作 者 :蒲晓辉,孙进,张鹏,王永军,孙英华,何仲贵
期 刊 :中国中药杂志 2011年 36卷 14期 页码:1959.
关键词:羟基喜树碱 ;纳米混悬剂 ;口服给药 ;药动学 ;高效液相色谱-荧光检测法 ;
Keywords:HCPT , nanosuspension , oral administration , pharmacokinetics , HPLC-FD ,
摘 要 :目的: 研究羟基喜树碱(HCPT)纳米混悬剂口服给药后在大鼠体内的药代动力学。 方法: 采用高效液相色谱-荧光检测法(HPLC-FD)测定HCPT的血药浓度,Diamonmsil-C18 色谱柱(4.6 mm×200 mm,5 μm),流动相甲醇-0.3%冰醋酸(用三乙胺调pH 5.0)(57 ∶ 43),流速1.0 mL·min-1 ;FP-920 型JASCO荧光检测器,激发波长363 nm;发射波长550 nm;柱温35 ℃;进样量20 μL。用DAS 2.0药动学程序处理HCPT的血药浓度-时间数据。 结果: 本测定方法HCPT在1~50 μg·L-1 线性关系良好,最低定量限1 μg·L-1 ;日内、日间精密度均小于4.3%;高、中、低3个血药浓度的提取回收率分别为98.94%,95.88%,102.7%,符合体内药物分析的要求。HCPT在大鼠体内的药代动力学符合二室开放模型,C max 为13.10 μg·L-1 ,T max 为0.75 h,t 1/2α 为8.242 h,t 1/2β 为136.122 h,AUC0~t 为116.77 μg·h·L-1 ,AUC0~∞ 为161.93 μg·h·L-1 。 结论: 本研究中的HPLC-FD专属性强,重复性好,操作简便,适合于研究HCPT在大鼠体内的药动学和药物浓度检测。本实验中的纳米混悬剂能加快HCPT口服吸收速度,为提高口服生物利用度提供了可能性。
Abstract:Objective: To study on pharmacokinetics of hydroxycamptothecine (HCPT) nanosuspensions in rats after oral administration. Method: The plasma concentrations of HCPT were determined by HPLC-FD. The analysis was performed on a diamonsil TM C18 column (4.6 mm×200 mm, 5 μm) with 0.3% acetic acid-triethylamine buffer (pH 5.0) and methanol (57 ∶ 43) as mobile phase. The flow rate was 1.0 mL·min-1 ; the excitation wave was set at 363 nm, and emission wave was set at 550 nm; the temperature was 35 ℃. All data of concentration-time of HCPT were treated with pharmacokinetics program DAS 2.0. Result: The concentration-peak area of this assay had a good linear relation in the range from 1 to 50 μg·L-1 , and the minimum limit of quantitation was 1 μg·L-1 . The inter- and intra-day precisions of HCPT were smaller than 4.3%, and the accuracy were between -5.59% and 5.59%. The recoveries of HCPT in three plasma concentrations including high, medial, low concentration were 98.94%, 95.88% and 102.69%, respectively, which was in line with the request of biopharmaceutical analysis. The plasma concentration time profiles of HCPT fitted in two-compartment models well, and the main pharmacokinetic parameters found for HCPT after oral administration were as follows: C max 13.10 μg·L-1 , T max 0.75 h, t 1/2α 8.242 h, t 1/2β 136.122 h, AUC0~t 116.77 μg·h·L-1 , AUC0~∞ 161.93 μg·h·L-1 . Conclusion: The HPLC-FD method was simple, with good specificity, reproducibility, and could be used to investigate the pharmacokinetics and determinate the concentration of hydroxycamptothecin. The nanosuspension in this study could accelerate the oral absorption rate of HCPT, and make improving bioavailability of HCPT possible.
