also results in the phosphorylati~n of Ser-79. In a cell-free ~ystem, however, cA PK, which as a rule mediates the actions of hormones that elev~te cyclic AMP, pho~phoryl~tes a different serine, namely Ser-77. This suggests that, in contra*~ction with generally ~ccepted knowledge, the action of glucagon on acetyl-C~A carboxylase is not mediated by cA-PK but by AMP-PK. Recently, to resol~e this co~tradiction, it has been proposed that, 3~L~L~
cyclic AMP and cA-PK exert their inactivating effect on SUBSTITUTE SIIEET

WO ~3/03734 2 1 1 5 ~ 7 ~j PCI/VS92/068~8 acetyl-CoA carboxylase not be a direct phosphorylatîon of the enzyme, but by an indirec:t mechanism, namely inhibition of the dephosphorylation of Ser-79 by protein phosphatase 2~ (Cohen ~ Hardies 10~4 Bioc._BloP. Acta 292, 199~
AMP PK inactivates ~M&-CoA reductase by phosphorylatin~ Ser-872 of the enzyme (Clarke & Hardie, ~
EM80 Journal 2439, 1990~. Thi~ phosphorylation is most likely re.ponsible for the inactivation of ~MG~CoA
lo reducta~e ~nown t~ o~cur when special precautions (e.q~
fr~eze-clamping) are not taken to avoid a ri~e of the concentra~ion of AMP after remoYing liver tissue~
AMP-PK phosphorylates Ser-565 of hormone-sensiti~e lipase. This phosphorylation inhibits subsequent phosphorylation and activation of hoxmone~sensitive lipase by cA PK (Gar on ~t al., 179 ~E _ 3~ h~ 249 , 1989 ) Phosphorylation of ho ~ one-sen~itive lipa~ by AMP-PK
might thus block the ac~ion of lipolytic ho~mones (and rel~ase of free fatty acids and glycerol from fa~ cells) which act by way of cyclic AMP and cA-PX.
AMP-PK ha~ also b~en reported to be itself regulated by phosphorylation, which activate~ the enzym~, and by dephosphorylation which lnactivates the enzym~. Nan~molar concentrations of f~tty acyl~C~A were shown to stimulate the ~MP-PK kinase', thus activating AMP-PK. Since the latter activation will result in inac~i~ation of acetyl~
CoA carboxylase, it provides a mechani~m whereby fatty acyl-C~A can exert feed-back inhibition on fatty acid synthesis.
Taken togeth~r, the studies of the ~ P-PK system indicate ~ha~ ik plays an important role in regulating the level~ of fatty acids and chole~terol in the body. That A~P-PK acts on both acety~.oCoA carbo~ylase and HMG-CoA
reductase most likely explains why hepatic f atty acid and cholester~l ~ynthesis are regulated in parallel in several situations (e.q., both synthetic pathways peak at the same SU~STITUTE SHEET

4 PCI/US92/~ ,28 2 l~6~

time of the day, both are inhibited by diets high in polyunsatllrated f atty acids ) .
As desc:ribed in de~ail below, we have discovered that the addition of the nucleoside, AICAriboside, to 5 suspensions of isolated rat hepatot::ytes provokes an inactivatioll o~ both acetyl C~ A carboxylase and HMGCoA
redut:tase ~ AICAriboside is ef f icient~ y c~nverted by phosphorylation intc~ the correspondil~g nuc:leotide, AIC:Aribotide or ZMP, in iss; lat~d rat hepato::ytes (Vinc~nt . .
10 et al., 40 ~k~ 125~ , 1991 , data not sho~n3 arld in vivo (data not shown). (ZMP can alss~ be formed from AICA
_" base administratic:n followed by L~i~
phosphoribosylation. ) Taken together these data indicate that AMP~actiYated protein kinas~ can be activated by ZMPp 15 which dlisplays striking structural .similarities with ~.
This discovery of ph~nnacological ~;timulators of AMP-activat~d protein kinase provi.des an un~que means to decrease concomitantly the synth~-si~ of fatty acids and o~
cholesterol in the liver~ Th~i discovery thus opens new 20 per6pectiv~s for the treatment of. hypertriglyceridemia and hypercholesterol~mia ~nd particularly of t:heir combined trea~ment, which often occurs and remains difficult to manage with presently available drugs ~Havel, 81 JO Clin.
Inves . 1653, 1988 ) .

