Dnoa
CI,n.
01....1_
Ph;lrma«>~lnCI
18 (51 365·)80 1990
OJI ~·596 .190 1()())S-O lI> 5, S08.00iO () 0\1)1'1
PfC'~
['m'\cd
All "ghl) rc~"cd CPI lind OncoI08), t-kdllmlschc Um\'crsi lalskllnlk . and [ns mu t fur Organlschc C hemic denpeocll\~
'" m
". J 77
\luo\anlronc. a q 1010\IC anlhracenedlon(' dl'rl\ a\l' e. has gl' en clinical e\ Idrncr of I!..'ndklal aCII\II~ m breast cancer. I}mphoma and leukaemia. &\('r31 dlfferenl mrchan Isms of aCllon ha,c ocen suggesli.'d 10 acrounl for thiS. In addilion 10 Inlercalallon. biological ctTl"CIS such as l'lrclroslaliC IIlleraCIiOnS ,,"llh DN A. DN"'prOlelll cross-lin ks. Immunosuppressl' e aell\ I\ICS. mhlbltlon of lopolwmerasc II. prostaglandin biOS) nlhCSIS and calCium rrlease ha\e oc('n d('$oCnocd \ anous methods of drug mOllitonng In blolOSlcal flUids and tissues are a' allable the hlgh("st SCnSIU\II) has bccn achlc'oo ,,"llh high performance hquld chromatOSnph~ ""Ith c1cctrochemlcal delcctlon. radlolmmunoassa) and enl)mC hnked Immunosorbent assa) Earl) pharmacolllleuc studies of mllO\antrone III e\penmental amma!5 uSing I7Idloac\I' C ma t("nal sho,,"oo an e:\lenSI'e !Issue dlSlrlbulion and a long term mal plasma half-11ft. Tht besl iii for the plasma conccn tr.:l\Ion·\lme rUT\t' III hunans IS achle\td m a 3companmenl modd ~II studies reponed a shon absorpllon half-life of bet""een 4, I and 10,7 millu tes. ,,"llh thc dlSlrlbu!lon phaSl,' oclng oct""een 0.3 and 3.1 hours. In contrast. till' ,alurs of thr t("rmillal half-11ft' arc qUlle 'anable. rangmg from 8,9 hours to 9 da)S [)Iffrrenctn.)1 pUI: Ull!d ICU l woP -qc ;)JJ" SIJJliJ SUll1lUq-.)sop :s.)nIC\
.Jrl\-
J1W;)1
-s \s puc ICJUOIlJ,xIC11U1 UJ.)"I.Jq JJUJJJjjlP IC11u.:>u
JJI)R PJ u,)Sqo .lJ.l" 1flin' ~)O SUOllCJ\
-od\')lJJ C p.)luJwnJOp sJlpms ljlO8 "\I,J\llJJdS,JJ
-U;);>UOJ wnJ~ "\Il'CJ IClpJCJUJd .lljl 01U1 3wOOZ
"lj -1 (3n' 001 pUI: 9Tf ,JJJ" uonellslulWPC IC,JUO\1
puc smOlj
000 8S:
;)P1S0U~qCJC ,JUISOI\J ljll" l.lljl;}301 P;}J,JISIUIWPC
-J,xlCJlUI JJIJC s.)rl\" JScJJ,\C ,Jljl puc 'lfliri
SR ..... SWl ,JUOJ\UCllOIIW 'cIW;)ClIn,JI snou;)30[J\w
0\ i:Si: WOJJ PJ~UCJ SUOIIClIUJJUOJ ICJUOluJdCll
.llnJc)o .)Sdel.lJ [BlpJRJUJd 4]1" IU;}l1ed I ul "1 (3n' OOS: 01
OO( JO ;}SUeJ
.lljl Ul II!IS .)J,)." SUO!IC.lIU')J UOJ
;)41 'SU!50P J,JIJe sm04 l'Z :p,)msc,)w .)J.)." 1fliw
Ol
01 dn suone.llu,J.lUOJ SnJp pmU ICUldSO.lqJ1;)J
>tc.x1 :(qL861) "IC IJ l;)SU~Ulj3
\q pJIJod.lJ .lJ,J."