全 文 :8;*U ¨Ü[©sª«|אַۯ°ÛǾ ]DEþ~ÇÊ][ ¹%º P#W # Ý» W # Eä W # .:e W # Ý) W # ¼½s W ! !Pg ü#A¯ K¯J # ü# SH ^[LMM^% Wg IJKIA¯ K¯J # >à IJ PPMMPX $ ; %! q & ©sª«| $.8@% אַۯ°ÛǾ]DEþ~[ÇôÊN] . ×=éIj{b ;  Ia% $.8B;JR% %Ô .8@ [QÇ< !R$I/)I0&; PN {b¢ $^gX IIpWMM II!L ! I%! ÙÊjÆo ;MgOi Ç@ $ j?ê U.LgM%$L[s^O%! ÙL PgM IB/I) QP &J8;_WM â 9=1T ÂIa%à ! Vx OXO )I& Hx LLM )I& ¢¬ OL l& Q· WM ! B R=1 WgM ÇÊ]PeÖ¥ .8@ [QÇ< ; 7() . ¤%Ô
.8@ ] P kLM ! */B QP n F:; ! JÔ·# P ! */B QP & ¥~ # ¥936 ^gOi& = # r # O QÇ<[µ#ÈH£6uê _Ng_^i! _LgNNi!PMWg[i! /Äþ~ÇÌ6[& .8@ ]DEþ~[ÇôÊN]/ÄüUCâ !4 I$K ê POgPM ! */B QP !E I$K ê Mg[L #!! PZW # ê NgW^W #!! PZW ê POXgPWW #!=, Mk! ê PPXg[[ ! */#/B QP !=, Mk y ê PXPg_O ! */#/B QP . ¤r[ .8B;JR «Y1 ! ij; ! èEyÉ ! LÄ .8@ ]DEþ~[ÇÊ]ÑÇÌ ¤§¨r[ï¬®Û ga^ .8@ ¯°GHL ! êµ=¯°ã̬µo>*g $ %! ©sª«| & ï¬®Û & ¯°ÛÇ & ÇÊ] & =éIj{b ; ÂIa% $ 8$f %!WMPMPWW[MMN $ E q % ! æ i - s © . W $ _[O % u v $WMM_7_OMOMM% $ > %! ! ·/0 ! ]^ ! &±×ÇÌÛâÑãÌÇÛ][ !@(&. $MW^%WO_NXOWP!-;I$&. #(`#/)**4\*I$&gG/I $ > %! YYß ! òñ ! &±×ÇÌÛâGªY·FG[ !@(&. $MO[N%ONNMXNM!-;I$&.U*#^WL\PXOgG/I !! ©sª«| $#A5%/KAG$IU2/2#(G)!.8@% õr ¯èQùöRSY_É̪« 4/.,!)!3-2/ /2?.&%/!/ rZè[| ãÌ| .8@ è.u[ | ! YrãÌ| ! Yr@ ! i@Y4Ú¥ & Y rl ! `xr ! ¯°GHC vhâã_ .8@ ,¯°ÜÛ\ ! Yj¤§¨Ù×Xw ; =MYÜ> .8@ ï¬®Û ! Z7±*g [GHã¥/T[
T ! á.>Jñ[ 8;*U ¨ Ü[ .8@ ï¬®Û M>A 8;*U ¨Ü[ .8@ אַÛ[ÇÛ]Y³K>`M[¼® ! ¤ §¨ü¬ $G/;W MNCâC
-]6~GH [è´ ]js©_ ! ¤Aª]DEþ~ÇÊ ]ÉêE>Q|D ! êƧ 8;*U ¨Ü[ .8@ אַÛgµ= .8@ [¯°ã̬µoù [¦) ]vhâã_?HI\ê ! Y j7«rA .8@ ÇÊ][`?ÛÇê ! fA .8@ ¯°ÛÇÇÊ][e ! ¢ªõ .8@ [¯°ï¬ÛÇFã[ÇÊ]Ûe P;^( Yj ! A .8@ ¯°ï¬ÛÇFã[þ~ÇÊ] wèi&[@Ø ! êTU .8@ ¯°ï¬ÛÇF ã]âã\[*gµoѦ) ! Á B;[MMM =éIj{bß $ ¥¤ .2$G# $ ! y èlÊQà %&J8;_WM â 9=1T ÂIa%à $ ¥ ¤6Ip}äÎ %&=d;P âIþ^LÄà $ øù x>ßàå %&@?B;PX %}ße $ _&>ö] ßàå %&..;W (u´¬l@ $ Î.àè# $ % ©sª«| $ Ùøc¿¥ÜÇ ! q o __i%& ª«| $ _&Ú½\N %&.8@ ï¬®Û $ lÜ %&.8<-LM $ _&¤9ÚwÀ÷Ñè# $ %&@8?1$ 9øl®¤rýÈÂå %& Æo $ {b q ! 8nÚöw\]çÛå %& jÆ $ {bq ! múU çÛè#$ %& ªççÛê6q u a02$% DE ! þi WOM kWLM *! ]KÝÇö D]ÊÌrßµo #