2 5 ~:xamples The follc~wing are specifit: non limiting examples of the ef f ects oî AICAribo~ide on synthesis of lipids .

Methods Experiments were perf ormed in isolated hepatocytes 30 prepared from normally fed ra~s. Measurement~ of fatty a ::id synthesis were performeid by incubation of the c2115 wikh 3H2û as described by Harris, 169 Arch. 13iochem.
BioPh~s O î~8, 1975 . Following irOcubation ~ two fractions were prepared. ~1~ A saponifiable lipid ~raction, which 35 contains the fatty acids ~both the free fat~y acids and ,::

SUBSTITUTE SHEET

WO S~3/03734 2 1 1 5 6 7 5 Pcrtus92/06828 those deriYed from the hydrolysis of triglycerides by the procedure). (2) A nc~nosaponifiable lipid ~Eraction, which contains mainly cholesterol, but also o~her steroids, terpenes, pros~agla~dins, also the ketone bodies ~minimal in the Eed ~tate~
In this method, addition of 3H20 result~ in the labelling of NADPH . Utilizativn of the latter by f atty acid syrlthetasQ re~;ults in the formation of labPlled fatty acids and triglycarid~s. Besides entering fatty acid synthesis, 3H2O can also enter the biosynthesis of cholesterol (at the level of H~qG-Co~ reductase).

Exam~le~ Effect of_AICAribosideg~

In isolated ~epatc~aytes from fed ra~s, fat:ty acid synthe~:is can proc:eed from endogenous substrates, glycos3en, and ketogenic amino acids. This endogenolls : . -f atty acid synthesis is completely inhibited by SOO ,uM
AICAriboside ( Fi~ . 2 ) 2 0 with qlus::ose .
AICAribc~side inhibits fatty acid synthesis from 15 mM
glllc:ose in a dose-dependent fashion (Fig. 3). Half maximal inhibition is obtained with about 50~LM ~:
AICAriboside .

~ :~:
~e AIC~riboside inhibits fatty acid sy~thesis from lactate 10 mM/pyruvate 1 mM (Fig. 4). Fatty acid '~
synthesis from lactate/pyruvate seems to be slightly less sanisitive to AIC~riboside ~han that from glucose (half-maximal inhibition with about 75 ~ AICAriboside). ~;~

SUBSTITUTE SHEET . ~:

W093/03734 P~T/US92~ ,2~
2 1 1`~

with ketoqenic amino acids Ketogenic amino acids enter f~tty acid gynth~is at the ~evP.l of ac~tyl-CoA, thus bypassing the transport of pyruvate into the mitochondria, and the enzymes pyru~ate dehy~rogena~e and pyruvate carboxylase. In vivo, ke~o~enic amino acids are thought to be first deaminated in muscle, and to be transport~d ther~after to the liver in the form of ketoaci~s. Thu~, fatty acid synthesis from 13 l~ucine was compared with th~t from its transam~nation product, 2-ketoi~ocaproic acid. As shown in Figs . 5 and 6, fatty ~cid synthesis with both substrate~ is compl~tely inhibited by AICAriboside 500 ~M.
, ~

