ISSN 1990-7508, Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 2008, Vol. 2, No. 4, pp. 373–380. © Pleiades Publishing, Ltd., 2008.Original Russian Text © V.A. Vavilin, N.F. Salakhutdinov, Yu.I. Ragino, N.E. Polyakov, M.B. Taraban, T.V. Leshina, E.M. Stakhneva, V.V. Lyakhovich, Yu.P. Nikitin, G.A. Tolstikov,2008, published in Biomeditsinskaya Khimiya.

373

INTRODUCTION

Hypercholesterolemia plays a key role in pathogen-esis of cardiovascular diseases associated with thedevelopment of atherosclerosis [1]. Morbidity and mor-tality of cardiovascular diseases still remain very highin Russia [2]. In this connection treatment of coronaryischemic heart disease (CHD) employes a cholesterollowering therapy at the level of total cholesterol (Chol)exceeding 5 mmol/l and LDL-Chol (cholesterol of lowdensity lipoproteins) exceeding 3 mmol/l [3]. At themoment inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA(3-HMG-CoA) reductase, known as statins, are the mosteffective drugs decreasing LDL-Chol and mortality of

atherosclerosis and IHD [4]. However, in most statinsthe effective daily dose of 20–80 mg causes appearanceof unwanted side effects, including hepatotoxicity,myalgia, myopathy, and in rare cases rhabdomyolysis[5]. This explains why search and development of newstatins of lower daily dose, exhibiting prolonged actionand effective with respect of lowering of atherogenicChol levels represent an important task.

One of the modern approaches for the developmentof a new drug preparations consists in employment ofknown pharmacons as complexes with natural com-plexons, particularly with glycyrrhizic acid (GA). Ear-lier it was demonstrated that complexes with GAincreased pharmacological efficiency of phenylbuta-zone, indomethacin [6], and nifedipine [7].

The Cholesterol Lowering Properties of the Complex Compound Simvastatin with Glycyrrhizic Acid (Simvaglyzin)

in Experimental Models

V. A. Vavilin

a

*, N. F. Salakhutdinov

b

, Yu. I. Ragino

c

, N. E. Polyakov

d

, M. B. Taraban

d

, T. V. Leshina

d

, E. M. Stakhneva

c

, V. V. Lyakhovich

a

, Yu. P. Nikitin

c

, and G. A. Tolstikov

b

a

Institute of Molecular Biology and Biophysics, Siberian Branch, Russian Academy of Medical Sciences, ul. Akad. Timakova 2, Novosibirsk, 630117 Russia; fax: (383)332-31-47; e-mail: drugsmet@soramn.ru

b

Novosibirsk Institute of Organic Chemistry, Siberian Branch, Russian Academy of Sciences, pr. Akad. Lavrent’eva 9, Novosibirsk, 630090 Russia; e-mail: anvar@nioch.nsc.ru

c

Institute of Internal Medicine, Siberian Branch, Russian Academy of Medical Sciences, ul. Bogatkova 175/1, Novosibirsk, 630089 Russia; e-mail: office@iimed.ru

d

Institute of Chemical Kinetics and Combustion, Siberian Branch, Russian Academy of Sciences, ul. Institutskaya 3, Novosibirsk, 630090 Russia; e-mail: root@ns.kinetics.nsc.ru

Received October 4, 2007

Abstract

—A molecular complex of simvastatin (SV) and glycyrrhizic acid (GA) (at their ratio of 1 : 4) hasbeen synthesized. The complex named “simvaglyzin” (SVG) was stable in aqueous and aqueous–alcohol solu-tions at GA concentrations exceeding 0.2 mM. In vitro SVG acted as an uncompetitive inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA (3-HMG-CoA) reductase (Ki of 94 nM). Appearance of this inhibitory activity is asso-ciated with cytochrome P450-dependent conversion of SVG, because the addition of 1 mM metyrapone to theincubation medium fully prevented the inhibition of 3-HMG-CoA reductase. SV and SVG (used at 300 nM con-centration) inhibited mevalonate synthesis rate by 39.15

±

8,27 and 38.85

±

3,04%, respectively. In vivo SVGshowed a dose-dependent cholesterol lowering effect. In rats the cholesterol lowering effect of SVG used atdaily doses equivalent to 66 and 100 mg/kg of SV was the same as the effect of SV administered at the dailydose of 200 mg/kg. The decrease in total cholesterol of blood serum was 7% and 9% (

p

< 0.05) and 8%, respec-tively. Myotoxicity of these SVG doses estimated by blood serum creatine phosphokinase (CPK) activity waslower than that of SV. In rats treated with SV the activity of CPK increased by 79% (

p

< 0.01), while in SVGtreated rats it decreased by 30% and 36% (

p

< 0.05). Any increase of the hepatotoxicity markers alanine ami-notransferase or aspartate aminotransferase in blood serum was not observed. The data suggest pharmacologi-cal synergism attributed to the SV-GA complex formation and increased safety of the resultant complex com-pared with a parent compound.

Key words

: simvaglyzin, simvastatin, glycyrrhyzic acid, 3-HMG-CoA-reductase inhibition, hypercholester-olemia in rats.

