Note: Descriptions are shown in the official language in which they were submitted.
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~
10
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 ~
- 12
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
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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
,::
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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). ~;~
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16
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 .
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17
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
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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