~g~yoL5~L bL:b ~Yl~
AcetyI-CoA carboxylase is the limiting step of fatty acid ~ynthesis. It is int~rcon~e~tible by phosphory~a~ion/depho~phorylation, the acti~e ~orm being ~:.
dephosphoryla~ed. ~etyl-CoA carbo~yla~e is activate~ by - :
a dephosphorylaking phosphatase alnd inactivat~d by s~eral kinases (including cAMP-dep~ndent protein kinase, under ~.
the influence of glucagon3. In add~tion, inacti~ation of acetyl-CoA carboxylase by an AMP-activated protein kinase ~:
has b~.en r~ported by Dr. Hardie's group. ~ig. 7 shows `~
25 tha~ acetyl-Co~ carboxylase (assay performed by th~ method :~
o~ BijleYeld &i Geelen, 91~ Bioc. Bio~. Acta 274, 1987~ is .
inactivated by the addition of AICAribosid~ 500 ~M. This suggests that ZMP act~ at the level of the AMP-activated .
protein.

Example 6: Effects_on the incorporation of~3H2 Q in the non-saponifiable li~d fraction .
In all experiments described above, incorporation of 3H20 also occurs in the non-saponifiable lipid fraction, ~ ;
~lthough to a smaller ex~ent than in the saponi~iab~e 35 lipid fraction. In all experiments al~o, AICA riboside .

SUBSTITUTE SHEET

WO 93/03734 2 1 1 ~ 6 7 ~ PCI/US92/06828 inhibits the inc:orporation of 3H20 in the non-saponifiabl2 frat:tion,. Figs. 8 and 9 illustra~e results in an experimen~ with 15 mM glucose. The e data indic:ate that the synthesis of cholesterol is inhibited by AICAriboside.
5 Hardie has shown that AMP-activated protain lcinase inac:tivates H~ CoA reductas~. Si~ilarly, Z~$P, by activating ~ activat~d protein kinase, inac:tivate~ both a~etyl-CoA carboxylas~ and ~MG-CoA reductase, the limiting enzyme~ o:f, resE~ectively , f atty acid and cholesterol 10 syn~hesis.

Example 7: _ Ef~ect of AICAriboside _on_ ~actiV~tV _o~

~ G~CoA reduc~se is the limiting step of ::holesterol synthl3sis., It is interconvertibl~ by 15 phospborylatlQn/dephosphorylation, the active form being depho~;phorylated. H~G-CoA reduc:tasa is acti~ated by the dephosphorylating phosphatas~s, protein phosphatase 2A and 2~, and inaGtivated by several prot~in kinases. These includ~ Ca~/calmodulin-depend~nt protein kinase, protein . .
20 ~ kinase C, and as also descr~bed by Dr. Hardie's group.
AMP-acti~ated protein kin2~se~ Fig~ 10 shows that ~he -~ ~:
intraperitoneal Injection of AIC~bosida at the do~;e of 500 mg/kg inackivates HMG-CoA reductase (assay performed . . .
by the method of Easiom & Zammit, 13iocheml. J. 220: 733-~ &
739-45, 1984) i~ rats in vivo. This suggests that ZMP ~ ~:
acts at the level of the AMP-activated protein kinase.
.~.

oeei 11~
Dr . Hardie and co-workers ( Davies et al ., Eur . J .
Biochem. 186: 123-8, 1989) have set up a specific and ~ ,:
sensitive assay of A~lP-a~ ivated protein kir~asie. It is based on the incorpora~ion of radioactivity from ty-32PJATP
into a 15-~mino ac:id peptide, termed the SAMS peptide, designed after the se~uence of acetyl-C:oA carboxylase `:~
surrounding Ser79, the site which is phosphorylated SUBSTITUTE SHEET

