Identification of metabolites of methylprednisolone in equine urine

755

CANlpsFLID.,1523w.3rd Aw2. B.C., Gmada, V&l lJ8 Received 9-2&-85 V-,

.

Wthylprednisolone and three metabolites, 17,2l,+hydroxy-6a -methyl-l+pregnadien3,11,2Gtrione, 6a-methyl-17,206,21-trihydroxyl,bpregnadiene-3,1Ldione, and 6o-mxhyl-118,17,20B,21-tetrahydroxyl,bpregnadier+3-one wx-e detected in equine urine after intraarticular administration of rrr?thylprednisolone acetate. All four ccrrpourds wre excreted both in the mconjugated form and as glucuronic acid conjugates. They were identified by carparing data obtained fran analyses by high performance liquid chramtography, thin-layer chranatography, ultraviolet spectroscopy and gas chranatography/mass spectranetry to those of the synthesized standards. The presence of trace amounts of a fourth mtabolite, 6o-n-ethyl-118,17,2Oo,21-tetrahydroxy-l,4-pregnadier+Sone, t-as indicated by high performance liquid chrcnmtography but confirmation has not been attained by the other methods.

Methylprednisolone diene-3,ZGdione) orally or

in

(L)

is tablet

a solution

of

and

its

saliun

succinate

action

is

the

blood is

phamecokinetics

Mthylprednisolone its in tion,

long the

horse, facilitate

of

prednisolone

August, September

of

ard

metabolism

(1113,17

1985

is

if of

between of in

of

these used

and

is

of

of

are n&as

water

horses

a study

race

The

large

tracks

XXI mg daily

for

local

its

metabolites.

dosages

to metabolize

of

imperative.

.

,21-trihydroxy-l,4-pregnadiene-1,2CI-dione),

the

given.

Canadian

expected

Steroids

dose

in

and

ester

anti-inflannatory

acetate

drugs

acetate

concentrations

racehorses

methylprednisolone horses

its

administered

at

availability 10

rapid

the

is

of

prolonged

in

currently

and

drug

an intravenous

corticosteroids

action

detection

If

and

injections

use

typically

Methylprednisolone

required,

local

acetate

duration

salt.

administered

then

extensive

are

The

as a suspension

succinate

in

,21-trihydroxy-l,bpregna-

glucocorticoid.

parenterally

salt

required,

The

a synthetic

form

methylprednisolone soluble

(6a-methyl-llB,17

due

to

required administra-

similarly in

LO jklich

S

756

WE=OXDI

the c-20 oxo group is reduced to a 2OBalcoho1, is oxidized [l].

to a ketone,

‘Ihe correspordiqg

hydranethylprednisolone pregnadien-k),

arid both C&20 reduction methylprrxlnisolom

the Gil

hydroxy fmctioo

amI C-11 oxidation

mtabolites

would be ZOe-di-

(2) (Q-methyl-1&l7,2O6,2l-tetrahydroxy-l,G methylprednisone

pregnsdiene-3,11,2Gtriooe)

(2) (17,2ldihydroxyAo+mthyl-1,G

and 2C&dihydranethylprednisone

(5) (6

-methyl-17,2~,21-trihydroxy-l,4-pregnadieoe-3,ll~ione).

and man [3] mthylprednisoloce vitro

studies

OCCUT

is oxidized

In rabbits

[2]

and -in [4] have shcxm that the G20 0x0 group of methylprednisolone

is reduced by dihydronicotinmide-adenioe presence of 3a,2Whydroxysteroid

to mthylprednisone,

dimcleotide

dehydrogenase

to yield

comzyme in the 2Wdihydrw

methylprednisolone.

Ihe trivial wanes ard abbreviations of the steroids are defined as follcws: Mp = tkthylprednisolone= Cxwrethyl-116,17 ,21-trihydroxy-l-&pregnadiene3,2O-diooe 2GCX-iMP= 2O-dihydrunethylprednisolone= Q-mthyl-116,17,20,21-tetrahydroxy-l+pregnadiewkme MRI = tkthylprednisone = 17,2l-dihydroxy&-mthyl-l,+regnadiene-3,11, 2Gtrione 2O-DHMFU= 2Odihydranethylprednisow = &nrsthyl-17,20,21-trihydroxyl&-pregnadiene-3,ll-dioue

Veterinary Depdledrol (Upjohn), a sterile suspension of Mp acetate used in the drug administration, was obtained fran ‘lko Products Carpany, Oraogeville, Ontario. MT was procured trun Upjohn Co., Kalamzoo, Michigan. hta-glucuronidase/arylsulfatase type H-2 fran Helixprmatia

S crude solution

was purchased

WDEOXDI

fran

Signs Chemical Co., Missouri.