IUJWIRJJI IC:hl4ICJIUI JJlJc RICP
-U[ "uollclnJJJ.) JIWJl5\S puc JnSSll mownl 1I1oq 01U1 uOlldJOSJJ Ul SJJu,J.lJ1JlP SIJJUJJ \lqcwnS;)ld lIJ!lI" 'Jlqc!lc,\
\,IJW,JJlX;) JJJ."
SlUJ!ICd Icnp~,\~p
-U! UI sJ JIJweJcd JI\JU I>t OJI:WJclj d "uol\n ,os s.J,J -SUIH)O JwnIO ,\
It
C UI ~w/'iIw
Or
pUll
s:
U,JJ"lJq
sJSop SU!lCICJSJ Ul UJ.\l3 UJJq sCli ,JUOJlUI:XOll~~
J~I.lUI>tOJCWJC4d
'(8861 "IC lJ luncQ -IljJOl8 :8861 "[I! I;) SUJqlvl pallsll
"JUOJ1UCX01!W JO UOneJ\Slulwpe ICJJljlCJIUI Jljl
-qnd UJJq J .\clj SluJlIcdjO JJqwnu JlqC1JplSUOJ C
sJ3cJnoJs!p {p,\IIJe J;)JnIJC)nUCw J41 UOSCJJ Slljl
Jnoql: I:lCp JI1JUl>tOJCWJClId puc "SII:UI
JO) puc '(886 [ "IC I,J [cS,JIS) IUJpuJd.lp .lSOp \[lc,J[J
UI p.)lpms UJJq SClj uO!ICJljddc II:JUOllJJdCJlU1
sc ..... 4JHI" PJq!JJSJp se."
\IlJ1XOIOJnJU
"(L86[
P.l\I!IJP
11 /1 ')slllid
"II: lJ SJ;)I,Jd) UOllClnJ
puc ApCJ 'S,J!pnI5 ICW!UC UI "(6861 "II! I.l lleH) SlooJ
-JIJ pJJlcdwl SUllSIX;rJJd JO SnJp pUOJJS C)O UOII
,),\JJ U )0 UO!lcuq,)\WJp p.)SnCJ "SIC,UJ IUI A[>tJJ."
-CJ\SIUIWpCOJ 5C lIJns "PJyllU;)PI Jq PlnOJ SJOlJllj
IC AIICJJljlClIUI UJ,\IS 'Sri S:"ll .lU01IUI!XOllW SC ..... 01
ICUOIIlPPC UOllJ;)rUl)O J\IS ,Jlj\ II: SJSOJJ,JU JO slJod
5C SJSO p 'SAJ>tUOW UI "SnJp J41 01 pJIcpJ \Iqlssod
-JJ ISOW U[ ".IJIlJq SI JUOJlUI:\O\IlU)O SJICSI:\CJ\X,J
SC ..... lI J! lj" UOllClOlJ:I\,JP [CJISOIOJn,JU R SC" .lJ.llII
JO JJUCJJIOI
S')sCJ ISOW Ul Inq "P,JAJ.)SqO SC,,, SIl,JJ JI \ SUldOJU Ul
P;lJUdIUO.) "PSS61
UOllJnp;)J V "(S:86 [ "IC I;) ;)lqcllnZ :S:86 I "IC I,J ,)).Iod
snOU,J\CJlUl \q \1,Jjl'>S P;)\':>llIJI: Jq IICJ ljJllj\\ sjJ,\JI
ICJOI ,'1I1 'SJUIP\JI:JljIUC J41
1I11 .....