WO 93/0373~ PCI/US92/ 28 2 ~ 7 ~ ~

exclusively by the AMP-activated protein kinase. Fig. 11 shows that in this assay, ZMP sti~ulates uj? to n~ar~y 8 fold the ae~tivity of rat liver AMP activated pro ein kinase, partlally purif ied up to the DEAE-Sepharose st8p 5 as desc:ribed by Da~ries et al . ( Eur ~ J ~ Biochem . 1~ 6:
123-8, 1989)~ ~alf~maximal stimulation is obtained al; 0.6 m~, and maximal stimulation at 4mM ZMP. Fi g. 12 shows that 2mPI Z:~P o~rxides th~ s~imulatory ef f ect of concentrations of ~NP below O.l mM, but is not additi~
10 and even slightly inhibitory at higher concentra~ion~; o~
ggesting that both nucl~otides bind to the sam~
site. Fig. 13 shows that the property of Z~P tG ~tim~llate ~ .
rat liver ~MP-activated pro~ein kinase i5 s~ared by a :-:
number of other AMP analog~;, namely tubercidin ~-~
15 monophosp3hates, dAtlP and ~a-AMP., The suc::¢inylated de~rivative of ZMP, SAICAR, ha~; nlD effect. Cyclic: ZMP and ~:
cyclic AMP ha~ littl~ or slightly inkaibitory ef~ect. ~;~
Other embodiments are within the f ollowing claims .

-;
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'';.'`'''.' .. .~.
",,,,..,:, ...
' ;~''.' `, ~,',.
';'" ,'.' SUBSTITlJTE SHEET

Claims (24)

Claims

1. A method for treating an animal, comprising the step of:
introducing into said animal a lipid-lowering amount of an AMP mimetic or pro-drug which stimulates AMP-activated protein kinase.

2. A method for treating an animal having an elevated serum lipid level, comprising the steps of:
identifying an animal having an elevated serum lipid level, and introducing into said animal a lipid lowering amount of an AMP mimetic or pro-drug which stimulates AMP-activated protein kinase.

3. The method of claim 2, wherein said identifying comprises chemically or biochemically measuring the serum lipid level of said animal and comparing said lipid level to a known desired maximum level of said lipid, wherein a serum lipid level above said maximum level indicates said elevated serum lipid level.

4. The method of claim 1, wherein said lipid is cholesterol.

5. The method of claim 1, wherein said lipid is triglyceride.

6. The method of claim 1, wherein said lipid is lipoprotein.

7. The method of claim 1, wherein said lipid is very low density lipid.

8. The method of claim 1, wherein said lipid is a chylomicron.

WO 93/03734 PCT/US92????28

9. The method of claim 1, wherein said lipid is a low density lipoprotein.

10. The method of claim 1, wherein said AMP mimetic is ZMP.

11. The method of claim 1, wherein said AMP mimetic pro-drug is AICAriboside.

12. The method of claim 1, wherein said AMP mimetic prodrug is AICA base.

13. The method of claim 1, wherein said AMP mimetic is an analog of ZMP.

14. The method of claim 1, wherein said AMP mimetic pro-drug is an analog of AICAriboside.

15. The method of claim 1 wherein said AMP mimetic pro-drug is an analog of AICA base.

16. The method of claim 1, wherein said AMP mimetic is a pro-drug of AICAriboside.

17. The method of claim 1, wherein said AMP mimetic is an analog of AMP.

18. The method of claim 1, wherein said AMP mimetic is an analog of adenine.

19. The method of claim 1, wherein said AMP mimetic is an analog of adenosine.

20. The method of claim 1, wherein said AMP mimetic pro-drug is an analog of adenosine.

21. The method of claim 1, wherein said animal suffers from a disease chosen from atherosclerosis, hyperlipidemia, hypercholesteremia, hypertriglyceridemia, coronary artery disease, transient ischemic attacks, stroke, angina pectoris, peripheral vascular disease and diabetes.

22. The method of claim 1, wherein said AMP mimetic pro-drug is a nucleoside which can be phosphorylated to form an AMP mimetic in vivo.

23. The method of claim 1, wherein said AMP mimetic pro-drug is a purine analog which can be phosphoribosylated to form an AMP mimetic in vivo.

24. The method of claim 1, wherein said AMP mimetic or AMP mimetic prodrug is selected from the group consisting of: tubercidin base, tubercidin nucleoside, tubercidin nucleotide, and analogs and prodrugs of said base, nucleoside and nucleotide.