The expected metabolites, 2Oo-and 2OWHMP, MKI and 2O&lIHMFO wzre synthesized fran MP , following chronic acid oddation and sodiun borohydride reduction procedures of related steroids [1,5,6,7]. The mthcxim-trimethylsilyl (t-&m) derivatives of MP and MFO, 1 and 6 respectively, were prepared fran mthoxylamine hydrochloride and N-tr~thylsilylimidazole (Pierce Chemicals Co., Rockford, Ill.) [8]. The acetonide derivatives of 2oDHW and 2O-DHNQ, Land 8 respectively, were prepared in acetone solution using p-toluenesulphonic acid as a catalyst and scdiun sulfate to remve water Eon-& during the reaction [6,7].

Sodiun acetate buffer was prepared by adjusting a 3&l sodiun acetate solution to pH 4.5-5.0 with concentrated acetic acid. Borate buffer ms prepared by adding a 1M sodiun carbonate solution to a 1M boric acidpotassium chloride solution until p+i 9.0.

Analytical ard preparative TLC was acccnplished on 0.25 mn precoated silica gel 60 F-254 plates (FM Laboratories, Damtadt, Cermwy). Chruttatogrm were developed to a height of 7 cm in 9:l (v/v) chlorofonm/mrx~ anol. carpounds were located by observation of fluorescence or quenching with IJV light at 254 III-I and xere visualized by spraying the plate heavily with phosphotungstic acid (PTA) spray reagent (1% w/v in ethanol) or tetrazolium blue (‘IZB) spray reagent (0.07% w/v in 1:2 v/v of ethanol/K% NaOH), follwd by heating the plate for 15 min. PTA reacts to produce a bright yellow color a,rd TZB produces a purple color. See Table 1 for details. For preparative TLC the appropriate zone was scraped off and the silica gel eluted twice with 2 mL of either methanol or acetone. Florisil

Cblum

Qrumtog&y

Four percent water deactivated Florisil (w/w) was made by adding 4 g of deionized water to 96 g of Florisil (W-100 mesh; Supelco Canada Ltd.), which had been previously activated at 170°C for 16 hr arrl cooled to room temperature in a desiccator. The mixture was shaken for 30 min and allowed to equilibrate overnight.

757

The colum of Florisil (1.1 cm internal diameter x 17011 gkss colum; 4g of 4% Florisil) ws washed with 10 I& of 95:5 dichloranethane /ethanol an3 was standardized by eluting Mf’ and the synthesized mtatmlites through it at a flaw-ate of l-2 drops/set, according to schme sham in Table 2.

Table

1.N

Chtxmtograplic bkthylprainisolaz

ad

37 20 77 71 54 34 84

MF 2Oo-an3 205DHMP 20 6DHMP~cetonide 20a-DHMP-acetonide Mm 2owmlFu 2W-DHWO-acetonide Solvent system: roan temperature. + sign indicates

Table

2.

Florisil prednisol~

Fractim # and wqx3sitirmb (VolmE collected) l/p/ (10 mL) 2/s% (10 mL) 3/16;/. ( 10 frL) 4/26;b (10 n-L) 5/20% (10 mL) 6/2cp/. (10 n-L) 7/20% (10 mL) 8/2d;b (10 d)

a&antitations colum is b0.5 mL of Carposition

Characteristerics HAxAites

9:l

(v/v)

spray

Cohn and

with

+ -

+ -

refractive

index

is

1.42

at

steroid.

&mmtography tMhylprednisolcm

percentage

bkthylprwhisol~

+ + + + -

chloroform/methanol; reacts

of

the

of

w

Mm

63 32 4 1

38 to 2 -

Umacteristics Eletabolites

Steroid

in

tsxbm4P

of

bthyl-

Fractiona 2om

-

-

-

3 55 42

1 61 34 3 1 -

4 52 36 8 -

determined by HPLC. Maxim amount of any steroid on the 15 pg. Steroids were added to the calm in three aliquots of 5% methanol in dichlorunethane. expressed as percentage of methanol in dichlormethane.