DOI:

10.1134/S1990750808040070

EXPERIMENTAL STUDIES

*To whom correspondence should be addressed.

374

BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY

Vol. 2

No. 4

2008

VAVILIN et al.

In this study we have developed a new hypocholes-terolemic compound by means of complex formationbetween GA and a known 3-HMG-CoA reductaseinhibitor, simvastatin, and investigated its inhibitoryactivity towards versus this enzyme in vitro and thecholesterol lowering (hypocholesterolemic) effect inexperimental animals in vivo.

MATERIALS AND METHODS

The following reagents were used in this study:dithiothreitol (DTT), glucose-6-phosphate (ICN,USA), glucose-6-phosphate dehydrogenase (Sigma,USA), NADPH (Gerbu, Germany), Tris-hydroxyme-thyl aminomethane, EDTA-Na, MgCl

2

(Serva, Germany),3-hydroxy-3-methyl-[3-

14

C]glutaryl-CoA (Amersham,United Kingdom), DL-3-hydroxy-3-methyl-glutaryl-CoA (Fluka, Germany), locally produced chemicals ofthe chemically pure grade. The complex compoundsimvaglyzin was developed using simvastatin (KRKA,Slovenia) and glycyrrhizic acid (KhimPharm, Kazakh-stan).

The complex of simvastatin (SV) and GA wasformed by mixing of aqueous–alcohol solution of simva-statin and GA at the molar ratio of 1:4 (using methanolor ethanol as an alcohol). Due to low solubuility ofthese substances in water they were dissolved in alco-hol and their initial 10 mM solutions were then dis-solved with distilled water (at the ratio alcohol : water1 : 5, w/w). The resultant preparation was dried. Theprocess of complex formation between SV and GA wasanalyzed using the methods of nuclear magnetic reso-nance (NMR) (a Bruker DPX-200 NMR spectrometer(Germany), proton resonance frequency of 200 MHz)and electron optical UV-Vis spectroscopy (a UV-2401-PCspectrometer). The complex stoichiometry was evalu-ated by the dependence of optical density of a SV solu-tion (measured at 247 nm) on GA concentration. A con-tribution of GA to absorbance was corrected by sub-tracting its own absorbance spectrum from the totalabsorbance spectrum. Measurements were carried outin a quartz cuvette using a water methanol mixture(20% methanol) at 0.1 mM SV.

Experiments on estimation of the hypocholester-olemic properties of simvaglyzin were carried out usingmale Wistar rats of 180–200 g. Animals (5 rats percage) were kept at free access to water and food (a stan-dard laboratory diet). Animals were acclimated to vivar-ium conditions for one week. After this period 1 ml oftheir blood was taken from a tail vein (point 0) underether anesthesia and formation of experimental hyperc-holesterolemia was started. 25 experimental animalsreceived a high-fat cholesterol diet for 4 weeks. This dietwidely used for the development of hypercholester-olemia and subsequent evaluation of the Chol loweringeffect of various compounds, including statins [8–10]. Itcontained 3% Chol, 5% animal fat, 0.1% 6-N-propyl-2-thiouracil (for suppression of thyroid gland functions)and 0.3% sodium taurocholate (for improved Chol

absorption) [11, 12]. After 4 weeks a blood sample wastaken as above (point 4 weeks). After that animalsreceived the standard laboratory diet again and weresubdivided into 5 groups (5 rats in each group). Ani-mals of group 1 received the standard laboratory dietonly and served as control. Animals of groups 2–5 received hypocholesterolemic compounds in aqueoussolutions (simvaglyzin, SVG) and suspensions (SV)added to their diet: group 2 received SV at the dailydose of 200

µ

g/kg (or 40

µ

g per rat); rats of groups 3,4, and 5 received SVG at the daily doses of 400, 665,and 1000

µ

g/kg (or 80, 133, and 200

µ

g per rat). In thegroups 3, 4, and 5 the daily doses of SV (calculated byits mass proportion in SVG of 0.1) were 40, 66, and100

µ

g/kg. After two weeks blood samples were takenfrom these animals again (point 6 weeks). A nightbefore the experiment rats were deprived of food buthad free access to water.

Lipid parameters of blood serum (total Chol, HDL-Chol and triglycerides, activities of serum aspartateaminotransferase (AST), alanine aminotransferase(ALT) and creatine phosphokinase (CPK, fractionCPK-Nac) were assayed using Biocon kits (Germany)and a biochemical autoanalyzer Labsystem (Finland).

Rats used for in vitro analysis of 3-HMG-CoAreductase inhibition by SVG were kept on the standardlaboratory diet only. Microsomes were isolated by theconventional method of differential centrifugation inthe medium: 20 mM Tris-HCl, pH 7.4, 0.15 M KCl,50 mM EDTA, and 2 mM DTT [13]. A final sediment,containing the microsomal fraction, was resuspendedin the medium containing 0.1 M KH

2

PO

4

, 20% glyc-erol, pH 7.4. Protein concentration was determined bythe method of Lowry. Microsomal metabolism ofHMG-CoA was studied as described by Kleinsek et al.with minor modifications. Microsomal suspension wasadded into the incubation medium containing 0.1 MKH