i0 # !`.8@ þ~6
[JK # !` !` {bFq !R$I/)I0&; PN {b¢ $ ^gX IIpWMM II!L ! I%& ÙÊjÆo ;MgOi Ç@ $ /_L_P/ { OX ï{ P^ WMPP ÷ [ ² !!!!!! ! ! ! ! ! !!!! ! ! ! ! S/&gOX! :004(!P^ 94&A! WMPP j?ê U.LgM% $L[s^O%& ÙL PgM IB/I) QP & J8;_WM â 9=1T ÂIa%à & Vx OXO )I& Hx LLM )I& ¢¬ OL l& Q· WM ! B ]j{ bFqª .8@ [a%#ê MgL )*/B QP # !` #` À+rI[±Ü ! P# .8@ ÈÂÇ WgL I*! 93P· ! Í PMM IB Ä{·br ! L·Æo rÖ>ä!Ô8Ñ ! æ ! & WL I*/B QP ¦Ý I 93·# P IB ¦ÝI8 WL IB ·br ! L· jÆä!8Ñ ! æ ! Ó& P I*/B QP [Ar I V6ArIjÆQÉFJä!&p P! W!L!PM!WL!LM ! */B QP [FJArI P# 8@^gM I*! 93P· ! Í PMM IB ·b r ! aÐÆoL·VÇÌrÖ ! aÆo8Ñ ! æ ! &Y·<ê ^M ! */ B QP [¦ÝI 93·#¦ ÝI P IB Í PM IB ·br ! ÆoÔ8Ñ ! & ^ I*/B QP [ 8@ ArI ÖV6Ar IQÉä!&pY·<ê PXM ! */B QP [~Àr I ²èrI&Í ^ l [0~A # !` $` Q0Ö¥ ! #DEQ0 PMM ! B! a PM ! B @ ! V OM 0! aÐ PMM ! B ~ÀrI $PXM ! * /B QP % G PMM ! B jÆ ! V O I)! Ù L I)! PL MMM %/I) QP ß PL I) #_óI WM ! B Q # !` %`
[«Y! #ctDEQ0 PMM ! B! MYaÐ~ÀrI ! ªç # !` $` uª
è E ! ó&ct{bd $ d P%& V PM ! */B QP [ .8@ ArI PMM ! B aÐp PMM ! B ctQ0 r ! MYaÆo PMM ! B ®ªç # !` $` uª
è E ! ó&j\[{bd $ d P%& #3çDEÛǾ[ Q0 PMM ! B! # !` $` uª
èE ! ó&{ bd $ d P% 0¼. ! Q0r~¬ÌYY¡X .8@ G~À[%Ô # !` &` À+Pn[`Ü ! #DEctQ0 PMM ! B 6uaÐ .8@ FJArI PMM ! B! Íj\Q 0ÇÌ<6uê P!W!L!PM!WL!LM ! */B QP ! # !` $` uèE ! ìYaÐ PMM ! B jÆ ! S%Ì< êóµÀ # S%Ìi~ÀÌ[TU_궵 À ! a¿J6ü¯QÉÈ9sP ! &[ûnÈ 9
PÓêÀ+Pn nÈ9
Pê FYMgMO >mMgMWN!(YMg__N O! ¿iF(ê PZ4 W .8@ ] P kLM ! */B QP n F : ; ! J Ô · # ê P ! */B QP =g ctQ0 &7g AÑ~À &g Q0 & $ g.8@& % g ~À 8@ d P!.8@ Z·%Ô{bd # !` `