"II: I;) SUJq1\,) UOIlCJ\SIUIlUPC
-1>1 :9861 "IC I.l IURlI>t1>1 :qL861 "IC I.) 1JSUlUlj3)
UOIICJIUJJUOJ JlII PUO\Jq d,)JIS SI .)\.InJ ;)SuodsJJ
sucwnll Ul p;)}Jod.lJ U,)Jq ,)\Clj SW t 01 I ,)U01IUCXOI
,Jsop JlIl pUI: (CS861 "[I: IJ SUJqI\') S.lnssn 01 \[J.\15
-!W)O U01\CJ\SlUlIUPC [CJJ41C1IUI)0 SJSCJ ..... JJ \'
-UJI\J ~pulq ihup J41 [IlJI3u[UIlllllJlllj ~I Ul)ILIlJIILI
"U;);)S SR," UO!ss,)JSoJd lJlj}Jn) ou 'SUOlsn)
-dl: J~W;)IS'S jO {l1J IXOI ~UI\IlU II ;)Sop Jljl ICljl SlJCJ
oj;) P;)U!RJp " 1[Clucd UI ".) Iq lssod sc ..... JSCUlClp .l1;)ld
JlII Su!pn[JuI "SUOSCJJ leJJ \;)S JOj P.l!Jl UJJq SClj
-WOJ WOll," UI SlU ;)!lcd IlU UI pJ ,\')lljJC SCM UOISS1W
JUOJIUeXOllW )0 UOllCJlS1UllUPC reuolS;)JOJOl
-;)J ;)1;)ldwOJ pUC 'Suo!IRJqd W OJ 10 uu:d lUCJylU~IS tn0lll~," pJICl;)IOI SC,,, UO!H:JISIUIWpC [clnJ[dcJ\u[
'(dCJJlI.L IcuOl'iI;)JOJOl
";)SOP ;)1I1 J O pJ!1I1-;)UO I noqc SC." Sn1p .lljl JO \I!pq
)0 s).}JdsV JI\;)UI>tOJCIU.lcljd S:"f
-1:1!c,\c J!WJIS,(S "JlnOJ snou,J\CJ lU ! .llj\ (q UOI\C1\ -S!U!WPC .IJljc Ullljl J.l~llj SJWll
OOt
lnoqc 'l (3n'
"\pnIS llCIUS Slljl IUOJJ PJ\U
u,),),,,IJq 1111s J1J" SUOlIC1\UJJUOJ
-;)P Jq 10UUIlJ uOIlJunj J,J\I[ p:ulCdlUI 1I11" SlUJ111!d
ICJnJ[dEJIU! slnolj t(" J.)ljc pUC '3wQ( 01 9 [ W01)
JOj sJlnJ uOllcJI[ddc leJJu.:>Q "\IIJcdcJ Jl[oqll\J1U
OOL [
pUC
OOs: [
"(L861
"IC I,) ljJSn~~1 CWOI[Jlj\05JW
[cnsn ;)1I1 )0 JUnolUC ;)lqCUC\ JWOS UICIJJ \t:41
ICJnJld puc JJJUCJ UC lJ C\O "JJJUI:J ISCJJq P,)S15CISC\
SIlJJ CWOU1JJI:J JClnll;)JOlCdJlI )0 JJUJs,JJd JlII .;q
paSUCJ 5;)sOQ
-JW U! sUOIsnjjJ [cJn.)[d)o lUJW\C;)JI ;)ljl JOj P;)ISJ\
PJU1Cld).,J Jq PlnoJ SJJUJJJlllp ;)S;)ljl IC41 pJ1SJS'ilns
se ..... JUOJIUCXOI1W)0 UOI\CJ\S!UIWPC IClnJ[dClIU[
SJOllJnc JlI.L 'sp\J[ Ulqrul[1Q ICIUJOU 1I11
"SJlpnl5 J;)ljunJ
\I
SlUJIlCd
pip UCljl .\l!JIXOlICJISOIOiCIUJClj JJJ\,)S .)JOW pJJUJ
JOJ P;)PU;)WIUOJ;)J UJJq selj ~wfliw Ot JUOJIUCXOI
-!JJdx.) cWfSlU 810 rl .IJljllJ JUOJlUCXO\IW u,J.\13
-!W jO ')sop ~U!UCIS e puc '.(\lJ! XO I JO 5[J.\JI ;)[qc
sjJ.\.)1 u!qnJl[lq P.lIC\JIJ ljll" SIUJllcd JJJUCJ \SC;)Jq
m
JUOJIUI!\OIl,,\' JO
s")IPUI~O")I~IUJl:lId
1I!JIUIl.)
374
and humans (dt: Dycker ('1 at 1988; Shepherd et al. 1987). Male rats rea:ived mitoxantrone ]mg eit her intravenously or injected into the carotid arlery. I nthe lalter group, brain tissue concen trations were 18 times higher than in the former. Infusions into the hepatic anery for primary hepatocellular carcinoma, and into the internal mammary artery for palients with advanced carcinoma of the breast. have betn tried with good clinical success. Systemic toxicity was reduced In companson with intravenous treatment, bu t pharmacokinetic data were not given.