2

PO

4

, pH 7.0, 4 mM DTT, 10 mM EDTA, 10 mMMgCl

2

, 1 mM NADPH, 4 mM glucose-6-phosphate,2 U glucose-6-phosphate dehydrogenase and preincu-bated for 2 min at room temperature. After addition ofequal volumes of SVG (final concentrations of 75, 150,300, and 600 nM) and a NADPH-regenerating systemthe reaction mixture was incubated during 8 min at37

°

C under shaking for generation a 3-HMG-CoAreductase inhibitor. After this incubation the labeledsubstrate, 3-HM[3-

14

C]G-CoA, was added at final con-centrations of 28 or 47

µ

M. Total volume of the reac-tion mixture was 100

µ

l, protein content was 0.24 mg.The reaction was carried out for 10 min and wasstopped by addition of 10

µ

l of 2.4 M HCl followed bysubsequent cooling of the reaction mixture in ice.Potency of inhibition of 3-HMG-CoA reductase reac-tion by the compounds studied was compared withthat observed in the presence of 300 nM SVG(equimolar to SV).

Sample preparation for a chromatographic proce-dure was started 1 h after termination of reaction; this

BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY

Vol. 2

No. 4

2008

CHOLESTEROL LOWERING PROPERTIES OF (SIMVAGLYZIN)

375

time interval was required for conversion of mevalonicacid into a lactone form [15].

After addition of 10 M KOH (1.5

µ

l) for partial neu-tralization of samples up to pH values of 3.5–4.5 theywere centrifuged at 3000 g for 5 min and supernatant(50

µ

l) was injected onto a column. Reaction product(mevalonate) and substrate (3-HMG-CoA) were sepa-rated by the method of ion-paired reverse-phase highperformance liquid chromatography described byScharnagi et al. [16] using a Nucleosil C

18

column(5

µ

m 250

×

2 mm) with a Vydac C

18

precolumn (30–40

µ

m 30

×

2 mm). Aqueous solution of methanol(53%, v/v) and 50 mM tetrabutylammonium phos-phate, pH 5.1, served as a mobile phase. A flow rate was150

µ

l/min. Detection was carried out by absorbance at254 nm. For quantitative characterization of the reac-tion product chromatographic fractions obtained duringeach 4 min were collected into vials and dried understream of air at room temperature. A dried residue wasdissolved in 7 ml of the dioxane scintillator containingPOPOP (300 mg/l), PPO (7 g/l), naphthalene (100 g/l).Radioactivity in samples was quantified using a Deltabeta-counter (Holland). Reconstruction of the radio-chromatographic profile and its overlapping to theUV-registered profile demonstrated that total time ofmevalonate elution was from 4 to 8 min and 3-HMG-CoAfrom 20 to 28 min of the chromatographic procedure(Fig. 1).

Inhibition constants were calculated using the Excelprogram. Statistical treatment employed SPSS for Win-dows (version 12.0) using Student’s criterion, correla-tion, linear regression analyses and dispersion analysis

50

40

30

20

10

0Abs

orba

nce

(0.0

2 op

tical

uni

ts o

ver

the

full

scal

e)

Cpm

(1

×

10

3

)3530252015105

min

0

20

40

60

80

100

1

2 3

Fig. 1.

Radiochromatogram of a sample of microsomalmetabolism of 3-hydroxy-3-methyl-[3-

14

C]glutaryl-CoA.A solid line shows UV behavior of standard solutions of 3-HM[3-

14

C]G-CoA and 3-HMG-CoA added to the incuba-tion mixture of microsomes. This chromatogram lacks asignal of mevalonate (from 4th to 8th min);

2

—unknownpeak;

3

—3-HM[3-

14

C]G-CoA. Columns designate radio-chromatographic behavior of a sample of microsomalmetabolism of 3-HM[3-

14

C]G-CoA;

1

—mevalonate;

3

—3-HM[3-

14

C]G-CoA.

CH3

O

H

OHO

OH

H3C

O

H3C CH3

H3C

H

COOH

HO

OO

OO

COOH

HO OH

COOH

OHHO

OH n

Simvastatin

Glycyrrhyzic acid

n

= 1–4

×

Fig. 2.

Structure of a chemical compound Simvaglyzin.

376

BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY

Vol. 2

No. 4

2008

VAVILIN et al.

(One-Way ANOVA) using Dannet’s criterion for multi-ple comparison. Differences were considered as statis-tically significant at

p

< 0.05.

RESULTS

Synthesis and properties of simvaglyzin (SVG)

. Anew chemical substance named as simvaglyzin (SVG)represents a molecular complex of simvastatin (SV)and glycyrrhizic acid (GA) (Fig. 2). Binding of SV andGA after mixing of their solutions was accompanied bychanges in absorbance spectrum of SV indicating com-plex formation of these compounds (Fig. 3). The UV-VIS spectrophotometric analysis of the mixture of SVand GA has shown that the increase in GA concentra-tion is accompanied by the change in the shape of SVspectrum due to complex formation (its maximumbecomes lower and shifts to a shorter wavelength region)(Fig. 3). At low concentrations of GA (up to 0.2 mM)no changes in the absorbance spectrum of SV wereobserved, however, subsequent increase in GA concen-tration resulted in significant change of the spectrum(Fig. 4). Calculation of constants characterizing com-plex stability and stoichiometry usually employs Ben-esi-Hildebrand equation (1) [17, 18]. In this study thestability constants for the complex of SV and GA wereanalyzed by changes of optical density of SV solutions(with its constant concentrations) with variable concen-trations of GA. Equation (1) is applicable for certainexperimental conditions (SV concentration < GA con-centration). In this case it is possible not only to esti-mate the complex stability constant (K) but also to

determine the stoichiometric ratio (n) within one exper-iment:

D

/

∆

D

– 1 = 1/[GA]

n

×

1/

K

, (1)

where

∆

D

=

∆ε

×

[SV],

K

is a complex stability con-stant determined for the reaction

SV +

n

GA SV–GA

n

as

(2)

At the segment of the increase (at various values of theparameter

n

) (0.2–0.6 mM GA, Fig. 4) the plot

D

/

∆

D

versus 1/[GA]

n

exhibited linear behavior only at

n

= 4.Thus, it was concluded that complex formation resultedin a compound containing one SV molecule and GAtetramer. The constant of complex stability calculatedfrom the slope

D

/

∆

D

versus 1/[GA]

4

using equation (1)was

K

= 3

×

10

14

M

–4

.The other conclusion, which follows from Figs. 3

and 4, is related to the dependence of the complexstructure on GA concentration. Based these results andknown literature data [18–21] one may conclude thatwithin the range of GA concentrations from 0.2 to0.8 mM cyclic associates consisting of 2–4 GA mole-cules are formed. Subsequent band shift of the complexabsorbance to the shorter wavelength regions observedat higher GA concentrations (Fig. 3) indicates that theincrease in GA concentrations about 0.8 mM and abovecauses enlargement of this complex followed by thechange in its stoichiometry. This results are consistentwith data on dynamic viscosity of aqeous ethanolicsolutions of GA [19], which sharply “jumped” at [GA]of 0.1 and 0.8 mM followed by subsequent smooth

K

KSV GAn–[ ]

SV[ ] GA[ ]n×----------------------------------,=

280260240220200λ, nm

2.5

2.0

1.5

0.5

0

1.0

A, optical units

GA = 0 mM

GA = 0.8 mM

Fig. 3. The dependence of the change in absorbance spec-trum of simvastatin (SV) on GA concentration. SV concen-tration was 0.1 mM (in 20% methanol). The lower spectrumline corresponds to absorbance spectrum in the presence of0.8 mM GA, the upper spectrum line corresponds to absor-bance spectrum in the absence of GA.

1.00.80.60.40.20[GA], mM

Experiment

–0.2

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

∆A 247

Fig. 4. The dependence of absorbance intensity of simvas-tatin (SV) on GA concentration. Absorbance was registeredat 247 nm, SV concentration was 0.1 mM.

BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol. 2 No. 4 2008

CHOLESTEROL LOWERING PROPERTIES OF (SIMVAGLYZIN) 377

increase; this is attributed to the increase of associatesand the position of bend points depends on alcohol con-tent in solution. Similar jump-like changes were earlierobserved for solubility of nifedipine (a known blockerof calcium channels) on GA concentration. In bothcases complex formation between the compounds stud-ied and GA was demonstrated [18]. Using changes ofspin-spin relaxation of GA T2 protons formation of GAmicelles was demonstrated at GA concentrationexceeding 0.8 mM [21]. Japanese scientists also inves-tigated micelle formation in the study on water solubleanalogues of glycyrrhyzin (for example, sodium sul-fates) [22].

Problems on the structure of GA complexes, partic-ularly on functional groups of GA involved into com-plex formation still represent points for discussion. Thepresent study of the complexes between SV and GAemployed analysis of changes in proton magnetic reso-nance (PMR) spectra. Addition of GA to SV solutioncaused the change in the GA PMR spectrum associatedwith proton line at the double bond located in the cen-tral part of GA near carbonyl group; this suggestsinvolvement of triterpene residue in the complex for-mation with SV. Changes in positions of PMR linesobserved for protons of SV naphthalene ring also sug-gest involvement of this particular fragments into com-plex formation (Fig. 2). Overlapping of carboxyl grouplines and methyl group protons in the PMR spectrum ofGA complicates subsequent analysis of putative candi-dates involved into association process of GA mole-cules and complex formation of this associate with SB.Thus, we should conclude that the NMR method oftenused for analysis of complexes with cyclodextrins inpharmacology [23, 24] does not provide exhaustiveinformation required for elucidation of GA complexstructure. Besides line overlapping this may be alsoattributed to poor solubility of GA in aqueous solutions.We do believe that the difference in sensitivity of PMRspectra to complex formation of cyclodextrins and GAmay be also attributed to an opened-chain structure ofGA molecule. Changes in chemical shifts of cyclodex-trin internal protons may be explained by displacementof water molecules from internal cavity during complexformation (this cannot occur in the case of GA com-plexes).

Thus, based on the data obtained we may concludethat the compound simvaglyzin (SVG) is a molecularcomplex of SV with GA (at their ratio 1 : 4); in solu-tions at GA concentrations exceeding 0.2 mM it is sta-ble. Our previous results [18, 25] indicate that GA com-plexes are stable not only in aqueous solution but alsoin other organic solvents: methanol, ethanol, acetoni-trile, DMSO. These complexes of GA favorably differfrom inclusion complexes of cyclodextrins.