S

S’DEOXDI

759

Five grams of Sephadex IX-20 (bead size:2>103 microns; Sigma Chemical Co.) was equilibrated in 95:5 dichlorcmthaw/methanol for 30 tin ad the gel poured into a glass colum (1.3 am internal diameter x 31 on). The Sephadex was further equilibrated overnight before being stardardized (see Table 3). With the stopcock fully open the flow rate was about 1 drop/set. After each elution the Sephadex was regenerated with Xl I& of 50:X1 (v/v) dichlorawthane/methanol follwd by 50 mL of 95:5 (v/v) dichlorawthane/methanol. Table 3.SephadaUK2OColumolnmatograPry~teristics prednisol~ aml kthylprednisolaw

of

Methyl-

IWabAites

Stervic?

v01lnle Eluted Solvent

ME0 16-24

the

2cb2a

W-mQ 36-46

2cb-mw 4U-52

(mL) composition

%xiTRPT~anxxnt. to

w 266

m-

calm

of in

is any three

95:5 (v/v) dichloranethane/methanol. steroid on the colum is 15 pg. Steroids aliquots of 0.5 mL of eluting solvent.

were

added

All analyses were performed using a Waters HPLC system consisting of a &45 pwp, Model UtK manual injector, M73Cl data module an3 a Waters Model 440 fixed wavelength detector set at 254 m. The steroids wre separated on a 10 micron Radial-PM C(H) colmm (8 m internal diameter x 10 cm) by three different mbile phases. See Table 4 for details. Ultraviolet

UV/VIS

(UV)Sp?ctroscqry W spectra were spectrophotcmeter.

measured

in methanol

with

a Perkin-Elmer

Lambda

3

M3s.s spectra were obtained via electron inpact ionization with a Finnigan O&I 1020 autmted CC/l% system equipped with an Incas data system. Steroids ware chraratographed on a 2 nm internal diameter x 31 cm glass colum packed with 3% OV-1 on SO-100 mesh Chrmsorb W (Chramtographic Specialties Limited, Brockville, Ontario). The tenperature

carrier gas was helix at was 220 C and the injection

a flow rate mode was

of 20 mL/min; the inlet splitless; the terrperature

S

760

WDEOXDI

of the separator an3 manifold colum oven t-rature uas 280°C at 25’C/min.

current kerosene acquired

wre 250°C and so”C, held at loO°C for 1 tin

respectively. ard then

Electron inpact ionization was performd at 7&V The mass spectrcmeter was calibrated of 500 PA. ard was tuned using decafluorotriphenylphosphine. fran 103 am to 700 am at 4 seclscan.

with with

Liquid Ummatography&xzteristics lhble4.Hi~Rxformarre prednisol~~*thylprednisol~MetabolitesanaC

MP 2ot?-DHMP 2co-DHMP 2OWXMP-acetonide 2CWIHMP~cetonide MFO 2cx3-DHMm 2WHMKkacetonide Mobile phase 1 mL/min. While phase at a flow rate labile pbse at a flow rate kbile phase 1 ml/rain.

Relative A

mtentimtime B

11.48

11.08

7.92 7.28

7.92 7.36

12.40 8.32

1% acetic

1Plplmie D

5.64 4.04 3.80 16.68 16.68 4.56 3.50 13.96

9.48 8.80 10.60

65:35

a flow

rate

of

B is of C is of D is

60:20:20 (v/v/v) 1% acetic acid/methanol/tetrahydrofuran 1 ml/rain. 60:20:20 (v/v/v) 1% acetic acid/rrethanol/tetrahydrofuran 2 ml/tin. 40:&J (v/v) 1% acetic acid/acetonitrile at a flew

rate

of

amiSmple

acid/acetonitrile

an mission perfluoroAll data were

A is

Drugkhinistratim

(v/v)

to

of plethyl-

(mi.n)inmbile C

8.96 6.60 27.20

The programed

at

Collection

One healthy standardbred mre was administered 100 mg of Mp acetate (Veterinary Depo-kkdrol) intraarticularly into the left carpaletacarpal joint. Urine sarrples wzre collected by indwllmg catheters before drug administration ard at 1, 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 30, 48, 72 and 96 hours post-administration. All samples were frozen until analyzed.