93i+/ ! #ctDEQ0 PMM ! B! # !` $` uª
±Ü # r # = O Y· < $ 6uê W!PM!^M ! */B QP % [Y·SÜ $e% ! QÉ X ¤6 ! d}%Ô O 5! .P e [< 0¼. !.8@ [¥~ # ¥93 31R 6 ^gOi! +/] LgL_i g~ ! 93i+/ :; $ ¼ P% ¼ P!.8@ QÇ<%Ô[93Ñ+/ç¨ $%YO% aз Z ! */B QP %&· Z ! */B QP ¥~93 31RZi ¥93 31RZi +/ 3-Zi W Pg_O PgLX Wg^W LgL_ PM PMgMO ^gOM Pg__ Pg_^ ^M O_g[ Mg^L PgP[ MgPM # !` (` RÔç¨ ! #ctDEQ0 PMM ! B! # !` &` uª
ÜÝ # = W Y·< $ 6uê W! ^M ! */B QP % [ ! 7<%Ô O ! 6u VQ0U¬ $WL l% ªÍ ^ #!QWM l Í OM 5! Ëñ O 5Ï ; Ö k*GÖ¥¾[Q0 Í W # [RÔ 0¼. !.8@ Q06u ]U¬ªÍ ^ #!QWM l Í OM 5! Ëñ O 5Ï ; Ö k*GÖ¥¾[Q0Í W # ¾RÔ $ ¼ W% # !` )` ÈH£ç¨ ! #ctDEQ0 PMM ! B! # !` &` uª[
ÜÝ # r # = O < $ 6uê W!PM!^M ! */B QP % [ ! 7<%Ô O & Z7#ct㥥l PMM ! B! # !` &` uªÖ ¥ ! Q6 ! ó&j\TU 7|< W Ö /MX_P/ { OX ï{ P^ WMPP ÷ [ ² !!!!!! ! ! ! ! ! !!!! ! ! ! ! S/&gOX! :004(!P^ 94&A! WMPP !! ¼ W! DEQ0r .8@ ]YZ Fqª[RÔ $%YO% Fq aз Z ! */B QP %&· Z ! */B QP 31R Zi 3- Zi U¬ $WL l% ªÍ ^ # W PgN_ WgLO QLgO_ ^M ^MgLN Og[O OgWL QWM l Í OM 5 W PgN^ PgXX Q[g_[ ^M O_gNO WgXO QOgXO O k* W WgPW ^gOP MgWN ^M O_g[ LgXM QLgXP µ#Ì]U¬ $WL l% ªÍ W # W Pg_O PgLX QOg^P ^M ^MgWO LgNM WgOM ¥
[TU_.Pµ#ÈH£ 0¼. ! .8@ Q0ÇÌ<] W!PM!^M ! */B QP 7[µ# ÈH£6uê _Ng_^i!_LgNNi!PMWg[i! ÈH£ :; $ ¼ O% ¼ O!.8@ QÇ<%Ô[ÈH£ $%YO% À½Y·< Z ! */B QP ÈH£ Zi 31RZi W _Ng_^ jPg__ WgM PM _LgNN jMgXL Mg[ ^M PMWg[ jPg^L Pg^ # #` ÛÇ
UÑ×® a02$% DE $L # %! §¨h%² PM #! l]Å l ! V .8@;)$)/040U()0/) PM I*/V* QP Û·® Û5DE 6uÛǾ MgMNO! MgWL! MgL! Mg[L!P!W!^!X!N!PW!W^!OX # #Qç MgL IB! ÍèÛ[ßç·r !PW MMM %/I) QP ß PM I)! 6Q0 ! QWM l 0rA # $` ÇôÊN]([%Ô PM I*/V* QP [Û·¯°ÛÇ`ÜQÇ< ; 7Pn $ d W% V§¨²&QÇ< ; 7() R=1 WgM ÇÊ]PeQÉÖ¥ ! Ó&O|ÇÊ ]( $ ¼ ^% $ , ¤JK> .8@ ¯°ï¬®Û[þ~Ç Ì<%Ô
!