Inhibition of 3-HMG-CoA reductase by simvag-lyzin. It is known that SV may undergo conversion intovarious products. Esterase or nonenzymtaic hydrolysisof SV results in formation of its hydroxyl acid [26, 27],

which is a more potent inhibitor of 3-HMG-CoA reduc-tase than the parent compound; the hypocholester-olemic effect of SV is attributed to formation of thisacid. Cytochrome P450 (3A4 and 3A5)-dependentmetabolism yields three main metabolites of SV(3'-hydroxy-SV, 6'-hydroxy-SV and 3',5-dihydrodiol-SV) and some other minor metabolites, which may befurther converted into hydroxy acids inhibiting3-HMG-CoA reductase [28]. For estimation of interre-lationship between inhibitory properties of the synthe-sized complex with preceded metabolism, we have per-formed experiments on inhibition of mevalonate syn-thesis in the persence of 300 nM SVG and increasingconcentrations of metyrapone, a cytochrome P450inhibitor. Results have shown that inhibition decreasedwith the increase of metyrapone concentration: withoutmetyrapone the inhibition was 37.7%, in the presenceof 10 µM metyrapone inhibition was 18.7%, and in thepresence of 1 mM metyrapone no inhibition wasobserved. Thus, inhibitory properties of SVG appearafter cytochrome P450- rather than esterase-dependentmetabolism.

Determination of type of inhibition and inhibitionconstants was carried out at two substrate concentra-tions of 28 and 47 µM and four SVG concentrations of75, 150, 300, and 600 nM. Type of inhibition analyzedusing the method of Cornish-Bowden (Fig. 5a) plots ofthe ratio of substrate concentration to the reaction rateversus inhibitor concentration yielded Ki value of94.18 nM and suggested uncompetitive type of inhibi-tion. Dixon’s reciprocal plot of the reaction rate versusinhibitor concentration yielded parallel straight lines

800–400 –200 0 200 400 600

1.8

1.2

0.6

–94.18

(a)

(b)

S/Vy = 0.0014x + 0.8478

y = 0.0003x + 0.7442

45

30

15

0

1/V

Inhibitor concentration, nM

700600500400300200100Inhibitor concentration, nM

Fig. 5. (a) Graphic determination of inhibition constant forinhibition of 3-HMG-CoA reductase by SVG by the methodof Cornish-Bowden. (b) Uncompetitive type of 3-HMG-CoAreductase inhibition by SVG: graphic analysis by the methodof Dixon. (�) substrate concentration 28 µM; (�) substrateconcentration 47 µM.

378

BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol. 2 No. 4 2008

VAVILIN et al.

(Fig. 5b) gave final evidence for the uncompetitive inhi-bition, because this type of inhibition is characterizedby the same changes in Vmax and Km.

Comparison of inhibitory potency of 300 nM SVGand SV on 3-HMG-CoA reductase (this is within therange of IC50 values from 100 to 300 nM reported forSV [29]) has shown that both compounds exhibit basi-cally the same inhibitory potency and decrease thisreaction by 39.15 ± 8.27 (SV) and 38.85 ± 3.04%(SVG).

This result is rather intriguing in the light of stabilityof the SVG complex under these conditions. Dilution ofaqueous or aqueous alcohol solution to concentrationsbelow 0.1 mM would cause partial dissociation of thiscomplex. Taking into consideration stoichiometry andstability constant of this complex it is possible to calcu-late its molar proportion in solution at 300 nM SVG:

We took into consideration that at complex stoichi-ometry of 1:4 [GA] = 4[SVG]. Complex functioning atsuch low concentrations means that its therapeutic activ-ity would significantly exceed activity of initial SV.

In vitro results suggest that SVG decreased Km andVmax values for mevalonate synthesis represented abasis for subsequent evaluation of its possible hypocho-lesterolemic effect in vivo.

The hypocholesterolemic effect in rats. The study ofparameters of lipid profile in rat blood serum indicates(Table 1) that maintenance of rats on the high-fat diet isaccompanied by the development of marked hypercho-lesterolemia: total cholesterol increased by 45% (p <0.01) compared with initial level. The effects of drugson the main parameters of lipid profile we estimated bytime-course of reverse development of hypercholester-olemia after transition of experimental animals fromthe high-fat diet to a standard laboratory diet. Sensitiv-ity of such comparisons increases due to reduced con-tribution of alimentary cholesterol. One can see thatmaintenance of rats on the standard laboratory dietcaused statistically significant decrease in total blood

SVG[ ]SV[ ]

---------------- 3 1014 GA[ ]4×× 6 10 10–×= =

Chol both in control and in all experimental groups(Table 1). Comparison of experimental groups withcontrol one has shown that in rats receiving SV at thedaily dose of 200 µg/kg total Chol in blood decreasesby 8% (p < 0.05). SVG exhibited dose-dependent effectand its doses of 665 and 1000 µg/kg (equivalent to 66and 100 mg of SV) caused the decrease of total bloodChol by 7 and 9% (p < 0.05). These results suggestmore pronounced hypocholesterolemic effect in vivo ofSVG compared with SV; this creates possibility of thedecrease of SV dose (based on SV content in SVG).