Isolatim

excreted

of Steroids To determine throughout

fnm ltrim the the

relative 96 hr

amounts collection

of unconjugated MF and metabolites period, 10 mL samples of urine

S

761

WDEOXDI

fran each of the 15 post-administration sampling intervals ware pooled, adjusted to pH 9.0 with borate buffer, saturated with scdiun sulfate an3 extracted twice with 150 mL of 9:l dichloranethane/isopropanol. After centrifugation, the aqueous phase was saved for cmjugate hydrolysis and the canbined organic extracts were filtered through whatman siliconetreated filter paper (previously soaked in dichloruasthane), concentrated --in vacua and evaporated to dryness in a 50°C water bath under nitrogen. The residue was extracted into 0.5 I& of 95:5 dichlorawthane/methanoanol three times and each aliquot was transferred to the top of a 4% Florisil The (4 g) colum, prewashed with 10 mL of 5% methanol in dichlorcmthane. steroids were eluted according to the schem in Table 2. Fractious 3-5, which contain greater than 95% of MF, MRI and 2OEDHMRl and greater than 5% of 206-DW, wre ccmbined. Fractions 6-g combined contain 20~ aw.d 2oB-Dw.

The residues remaining after removal of the solvent were extracted into 0.5 n-L of 5%mthanol in dichlorawthane three times ard further ‘hm fractions were collected: chronatographed on 5 g of Sephadex IA-20. fraction 15-29 mb contains MFO, ZOfc-DI-MQ and about 30% of t-P,and fraction 29-52 mL contains 20a- and ZOB-DHMPand about 7CfL of MP. After evaporation of the solvent the residues wre chrmatographed and the steroids quantified by HPLC. To determine the relative amunts of conjugated MP and metab olites excreted, the renaining urine uas adjusted to pH 4.5 with acetate buffer ard incubated with 1.5 mb of B-glucuronidase at 37’C for 16 hr. The pH was increased to 9.0 by adding 5cp/,NaOH and borate buffer ard work-up proceeded identically to the unconjugated samples.

MP and three jugates

were excreted

intraarticular

in urine

ware first for

and their sqles

cleaved

analysis

identification.

tization

to

those

not been confirmed observed.

A

fourth

and acetonide

by the other

acid cm96 hr after

glucuronic

acid

enzyme to release

the free

were identified

as MPD,

frcm HFW, TLC, IJV and

standards.

of ZCB-DHMFCand 2@-DHMP and K&S

gested by HPLC analysis

The

data obtained

of the synthesized

glucuronic

fran 0 through

The three rmstabolites

also aided the identification.

metabolite

collected

by B-glucuronidase

2C&DHMKI and 2@3-DHM?by curparing E/MS

respective

of 100 mg of MF’ acetate.

injection

conjugates steroids

metabolites

Acetonide

derivatization

metabolite,

derivatization,tut

deriva-

of MP and MH)

2oO-DHMP, was sugits

methods due to the 1~ levels

identity of the

has

S

‘762

The results

T-BOXDI

v.ere expected because prednisolone

follcms

similar

hhhhh reversible -DW

metabolism

is further

to MKI [2].

supported

The presence of trace

because trace

of 2C&dihydroprednisone

amunts

(17,2Oa,21-trihydroxy-1,4-pregnadiene-3,ll-dione) treatmsnt

of hmms

11,20-trione)

with prednisone

are inferred

(17 ,2ldihydroxy-l+pregnadiene-3,

af I&hylprednisolaneaditsI42tablites

HPLC was used to estimate bolites

excreted

bolites

were prepared

equal response

throughout

the relative

only in analytical

factors

mking

the values

purely

(36.7%) follmd

t@ (2UX).

by uncharged

at 96 hr post-administration.

drug being excreted

in excretion in higher

administration

of MFU to rabbits

the metabolite

Mp [2].

Table 5.lklati~2~~ metabolites-tedas steroids

[5].

results

mount

Mp was still metabolite

prednisone with

On the other in higher

plasm

detectable besides

.