]¨Æ¼.6
^L # f B # +/ # iR; ! LÄ .8@ ¯°ï¬®Û[ þ~ÇÊ]ÑQÇ vhA .8@ [ a%
è .8B;,S!.8B;JR P!L;X( !B;<1 [( ! ªr .8B;JR yÉ # Ëy # fB ! Yj\ê6 úÙÊjèÆo ;871 [( # jÆ ; Ç@¥¦ I X( ! f]¤§¨ç¡Æo ;871 EêÙÊj ! !! d W!.8@ ï¬®Û PM I*/V* QP [Û·¯° ÛǾ]DEþ~[Ç ; 7Pn $%YL% ¼ ^!.8@ אַۯ°ÛǾ]DE þ~[ÇÊ]( $%YL% (
_ =, Mk! Z ! */#/B QP PPXg[[ jOLgW^ =, Mk y Z ! */#/B QP PXPg_O j^ LgXN ! I$K Z# Mg[L jMgMM 4 I$K Z ! */B QP POgPM j^ g^W ! PZW # Z# NgW^ jPMgLX ! PZW Z# POXgPW jWOOgW^ BZJZB/# QP /V* QP XLg^_ jPXg_P N PM Z# QP MgMXW jMgMW^ N PW Z# QP MgXW jMgXM N WP Z# QP MgWX jMgWO N $ Z# QP [g^L jPPgLN U PZJ ZB/V* QP P XXNgNX j^ MLgLN 0RFã¤ì¡= ! Í .8@ [nfD ! V .8@ [t\? 871 r¥<[af`Úª ÙÊjjÆ ; Ç@¥¦I ! >ª¡ç¨Ú ¤ ! Jñ/ÔÆo ; Ç@¥¦IEêÙÊj ! Ó Æo ;MgOi Ç@ $ j?ê U.LgM!L[ s^O% ]jÙÊjª ! 6; ! a%#*kp MgL )*/B QP ! g|A .8@ QÉÇÊ][a% & Q0Ö¥7 ! EFI ; IW#Ñw t7RI ; IW#[6 ! ìÈH£ & fwt[ÈH£= # èEyHR7 é ¾P>twÛÆo # jÆ # Æo ; jÆ $Ps P%! 0RjÆwt[ctQ¤ìJ # ÈH£J= ! YjJñ´µjÆEtwÛ .8@ jA6ZY·ê OX^gO^!&/*, ê PgM[! ª lrÑr&Y; ! ]j .8@ *9¨êã ÌÇÛ]6¨Fãr[{ ^ ¨ .8@ ¯°ã̬ õªxÚ¯°ÜÛ[bcD ! Yjâ /PX_P/ { OX ï{ P^ WMPP ÷ [ ² !!!!!! ! ! ! ! ! !!!! ! ! ! ! S/&gOX! :004(!P^ 94&A! WMPP ã_¯°ÜÛ4,\ vh7«1l[ .8@ ¯ °ÜÛèùþl+# L!Xã0ÔIئ # lX + # rÛ # Ø¦Û # 0ÔIXãÑ-h½X[ï ¬` P;^!N;_( ! ¿ÜÛ[ã̬¿YjZ ! þ CEÑç¨CE&D ! VئÛÑrÛ[(A ã̬&] OMi ^ ÐZ§.&Æ.> .8@ ¯°ã̬C אַÛwèµ=ÇÌrÖ # rKL£GaÇ̯°¬[C- ! vhÏ _S[אַÛ꯰ÜÛ ! ûöׯ # ): Y$ $%$U$I4)(%# b%%ù $(I()5% PM( Yj ! ¤ÜÝ> .8@ ï¬®Û ! >AªÇÊ]É êQÉ> ! TUªõ)*µ= .8@ ¯° ã̬ §¨0¼. !.8@ ¯°ï¬®Û]DE þ~[ÇÊ]¡P/ÄüUCâ !.8@ ï¬ ®Û¯°ÛǾ N $ N PM D PMM ý^ ! *í .8@ QÐþ~¾GH^ ! f±rCU[LM& 0 &! I$K õ ^L I)! f7«A¯° .8@O I*/ V* QP [6EQÉÇÊ][u½ .8@ [ ! I$K 6 uê O # O( ! Z7i7« ^( [j !.8@ ï¬ ®Û¯°ÛÇÈÂǯ°ÛÇGH^ ! ® ! ¤ dDÙm¡i .8@ X¬®Û¯°¾DEþ~[ ÇÊ]( ! `Æ.