3.6 Discussion of Phannacokinetic Data in Huma ns
All of the studies reviewed above demonstrated a vel')' short t 'hl.l (range 4. 1 to 10.7 minutes). Serum
mitoxantro ne concentrations 31 the end of infusion were between several hundred ,.glL and I mg/l and declined to below 10 ~81L. which was in the ra nge of the assay sensi tivity of several methods, within 8 to 24 hours. T hus, mitoxantrone rapid ly leaves the plasma compartment, and binds to endo thelial surfaces and penetrates into the formed elemen ts of blood (Albens et al. 1983, 1985a, b, 1989; Savaraj et al. 1982b). T he d is tribution phase (tYl.lo2) is given at between 0.3 and 3.1 hours. Many of the reported half-life values o f the elimination phase (tYl.lo}) a re between 10 and 40 hours, but 3 authors reponed much longer values (between 7 and 12 days). It is possible that in the present autho rs' own study the adm inistration of cyclophospha mide might have altered the e li mination ofmitoxantrone, altho ugh in a study in leukaemia patien ts wi th si ngle agent treatment, the same values were achieved. In addition, the authors' concept is supported by pharmacokinetic studies in animals and the data from autopsy studies with high tissue coocen tralions a variably long time after dosing. Variabilities in the estimated pharmaco. kinetic parameters in these studies may also be due to the differences in data poin ts beyond 24 hours, weight ing a nd assay sensi tivi ty. Concentra tions at day 21 after administration were between 290 and 1000 n8lL as measured by RIA ; none o f the HPLC
methods ISsenSItI ve enough to measure mLloxantrone concentratIons in the lower range. The auth· ors' combination of the measurement of mitox· an trone concentra tions in plasma and unne mIght help to overcome these problems panly, Since the solid phase extractIon method permItted the load· ing o f large volumes of urine and resulted In a sen· sitivity of 0.2 I181L murine. In summary. the foll owing consensus may be suggested: mItoxantrone ex hibits arapid inlllal dIS' tribution phase followed by a relatively slow eltm· ination phase. The results suggest a deep tIssue companment with a slow release. The very large volume of distribution shown by all studies sug· gests that much of the drug is sequestered in the tissues. All tissue measurements showed that mItoxantrone persists m human tissues for prolonged pe. riods. However, concentrations of drug in tIssues in which a tumour was located generally did not correlate with concentration of drug In the tumour itself, although exceptions to this were the lIver and brain (Stewart et al. 1986). The calculated mea n renal clearance values were between 0.9 and 2.7 L/ h/ m2 and accounted for about 4% of the dose in 48 hours. The observation in humans that 28% of the dose is excreted in the faeces within 5 days. as well as the high rate of biliary excretion in animals. supports the concept that bile may be the major route for the ehmmation of mitoxantrone.
4, Elimination, Metabolism, Metabolites 4. 1 Elimi nation and Metabolism Investigations with bi le--duct cannula ted rats. rabbi ts, dogs and monkeys, and isolated perfused rat liver (Batra et al. 1986: Chiccarelll ct al. 1984; Ehninger et al. 1984; Lu el al. 1984: Richard et al. 1989). showed that biliary excret io n is the major excretory pathway for mitoxantrone and ItS me· tabolites. In rats 56% of the radioacti vi ty of 14C·labelled mitoxantrone was excreted in 120 hours via the bile, and in to faeces of non-cannu1ated rats; an avo erage of 35% o f administered radioactivity was