Administration of both SV and SVG caused (statis-tically insignificant) increase in HDL-Chol by 3–5%(p > 0.05) and blood tryglycerides by 3–18% (p >0.05). According to other authors statistically signifi-cant changes of these parameters would be achievedusing SV doses one order of magnitude higher thanthose used in this study. For example, in rabbits withhypercholesterolemia administration of SV at the dailydose 2.5–10 mg/kg for 4–6 weeks was accompanied bythe increase in HDL-Chol by 5–15% and the decreaseof tryglycerides by 10–30% [8, 33]; interestingly, in thestudy [30] statistical significance was not achieved.Estimation of SV effect in 4444 patients with IHD dem-onstrated that the level of HDL-Chol increased by 8%after administration of SV at the daily doses 20–40 mg[31]. The effect of SV on blood triglycerides is indirectand is associated with the decrease in formation andsecretion of very low density lipoproteins (VLDL)(containing about 30% of triglycerides) by hepatocytesand increased binding and catabolism of VLDL rem-nants by hepatocyte apo-B,E receptors [32, 33].

Hepato- and myotoxicity in rats. It is known thatbesides valuable therapeutic hypocholesterolemiceffect statins have some side effects, including hepato-and myotoxicity. So it was necessary to estimate hepa-totoxic and myotoxic properties of the investigatedcomplex. We did not find any increase of hepatic ASTand ALT in blood of rats treated with SV and SVG; thissuggests lack of hepatotoxicity of SVG and SV dosesused in this study (Table 2). Moreover, earlier it wasdemonstrated that GA exhibited a hepatoprotector

Table 1. Time course of total cholesterol in blood (mmol/l, mg/dl) of rats treated with SVG

Time pointsof blood sampling Control SV 200

µg/kg/day

SVG 400(SV 40)

µg/kg/day

SVG 665(SV 66)

µg/kg/day

SVG 1000(SV 100)µg/kg/day

0 weeks 2.52 ± 0.10 mmol/l, 97.4 ± 4.1 mg/dl

4 weeks 3.66 ± 0.15 mmol/l, 141.6 ± 5.9 mg/dl (+45%)

6 weeks 2.78 ± 0.13,107.5 ± 4.9*

2.49 ± 0.12,96.5 ± 4.4**

2.66 ± 0.12,103.1 ± 4.5**

2.53 ± 0.13,98.1 ± 4.7**

2.46 ± 0.10,95.3 ± 4.0**

Absolute difference –24% –32% –27% –31% –33%

Difference with control –8%^ –3% –7%^ –9%^

Note: * Comparison with the point 4 weeks, p < 0.05; ** p < 0.01; ^ comparison with control, p < 0.05. Here and in Table 2 data representmean ± SEM.

BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol. 2 No. 4 2008

CHOLESTEROL LOWERING PROPERTIES OF (SIMVAGLYZIN) 379

effect associated with the decrease of transaminaseactivity and lipid peroxides in hepatocytes after treat-ments with such toxins as allyl formiate and carbon tet-rachloride [6]. The level of blood CPK increased in ratstreated with SV and SVG for two weeks. However, inrats treated with SV this parameter increased by 79%(p < 0.01), whereas in rats treated with SVG thisincrease was just 30–36% (p < 0.05). Taking into con-sideration proportion of SV in SVG one can see thatboth preparations exhibit similar myotoxic effect.However, more pronounced hypocholesterolemiceffect provides safer application of SVG comparedwith SV.

DISCUSSION

Earlier it was demonstrated that SV is a competitiveinhibitor of 3-HMG-CoA reductase [28, 34]. This andother statins occupy 3-HMG-CoA binding sites andcomplicate access to the active site of this enzyme [35].Change of the mode of the inhibitory action from com-petitive (typical for SV) to the uncompetitive detectedfor SVG is probably associated with the increase in thesize of SVG molecule compared with the initial mole-cule (SV) and also with the presence of charged func-tional groups in GA residues potentiating the interac-tion. The latter probably causes the increase in dissoci-ation time for the complex enzyme-product, whichrepresents a kinetic basis for uncompetitive inhibition[36]. The expected pharmacokinetic consequence ofthis effect would consist in the decrease of the rate ofcomplex elimination compared with SV.

It is principally important that the inhibitory effectof SVG on 3-HMG-CoA reductase activity in vitro iscomparable to the effect of SV, whereas in vivo the cho-lesterol lowering effect of SVG exceeds that of SV. Thissuggests pharmacological synergism due to complexformation between SV and GA. Similar manifestationsof complex formation were earlier found for antiar-rhythmic and hypotensive preparations [6, 7]. Takinginto consideration chemical structure of SVG onewould suggest the existence of more molecular targetsresponsible for manifestation of its hypocholester-olemic effect in vivo compared with SV. It is possible