> .8@ אַۯ° ÛÇX¬®Û¯°ÛÇGH^ ! Vã̬ `è.uµ= ìõ .8@ אַۯ°ÛÇ[ =, Mk! ê PPXg[[ ! */#/B QP ! %7«r1l [lX+Ñ0ÔIئ[ =, Mk! W( ! f6 7«1l[ $P^g^[ jMgN[% ! */#/IB QP $ §¨A Øê6E ! ÛÇÛ·ê WM I*/V* QP % O( #$X_OgN j WOgL% ! */#/B QP $ §¨AØê± ! ÛÇÛ·ê PW I*/I QW % ^( ! Ðè*gõ] .8@ אַۯ °ÛǾ]r^LrK ! QÐ6¾Y U. * [D\rÖ !.8@ PiK|ÚX¬` ZfÒeGH ! JFõYêç¨Ì [YZGY Z§¨Ug[ç¨-C`Ú[ wþÈYèS Q|D ! Z7 .8@ ¯°ï¬®Û`èS Q|D[á.ÑC $ % P (! ¿Yó ! ¹¨© F ©sª«|lX+[ÜÝGDEþ~ÇÊ ] 9(F rÇöD]]1 ! WMMN! O_$W%! POWg W (! ¿Yó ! ¹¨© F ©ª«|ùþl+\ÑL!Xã0ÔI ئ[DEþ~ÇÊ] 9(g Ç]iâã ! WMMN! PX $^%. WLOg O (! [W$ ! ¹Z! F ©sª«|ئÛ]6Eþ~[ÇôÊN ]GÙ56 9(F rÇ! WMMP! PW$PM%. LN_g ^ (! ï ! ¹N ! ·º ! F ©sª|r]æ$áFþ~[( Aã̬ 9(F râãÇ¥]£¤ ! WMML! WP$P%. ^Xg L (! [! @þ ! ÏJ4 ! F ?H﬩sª«|[ÇôÊ N]ÑÙ56 9(g ræ$âã ! WMML! OW$P_%. PPPXg X (! ] ! ±Ý^ ! ïà ! F ©sª«|= YZÛÇfg[Ç Ê] 9(g rÇöD]]1 ! WMML! OX$X%.LOg [ (! ¹_A ! ¬å ! ?×Í ! F hjüo½X[©sª«|ï¬ Y¶þ[ÜÝGª6EÙ56 9(F Ç]]1 !WMMN! ^O $P%._Pg N (! ` !
! Qta ! F YZhjüojA6ZY·Aw¶ ©sª«|[ 8-?;8.R= ﬦ§þ®!Ñþ~ÇÊ] Éê[st 9(F rÇ]£¤ ! WMM^! ^M$WP%. PX^Og _ (!B4 C! 14) 9! a$)*d9! (2$&FB]45 G#%/I$2/*%$U#A;(&(G2%/0; U%$A/)`$2/) I$000U(G2%/I(2%GI(2#/5 6/%2#(5(2(%I)$2/) /6 #A5%/KAG$IU2/2#(G) ) %$2U&$0I$9(F#%/I$2/*%$U#$! WMMN! X[$_ZPM%.NOOg PM(!B4 7! c#$)*ceF>/D(&G/&/);0U(G6GIG%/0U#(%(0E2# #*#&A 50U(%0(5 #A5%/KAG$IU2/2#(G) G/%(02#(%U%(U$%$2/)! %(&($0( b(#$D/%! $)5 2#(%$U(42G(6G()GA$*$)02G/&/)GG$)G(%9(F9 8#$%I1G! WMMX! _L$PW%.WXP_g /WX_P/ { OX ï{ P^ WMPP ÷ [ ² !!!!!! ! ! ! ! ! !!!! ! ! ! ! S/&gOX! :004(!P^ 94&A! WMPP =DIHF BCME/G./TB[0CAD0TKBL32O?C/CBKIKMACK0BCKV0DE/N010DF BL0CE0N0D0CU O-UM0CG/DK/LDAGBG/1/H.0C0KDG/D0BC 8,+$/#4 P! W ! 1,>9) W ! c.=>?8()* W ! a=>?d/)*H4) W ! 1,>d)*#4$ W ! .-c#/)**4 W ! $Pg$3/(./2-?!&2/04)0-=-)8@-%/% B%&-(#&!7! N/&8-%= ^[LMM^! 43&%/& W*:23))0)8$3/(./27! :3-%7/%= $3/(./2-?!