found In fa{'c{'s dunng Ih{' firSI 48 hours aft{'r dosIng (Balra ct al. 1986: Chl{'carclli el al. 198-1 ). StudIes '>11th mo n lo.{'~s (Batra {'lOll. 1986) and dogs (Lu el al. 198-1) also sho'>lcd that 47.3 and 50.5%. respectJlel~. ofdrug-rclated radlOaClI\II~ '>las ellmmOIled Iia Ihe blk III\h1l1 110 hours. I n humans. Ihe mean recoler~ of radlOaCllllt~ In faeces oler 5 da~s '>las 18.3000. Indleal1l1g that. as '>11th animals. blliar~ delollflcation IS Ihe major excretor) palh'>Ia~ In humans (o\lberts CI al. 1985al. Urlnar~ excrellOn of radioacllll\Y was found to be comparalllciy 10'>1 In all SpeCiCS cxamlned. I n rabbits. kss Ihan 6 10 8% of Ihe dosl' '>I as excreted during a 6-hour obsenalion period; 10 rals and dogs. 6.7 or 4.9% of radlOaclil'lI~ has been found In Ihe same collectIOn IImc, Increasing to 13.6% of Ihe admlnislered dose in rats when the lime of colleClion '>las e.\lended to 120 hours, Following a single Intralenous dose of l~C-label!ed mllOlanIro ne. renal e\cr..:l1on of radlOaClllll~ In cancer palients lIas similar 10 Ihal 10 animals. Thest' dt'lermlnallOns of bll1ar~ or rl'nal c\cre· lion '>Icrc PI-'rfoTrlll'd b~ mcasurt'nll'nt of th..: 101;11 radloactll II~ of blk and unne. Some Inl cSllgators hal{' abo c\anllncd IhL' collected bod~ flUids b~ H PLC monlloTlng lighl absorpllon al 658nm. 10 delermlne Ihe fracllon of radioacil I H~ belonging 10 the unchanged drug. The resulls are summarised In lable I for animal studies and lable II for studies 10 humans. The signlficanl differences demon· strale thaI up 10 511\1 (dogs). n.5 10 25% (rabbits) and -10000 (rOllS) of Ihl' IOlal ncn:ted radlOactllll~ mu~1 be allnbUlcd to melaholllcs of mllo\antrone (Alberts el a1. 19S5b: Balra et al. 1986: Chiccarelll el a1. 198-1: Lu et OIL 198-1: ~·l acph..:rson e\ a1. 198-1: Salaraj el al. 198101). Three mon.' polar melabolites hale been delected In an Isolated perfused rat Iller model after separation yia H PLC (Ehnlnger el a1. 198-1). H PLC Inlesllgallons of unne. bile. and plasma of pal1ents showed up 10 -I melaboll t('s (Alberts el aI, 1985a: Chiccarelll el al. 1986: Czej ka & Georgopoulos 1988: Ehnlnger 1'1 al. 1985a.b: Lu et al. 1984: Macpherson 1'1 a1. 1984: Pa~el et a1. 1988: Sm~th 1'1 a1. 1986: Van Belle el a1. 1985. 1986) '>I hich were also obsc-rved 10 Ihe unne and bile of rals (Al'ramlS 1981: Chlccarcill el al. 198-1: Wolf el
r (Rich. al. 1986). dogs (Lu e l al. 1984) a nd abbits ard et al. 1989). However. the amount of the melaboilles delected by lighl abwrpl ion b) no means equalled Ihe radIoactIvity nOl bound 10 the pare nt compound (EhnlOger et al. 1985b). -I.:! Structural .\nal)sis of ~lelaboliles
Fell a\tempts have been made 10 e1ucida le Ihe metabolism of mlloxantronc. and on I) 1 laboratories hale reponed efforts 10 Ihe elucidation of the chemical slructure of melaboliles deleCled by sel'eral IIll'Cstlgators (Chiccarell i e l a1. 1986: Ehninger el al. 198-1. 