that SVG realizes all pharmacological properties of SV:inhibition of 3-HMG-CoA reductase (this results in thedecrease of Chol synthesis in hepatocytes); stimulationof synthesis of apo-B,E-receptors for LDL on hepato-cyte plasma membrane, the increase of blood LDLuptake by hepatocytes and the decrease of cholester-olemia [37]; inhibition of apo-B synthesis followed bythe decrease of LDL and VLDL formation by hepato-cytes and the increase of apo-B,E-receptor binding andcatabolism of VLDL [33]. GA may also exhibit ownhypolipidemic effect acting as phospholipase A2 inhi-bitor [6]. Possibiliy of GA action on cytochrome P450and the whole set of microsomal monooxygenases is ofspecial interest. It is known that GA induces cyto-chrome P450 and monooxygenases [38], whereas invitro inhibitory effects of GA on cytochrome P450 andNADPH-CYP have been registered [39]. It is also pos-sible that SV and SVG exhibit different pharmaco-kinetic behavior due to physico-chemical differencesrelated differences in their transport and biotransforma-tion. This requires special investigation.

Summarizing results of the present study we mayconclude:

1. A compound synthesized from simvastatin (SV)and glycyrrhizic acid (GA) is the molecular complex ofSV and GA (at their ratio of 1 : 4), which is stable insolution at GA concentrations exceeding 0.2 mM.

2. Simvaglyzin is an uncompetitive inhibitor of3-HMG-CoA reductase.

3. Experiments on rats have demonstrated that sim-vaglyzin is more effective and safer hypocholester-olemic agent than simvastatin.

ACKNOWLEDGMENTS

This work was supported by a grant from FederalScience and Technology Program of Ministry of Indus-try, Science, and Technologies of Russian Federation(project no. IB-37/02, State contract no. 43.004.11.2535,2004) and the Integration project of Siberian Branchesof Russian Academy of Sciences and Russian Academyof Medical Sciences (no. 53, 2006–2008).

Table 2. Time course of total CPK in blood (U/l) of rats treated with SVG

Time pointsof blood sampling Control SV 200

µg/kg/day

SVG 400(SV 40)

µg/kg/day

SVG 665(SV 66)

µg/kg/day

SVG 1000(SV 100)µg/kg/day

0 weeks 432.7 ± 23.9

4 weeks 373.1 ± 21.6 (–14%)

6 weeks 384.1 ± 15.7 667.6 ± 26.0** 501.0 ± 19.7* 484.6 ± 21.0* 506.4 ± 21.8*

Absolute difference +3% +79% +34% +30% +36%

Difference with control +76%^^ +31%^# +27%^# +33%^#

Note: * Comparison with the point 4 weeks, p < 0.05; ** p < 0.01; ^ comparison with control, p < 0.05, ^^ p < 0.01; # comparison withthe groups SV, p < 0.05.

380

BIOCHEMISTRY (MOSCOW) SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol. 2 No. 4 2008

VAVILIN et al.

REFERENCES

1. Anderson, K.M., Castelli, W.P., and Levy, D., J. Am.Med. Assoc., 1987, vol. 257, pp. 2176–2180.

2. Oganov, R.G. and Maslennikova, G.Ya., Kardiovask.Ter. Profil., 2002, vol. 3, pp. 4–8.

3. Diagnostika i korrektsiya narushenii lipidnogo obmena stsel’yu profilaktiki i lecheniya ateroskleroza. Rossiiskierekomendatsii VNOK., Kardiovask. ter. profil., 2004,Suppl. 2, p. 36.

4. Expert Panel on Detection, Evaluation, and Treatment ofHigh Blood Cholesterol in Adults: Executive Summaryof the Third Report of the National Cholesterol Educa-tion, and Treatment of High Blood Cholesterol in Adults(Adult Treatment Panel III), JAMA, 2001, vol. 285,pp. 2486–2497.

5. Puddu, P., Puddu, G.M., and Muscari, A., Acta Cardiol.,2001, vol. 56, pp. 225–231.

6. Tolstikov, G.A., Baltina, L.A., Shul’ts, E.E., and Pok-rovskii, A.G., Bioorgan. Khim., 1997, vol. 23, pp. 691–703.

7. Tolstikova, T.G., Sorokina, I.V., Bryzgalov, A.O.,Dolgikh, M.P., Lifshits, G.I., and Khvostov, M.V., Rat-sional’naya Farmakoterapiya v Kardiologii, 2006,vol. 1, pp. 55–58.

8. Kobayashi, M., Ishida, F., Takahashi, T., Taguchi, K.,Watanabe, K., Ohmura, I., and Kamei, T., Jpn. J. Phar-macol., 1989, vol. 49, pp. 125–133.

9. Ishida, F., Watanabe, K., Sato, A., Taguchi, K., Kakubari,K., Kitani, K., and Kamei, T., Biochim. Biophys. Acta,1990, vol. 1042, pp. 365–373.

10. Hayashi, T., Rani, P.J.A., Fukatsu, A., Matsui-Hirai, H.,Osawa, M. Miyazaki, A., Tsunekawa, T., Kano-Hayashi, H., Iguchi, A., and Sumi, D., Atherosclerosis,2004, vol. 176, pp. 255–263.