Mp

or prednisolone the administered hand, intravenous concentrations

of

ofrlrShylpn?dnisolaleandmethylprednisolone lmcon-ted ad glucuronide lxmwted

Conjugated

36.7

1.4

-

Unconjugated

20.0

4.5

0.4

aCslculated fran HPLC area responses each ccrrpound. The most efficient

assuning

method of isolating

an 8 or 10 hr post-administration

All metabolites

Nevertheless,

in the greatest

of both steroids,

anmnts

were based on

qualitative.

In fact, either

Since the meta-

estimations

The most abmdant

wes ZCf!-DHMIo. In humns, results

period.

amunts,

t@ was excreted

administration

amounts of MP and its meta-

the 96 hr collection

Table 5 shaJs that conjugated

tractig

by TLC after

[5].

Relati~kxmts

glucuronide

amounts of 2Ca

ware isolated

in identifiable

1.4

3.5

14.2

17.8

equal response factors

the n-etabolites

sarrple of unhydrolyzed

for

was exurine.

amounts fro-n 100 mL of urine.

763

Tentative collectiq

identification

the appropriate

carparirlg

‘IX Rf values

concurrently,

of the mtabolites

Florisil

ard Sephadex fractions

and HPLC retention

to those obtained

sumsrise

the chrcmstcgra&ic

retention

times of the steroids

fran

star&t&

(Table

1).

plates

were remved,

icularly

by ‘IT.L

of the steroids. 206-DHMFU (Table

recovery

by tiss

fran

corticosteroids

2GDHMP, regardless For further each Sqhadex

In this

study,

of whether

and the steroid

chrcm-

were hindered, wre

separated

The steroids

also

partpurified provided

fran MFO,

separated

well

on

to that

for differentiating

colour

the

with

after

the a-ketol

heating,

whereas

howaver,

PTA reagent

reacted

only with

such as MP and

a C-20 Ret0 or hydroxy group is present. residue

cmtainirg

unlike

reacts

a C-11 hydroxy group,

fran

one-half

of the eluate

2GDRMP and 2O-DHMpDwas converted

‘Ihe 2C!o- and 20R-acetonide

uare stable

and chrcmtographed

the s&es

‘IZB reagent

identification,

using 60:40 acetonitrile/l%

zones on the TLC

as in 2O-Dl4P and 2O-DRMF0, prevents

possessirg

fraction

+ 0.05 set

Sephadex chrmtography

very useful

The camm

9:l chloroform/methanol,

derivatives

unless

the

[l].

of the C-20 keto group, with TZB.

with methanol

to produce a purple

those corticosteroids

All

gel was about 60%, similar

wre

studied.

group of corticosteroids

acetonide.

3).

I-4

+ 0.04 of the

the appropriate

MP kas clearly

silica

ard Rylance

TII: spray reagents

within

RPLC ard TLC analysis

clean-up.

TLC but their

wre

RPLC peak MS collected

Frequently

and Sephadex colum

and 2O-DHMPfran

reaction

eluted

samples,

the best separation

reduction

either

the steroids

Tables

of the standards.

the Rf values

Additiooslly,

run

standards,

samples.

by

anl then

fran urine were within

with enyme hydrolyzed

by Florisil

observed

the urine

isolated

(Table 4) and all

atographed

times of the

characteristics

of the standards

by RPE or the appropriate

was accomplished

their

acetic to hydrolysis

isaners

could be separated

of

to the

by TIE in

underivatized

anakgues,

or by HPLC

acid as the nobile

phase.

‘Ihe acetcmide

in the acidic

mobile

phase.

Ultraviolet

Spectro6mpy ‘Ihe W spectra

shift

the C-11 oxo fran

the C-11

Both Ml?0 ard 2Os-DHMpDabsorb at 237.5 nn whereas

hydroxy steroids. reduction

could only differentiate

of the C-11 0x0 group to an hydroxy group produces a bathochranic

to 242 na for W and to 243.5 ms for ZO-DHMF’.

c&i QlrcNmtm

spectwtry

Mass spectranetry steroids

synthesized

tra and retention

wds required

fran IQ and those isolated

tinxas of the steroids

to those of the pure standards. to prevent

to confirm

thermal

the thermally

labile

acetonide

derivatives.