1985a.b: Macpherson el al. 1984: Richard el al. 1989: SmYlh el OIL 1986: Van B elle et al. 1986: Wolfet oIl. 1986). The main reasons for Ihis gap In struct ural Io.no'>lledge seem to be Ihe veT) low occurrence of uri naT) melabohles and diflieullics III finding chromatographic condlllOns. allowmg their preparatllc separallOn. The anlhraqulnonoid mOlel~. coupled with Ihe high polan I) of the molecule. causes leT} low 1'01alllll~. malo.lOg gas chromalographlC separalions IOlpQsslble and complicatlOg mass spectromelric inlesligallons. Mass spenromclric delection of lhe molecule Ion o f mitoxantrone 10 unnar) cxtraCIS IS I ef) dlflicult and requires negatlle Ion deso rptIOn chcmlcallOntsa tlOn or Ihcrmospray IOnisa tion (Blanz 1989). The strong basic aminO grou p ofl he Side chams necessnales aCidiC chromatograph ic condlllons 10 nrcumlcnt 10rl l(' in teraCtions wil h uncapped Sllanol groups of reI crsed-phase Silica gel. Less acidic cond1\1ons can be applied bu t require Ihe usc ofhlSh sail conccnlral10ns, Ihercb~ limni ng Ihelr appll('ablllt~ to prepara\lle separallon. So far Ihere ha\e been no repons of effons to overcome these separallon problems b} uSing macrophasc re\ersed-phase pol~mers lackmg the potenl1al for 10ntC IO leraCllons. All de\eloped chromatographic mel hods In\olle strong changes In pH. so Ihal chemical denalunng of pH.senslllle metaboliles. especiall~ of conjugates. mUSI be e\JX'clcd. In general. the 10'>1 degree of solubllit~ of Ih(' parent drug and metabolites after ennchment IS surpnSlOg. and represe nls
Om Pharmacoklrl('/ 18 ($) 1990
J76
a major problem in elucidation of the chemical struclure. The low solubility could be related to chemical denaturing during i50lation or to ionic or hydrophobic interactions with components ofbiological origin, especially proteins. which denature during enrichment procedures. The identification of metabolites as drug-related material has been achieved either by application of a radioactive now detector monitoring the radioactivity of the eluate after adminiSU'ation of 14e. labelled mito;\antrone (Richard el al. 1989). or by monitoring the typical light absorption of the ch romophoric system al 658nm . Two metabolites of mitoxantrone isolated from the urine ofpalienls have been characterised as the mono- and dicarboxyiic acid derivatives resulting from the oxidation of the terminal methylene groups of the side chains (Chiccarelli et al. 1986). The structural el u-
T.~
I. BtIi'ry.Nl urinary '>lCtIIIOn
""""
....
R........
.... U....
""
Urino
"C 1%"
...
28.5
A. I.
""'"
...-'Y'
U_
• b c d
Unchanged
Oruo
"""
"'"
19.5 4.5".2
SO.5"
3.7
~.
0'rldioactMty loIIowIng intrlvenout
'"
•••
...
cidations have been performed by negative Ion chemical ionisation mass spectrometry and comparison of mass spectrometric fragmentation and HPLC retention times of isolated metabolites and synthesised referentt substantts. The synthesis of 5.g·bIS-(I-IJcH(2-«2·hydroxyethyl)amino)ethyl)amino )-I.4...·rl\ D ...
P~n~
Pharma,·ol~~
318
Cf/ll. Phur",uml../IIf"/ M (.~) /WO
.n,sm of k llon and p/'La~Hltl," of no"nl~ In In\1'1I_
'cnous and 'nlf1lpmIOM1lII~py In Collman (Ed ) Thr (Uf"'i'lL n.lus Dfno'·.nlro~. pp I So2 I. John W,k,. Ne ..... Yon.
""
"Ibens OS,
Pr", Viol. Bo...·(kn GT. M lK'kel C. o.llon \\IS
Mech-
anIsm of Kllon and p/'Larmaeoklnclln of no~an!roM ,n ,n....
' '1'Il0\l1 .nd IMlra~ntonoell C'hcmolht1'1lp) 1S-21. 1989 Alben. OS. ~n, YM. u'ch S.
t1\(,1'1Ip)
Ad"1l(n In ("!lttr
0."" TI'. \\ood_rd DL DIspm,tlOn of m,'ountronoe ,n pallen,s, ("nttr Treatment Re"~"'I 10 (B); 21-27, 198) Albtns Pt'na YM. u .." s. TP. Woodward OL 0.,.. POSItIOn of mllOUnlrOM In nllKff pfllJCnl1. C'!Kff RncaKh
0.",
os.