11. Swift, L.L., Soule, P.D., and LeQuire, V.S., J. Lipid Res.,1982, vol. 23, pp. 962–971.

12. Salter, A.M., Hayashi, R., Alseeni, M., Brown, N.F.,Bruce, J., Sorensen, O., Atkinson, E.A., Middleton, B.,Bleackley, R.C., and Brindley, D.N., Biochem J., 1991,vol. 276, pp. 825–832.

13. Kleinsek, D.A., Ranganathan, S., and Porter, J.W., Proc.Natl. Acad. Sci. USA, 1977, vol. 74, pp. 1431–1435.

14. Kleinsek, D.A., Jabalquinto, A.M., and Porter, J.W.,J. Biol. Chem., 1980, vol. 255, pp. 3918–3923.

15. Jemal, M., Schuster, A., and Whigan, D.B., Rapid Com-mun. Mass Spectrom., 2003, vol. 17, pp. 1723–1734.

16. Scharnagi, H., Marz, W., Schliak, M., Loser, R., andGross, R., J. Lipid Res., 1995, vol. 36, pp. 622–627.

17. Lopez, E.A., Bosque-Sendra, J.M., Rodriguez, L.C.,Campana, A.M.J., and Aaron, J.J., Anal. Bioanal. Chem.,2003, vol. 375, pp. 414–417.

18. Polyakov, N.E., Leshina, T.V., Salakhutdinov, N.F., andKispert, L.D., J. Phys. Chem. B., 2006, vol. 110,pp. 6991–6998.

19. Romanko, T.V. and Murinov, Yu.I., Zhurn. Fiz. Khimii,2001, vol. 75, pp. 1601–1604.

20. Sangalov, E.Yu., Zhurn. Obshch. Khimii, 1999, vol. 69,pp. 667–670.

21. Gusakov, V.N., Maistrenko, V.N., and Safiullin, P.P.,Zhurn. Obshch. Khimii, 2001, vol. 71, pp. 1382–1386.

22. Saito, S., Furumoto, T., Ochiai, M., Hosono, A.,Hoshino, H., Haraguchi, U., Ikeda, R., and Shimada, N.,Eur. J. Med. Chem., 1996, vol. 31, pp. 365–369.

23. Polyakov, N.E., Leshina, T.V., Hand, E.O., Petrenko, E.,and Kispert, L.D., J. Photochem. Photobiol. A: Chem.,2003, vol. 161, pp. 216–223.

24. Polyakov, N.E., Leshina, T.V., Konovalova, T.A.,Hand, E.O., and Kispert, L.D., Free Radicals Biol. Med.,2004, vol. 36, pp. 872–880.

25. Polyakov, N.E., Khan, V.K., Taraban, M.B., Leshina, T.V.,Salakhutdinov, N.F., and Tolstikov, G.A., J. Phys. Chem.B., 2005, vol. 109, pp. 24526–24530.

26. Prueksaritanont, T., Gorham, L.M., Ma, B., Liu, L.,Yu, X., Zhao, J.J., Slaughter, D.E., Arison, B.H., andVeas, K.P., Drug Metab. Dispos., 1997, vol. 25,pp. 1191–1199.

27. Prueksaritanont, T., Ma, B., and Yu, N., Br. J. Clin. Phar-macol., 2003, vol. 56, pp. 120–124.

28. Vickers, S., Duncan, C.A., Vyas, K.P., Kari, P.H.,Arison, B., Prakash, S.R., Ramjit, H.G., Pitzenberger, S.M.,Stocker, G., and Duggan, D.E., Drug Metab. Dispos.,1990, vol. 18, pp. 476–483.

29. Dansette, P.M., Jaoen, M., and Pons, C., Exp. Toxicol.Pathol., 2000, vol. 52, pp. 145–148.

30. Plosker, G.L. and McTavish, D., Drugs, 1995, vol. 50,pp. 334–363.

31. Scandinavian Simvastatin Survival Study Group: Ran-domised trial of cholesterol lowering in 4444 patientswith coronary heart disease: the Scandinavian Simvasta-tin Survival Study (4S), Lancet, 1994, vol. 344,pp. 1383–1389.

32. Alberts, A.W., Cardiology, 1990, vol. 77, pp. 14–21.33. Ginsberg, H.N., Le, N.A., Short, M.P. Ramakrishnan, R,

and Desnick, R.J., J. Clin. Investigations, 1987, vol. 80,pp. 1692–1697.

34. Hoffman, W.F., Alberts, A.W., Anderson, P.S., Chen, J.S.,and Willard, A.K., J. Med. Chem., 1986, vol. 29,pp. 849–852.

35. Istvan, E.S., Amer. Heart J., 2002, vol. 144, pp. S27–S32.

36. Kornish-Bouden, E., Osnovy fermentativnoi kinetiki(Principles of Enzymatic Kinetics), Moscow: Moskva,1979.

37. Lennernas, H. and Fager, G., Clin. Pharmacokinet.,1997, vol. 32, pp. 403–425.

38. Paolini, M., Barrilari, J., Broccoli, M., Pozzetti, L., andPerocco, P., Cancer Lett., 1999, vol. 145, pp. 35–42.

39. Liu, K.H., Kim, M.J., Jeon, B.H., Shon, J.H., Cha, I.J.,Cho, K.H., and Shin, J.G., J. Clin. Pharm. Ther., 2006,vol. 31, pp. 83–91.