All

the basis of their under electron

inpact

of the Ihe mass spec-

fran urine were identical

the Cc/t%.

required Suitable

derivatization derivatives

C-17 side chain are t%I-‘lW and G20, the derivatized

molecular

fran urine.

isolated

All the steroids

breakdown inside

stabilize

the identity

wzights

conditions

steroids

which 21-

were characterized

on

even though they responded poorly

ard the spectra

showad only

1a.1 intensity

ions in the high nass region. Ihe msss spectra very similar tm atanic

of 2OPDHMRl acetonide

in the 1~ rrass region mass units

duce molecular

(l-270

appear in the high mass region.

Some of the sarrples analyzed

mass spectrcmetry ferent

difficult.

pro-

and diagnostic

contained

Houaver,

small anounts of steroid making confirmation

their

Cc retention

so

based on

times are dif-

(11.00 min for 206-DHMFU and 12.13 min for 2OWHMP) thus aiding identification. ‘lhe mass spectra

useful

are by

(<3X of the ccnnon base peak m/e

that these ions were absent in the spectra,

their

Both steroids

ions (M+ at m/e 416 for 206-DHMRI acetonide)

(M-15)+ ions all of very low intensity 135).

and ZOE-DHMPacetonide

Amy) but peaks differing

for explaining

hydrochloride the sterically

of the MD-‘It% derivatives

their

structure.

to produce only a bis-oxime hindered

with trimethylsilylimidazole which shows the molecular

Cl1

of MP and Mpo were

MFO reacted

with methoxylamine

derivative.

Like prednisone

0x0 group does not react. gave the 2M&2lt%

derivative,

Further

[9],

treatment

the spectnm

ion (M+ m/e 574; 5% of the base peak m/e 229))

of

S m+ow1+;m/e base

peak).

543),

m/e

646;

2% of

the

the

peak

base of

silylimidazole is

gave barely

diagnostic m/e

(6X

the

the

m/e

base

present.

Negative

sensitive

and

is

enol

methoxylamine

323

of

(96/.

in

the

trimethyl-

ether

hydrochloride

derivative. than m/e

The

1% of

6171,

corticosteroid

only ion

m/e

derivative

(M+

the

and

trimathyl-

molecular

base

ion

peak

m/e

([M-Obk-HCEik3]+;

(M+ m/e Other

163.

m/e

527)

and

peak).

analyzing

method

and

derivative

272).

less

the

453)

2f+2R1s

trimthylsilyl

2l4?-3’IX

([M-QI+]+;

765

m/e

the

Cl1

MP with

are

of

As for Cc/a

of

discernible,

ions

276

heating

prccluced

Reaction

648)

([MXX&-HOSiMe3]+;

Extended

silylimidazole

TPEIEOXD131

useful chemical

selective

if

derivatives microgram

ionization

by electron

quantities Cc/E

has

of been

iqxct

steroid shaJn

are to

be rrore

[8].

This study was funded entirely by the Race Track Division of Agriculture Canada as part of their Equine Drug Evaluation Program. We thank Dr. M. Weber and the staff of Agriculture Canada’s Equine Center for supplying the samples and A. Stevenson, supervising chemist of Race Track Division, for organizing the equine drug research program and for editorial assistance.

1. 2.

Moss, Ebling, lJ,

3. 4. 5.

7.

8. 9.

296

H.J., J. ENDRIXIIN. S.J. and Jusko, W.J.,

7, 129 (1967). DRUG METAB. DISKIS.

(1985).

Szefler, S.J, Rose J.Q., Ellis, E.F., Spector, S.L., Green, A.W. and Jusko, W.J., J. ALLERGY CLIN. IMEmL. 66, 447 (1980). Wagner, J.G., Disanto, A.R., Gillespie, W.R. and Albert, K.S., RES. COW. CHR% PATH. PHARMACCL. 2, 387 (1981). Gray, C.H., Green, M.A.S., Holness, N.J. and Iunnon, J.B., J. ENIXXIN. 14,

6.

M.S. and Rylance, W.F., Szefler,

146

(1956).

Bailey, E., STEROIDS lo, 527 (1967). Lewbart, M.L. and Schneider, J.L., J.ORG. CtIEM. y’, 3513 (1969). Houghton, E., Teale, P., IhoMsia, M.C. and Wellby. J.K., BIOMED. SPECIROM. 2, 459 (1982). Thenot, J.P. and Homing, E. C. ANAL. LETT. 1, 21 (1972).

MASS