45' 1179.18&Too:ftdmp 4) 345. 1984 Chlebo ..'Sk, RT. Ton, M. Bulca ..1t L \!o oodwud DL. Mno~. Intron .. In h.. p.:alle dysfuIKIlon: faclon Ifll""I\(InIIO\I("II) Ind n:spon5f. Abslract PrQ(ft(j,np of lho: Am"'lC1In SocI .. I) of C'*MlC'DIOncolos) 5: 46. 198b CIIoi KE. Sm~ulc- JA. llan) OS. McGralh SC. 11lI1) KM CI al HIJh·ptrformanC\' hqu,d ehrom.IOSl'1lpi'll(" 1)101) for m,IO~' In lroM In pluma UIJ"I clcctroc~.c-mlCllllkl«hon JO' \ \Hand hrCdl' In klll-n'm, ,.'11> f1f1ll,h Journ,1 "t Itd.'mawl',!!'~' "-" 1 ul J",Ilum"r urug, amrlam",n, Jnd 01" lO.am,oO,·(OU'JOlr,)O,·1 anu Ih,'lr ab,,,,, lU ."n.!,'n...' oud.·" alld, f>r, ...·....-..Jln~' 011he '~.",nJI \.-a.!,·m, ,,( .... I.'",., ~1 hIli:. /\1.(111 Iarl\n~''''''''1 I T.apn", I \klan.,.,J 11.11. l'lIc.aU,on, ", J n,," ~nlliumuur ~nL I.J-d,h,d.",,·~ ~·II, .. :[~_ II ~-h>d"" ... (h, llam "'o[~lh' 'lam,"o-~ In.J n. h.a,cnedHln,' ""h n""le .. ;KId, 1I,0....... ml(';ll Pharm~_ ok'!!' 11, ~11·~.w'. I'I~I ,..har:uh 1.1) ',,,~lll. ~ Inhlblhon of ~u"an.,,'n ,"ml.l\~"'.l ml n~mll "I'ld pc'O"dlloon b, mOlO,~nl"'''''' ~rId 3m,'IanITO~, 1"0 nc.-" ~n!h~n~ j-HIII~~ I\lH5 Lalhano , ... lU'labk -\(i, Polla.d (\, \I"n.· \1 ,,'I,'J,,'n J_ CI al P~"'l'klla afu" 'mr.llh,...·al nmuIJn""n.· I In.~1 ~ 1.1nu.-lc" a"d, "uul ~~ -'11~8·"OJS, 1985 \ u ... ~'3r.lJ' l,)Q IT I'ha.maeol""nl dl~po~'"0n 01 '''_ dlh~. l\lb5 ">'J~ RI "'hJrJ,h I D \ ,tountronc I propen,'" tOI fr,-': rad· Kal (."m3I1lln an.! 101'1
CI,n.
01....1_
Ph;lrma«>~lnCI
18 (51 365·)80 1990
OJI ~·596 .190 1()())S-O lI> 5, S08.00iO () 0\1)1'1
PfC'~
['m'\cd
All "ghl) rc~"cd CPI lind OncoI08), t-kdllmlschc Um\'crsi lalskllnlk . and [ns mu t fur Organlschc C hemic denpeocll\~
'" m
". J 77
\luo\anlronc. a q 1010\IC anlhracenedlon(' dl'rl\ a\l' e. has gl' en clinical e\ Idrncr of I!..'ndklal aCII\II~ m breast cancer. I}mphoma and leukaemia. &\('r31 dlfferenl mrchan Isms of aCllon ha,c ocen suggesli.'d 10 acrounl for thiS. In addilion 10 Inlercalallon. biological ctTl"CIS such as l'lrclroslaliC IIlleraCIiOnS ,,"llh DN A. DN"'prOlelll cross-lin ks. Immunosuppressl' e aell\ I\ICS. mhlbltlon of lopolwmerasc II. prostaglandin biOS) nlhCSIS and calCium rrlease ha\e oc('n d('$oCnocd \ anous methods of drug mOllitonng In blolOSlcal flUids and tissues are a' allable the hlgh("st SCnSIU\II) has bccn achlc'oo ,,"llh high performance hquld chromatOSnph~ ""Ith c1cctrochemlcal delcctlon. radlolmmunoassa) and enl)mC hnked