&2/0B%&-(#&!7! :3-%7/%= PPMMPX! 43&%/% 6NKDG/TD(8NWATD0RA. @/0245A/) U#$%I$G/V)(2G0/6#A5%/KAG$IU2/2#(G)($.8@% )$)/040U()0/)0) %$20$62(%/%$&$5; I)02%$2/)F>ADEBH. @#(U&$0I$G/)G()2%$2/)0/6.8@E(%(5(2(%I)(5 bA.8B;JRF@#($)$&A00E$0U(%6/%I(5 /) $5$I/)0& @< PN G/&4I) $^gX IIpWMM II! L ! I% E2# MgOi $G(2G$G5;2%(2#A&$I)(b46(%$U.LgM% $)5 I(2#$)/&$L[s^O% $0I/b&( U#$0(F@#(6&/E%$2(E$0PgM IB/I) QP & 2#((KG2$2/) E$D(E$00(2$2OXO )I! $)5 (I00/) E$D(E$00(2$2LLM )I& 2#(2(IU(%$; 24%(E$0OL lF=&5$2$/6G/)G()2%$2/);2I(/6.8@E(%(2%($2(5 E2# U#$%I$G/V)(2G0U%/*%$IR=1 WgMF:AKI1D. @#(G/)G()2%$; 2/);U($V $%($/62#0$00$A#$5 $*//5 &)($%%(&$2/) ) 2#(%$)*(6%/IP 2/LM ! */B QP ! $)5 2#(I)I4I&I2/6]4$)22$2/) E$0P ! */B QP F@#()2(%;$)5 )2%$;5$AU%(G0/)0/6.8@E(%(0I$&(%2#$) ^gOi! $)5 2#($GG4%$GAE(%(b(2E(() QLgL_i $)5 LgL_iF@#(%(G/D(%(0/6.8@) 2#%((U&$0I$G/)G()2%$2/)0)G&45)*#*#! I(5$&! &/EG/)G()2%$2/) E(%(_Ng_^i! _LgNNi $)5 PMWgX_i! %(0U(G2D(&A! E#G# E$0) &)(E2# 2#(%(]4(02/6b/U#$%I$G(42G$&$)$&A00F@#(U&$0I$G/)G()2%$2/) 2I(U%/6&(0 /6.8@62(5 ) 2E/;G/IU$%2I()2I/5(&0E(&! $)5 2#(I$) U#$%I$G/V)(2GU$%$I(2(%06/4)5 6/%.8@$62(%/%$&$5I)02%$2/) E(%($06/&/E0. 4 I$K POgPM ! */B QP ! E I$K Mg[L #! ! PZW # NgW^W #! ! PZW POXgPWW #! =, Mk! PPXg[[ ! */#/B QP ! =, Mk y PXPg_O ! * /#/B QP F2BCT1IK0BC. @#(.8B;JRI(2#/5 E$00IU&(! E2# *//5 0U(G6G2A! %(U%/54Gb&2A! $)5 G/4&5 b(40(5 2/)D(02*$2( 2#(U#$%I$G/V)(2G0$)5 5(2(%I)$2(2#(G/)G()2%$2/) /6#A5%/KAG$IU2/2#(G)F@#()$)/040U()0/) ) 2#00245AG/4&5 $GG(&(%$2(2#( /%$&$b0/%U2/) %$2(/6.8@! $)5 I$V(IU%/D)*b/$D$&$b&2A/6.8@U/00b&(g XAF VBGHK(!.8@& )$)/040U()0/)& /%$&$5I)02%$2/)& U#$%I$G/V)(2G0& .8B;JR HB0.!+]%#)QTWT..#+!!!%#& $
$Q ! ¦§¨ % /OX_P/ { OX ï{ P^ WMPP ÷ [ ² !!!!!! ! ! ! ! ! !!!! ! ! ! ! S/&gOX! :004(!P^ 94&A! WMPP