Immunosorbent assa) Earl) pharmacolllleuc studies of mllO\antrone III e\penmental amma!5 uSing I7Idloac\I' C ma t("nal sho,,"oo an e:\lenSI'e !Issue dlSlrlbulion and a long term mal plasma half-11ft. Tht besl iii for the plasma conccn tr.:l\Ion·\lme rUT\t' III hunans IS achle\td m a 3companmenl modd ~II studies reponed a shon absorpllon half-life of bet""een 4, I and 10,7 millu tes. ,,"llh thc dlSlrlbu!lon phaSl,' oclng oct""een 0.3 and 3.1 hours. In contrast. till' ,alurs of thr t("rmillal half-11ft' arc qUlle 'anable. rangmg from 8,9 hours to 9 da)S [)Iffrrenctn.)1 pUI: Ull!d ICU l woP -qc ;)JJ" SIJJliJ SUll1lUq-.)sop :s.)nIC\
.Jrl\-
J1W;)1
-s \s puc ICJUOIlJ,xIC11U1 UJ.)"I.Jq JJUJJJjjlP IC11u.:>u
JJI)R PJ u,)Sqo .lJ.l" 1flin' ~)O SUOllCJ\
-od\')lJJ C p.)luJwnJOp sJlpms ljlO8 "\I,J\llJJdS,JJ
-U;);>UOJ wnJ~ "\Il'CJ IClpJCJUJd .lljl 01U1 3wOOZ
"lj -1 (3n' 001 pUI: 9Tf ,JJJ" uonellslulWPC IC,JUO\1
puc smOlj
000 8S:
;)P1S0U~qCJC ,JUISOI\J ljll" l.lljl;}301 P;}J,JISIUIWPC
-J,xlCJlUI JJIJC s.)rl\" JScJJ,\C ,Jljl puc 'lfliri
SR ..... SWl ,JUOJ\UCllOIIW 'cIW;)ClIn,JI snou;)30[J\w
0\ i:Si: WOJJ PJ~UCJ SUOIIClIUJJUOJ ICJUOluJdCll
.llnJc)o .)Sdel.lJ [BlpJRJUJd 4]1" IU;}l1ed I ul "1 (3n' OOS: 01
OO( JO ;}SUeJ
.lljl Ul II!IS .)J,)." SUO!IC.lIU')J UOJ
;)41 'SU!50P J,JIJe sm04 l'Z :p,)msc,)w .)J.)." 1fliw
Ol
01 dn suone.llu,J.lUOJ SnJp pmU ICUldSO.lqJ1;)J
>tc.x1 :(qL861) "IC IJ l;)SU~Ulj3
\q pJIJod.lJ .lJ,J."
IUJWIRJJI IC:hl4ICJIUI JJlJc RICP
-U[ "uollclnJJJ.) JIWJl5\S puc JnSSll mownl 1I1oq 01U1 uOlldJOSJJ Ul SJJu,J.lJ1JlP SIJJUJJ \lqcwnS;)ld lIJ!lI" 'Jlqc!lc,\
\,IJW,JJlX;) JJJ."
SlUJ!ICd Icnp~,\~p
-U! UI sJ JIJweJcd JI\JU I>t OJI:WJclj d "uol\n ,os s.J,J -SUIH)O JwnIO ,\
It
C UI ~w/'iIw
Or
pUll
s:
U,JJ"lJq
sJSop SU!lCICJSJ Ul UJ.\l3 UJJq sCli ,JUOJlUI:XOll~~
J~I.lUI>tOJCWJC4d
'(8861 "IC lJ luncQ -IljJOl8 :8861 "[I! I;) SUJqlvl pallsll
"JUOJ1UCX01!W JO UOneJ\Slulwpe ICJJljlCJIUI Jljl
-qnd UJJq J .\clj SluJlIcdjO JJqwnu JlqC1JplSUOJ C
sJ3cJnoJs!p {p,\IIJe J;)JnIJC)nUCw J41 UOSCJJ Slljl
Jnoql: I:lCp JI1JUl>tOJCWJClId puc "SII:UI
JO) puc '(886 [ "IC I,J [cS,JIS) IUJpuJd.lp .lSOp \[lc,J[J
UI p.)lpms UJJq SClj uO!ICJljddc II:JUOllJJdCJlU1
sc ..... 4JHI" PJq!JJSJp se."
\IlJ1XOIOJnJU
"(L86[
P.l\I!IJP
11 /1 ')slllid
"II: lJ SJ;)I,Jd) UOllClnJ
puc ApCJ 'S,J!pnI5 ICW!UC UI "(6861 "II! I.l lleH) SlooJ
-JIJ pJJlcdwl SUllSIX;rJJd JO SnJp pUOJJS C)O UOII
,),\JJ U )0 UO!lcuq,)\WJp p.)SnCJ "SIC,UJ IUI A[>tJJ."
-CJ\SIUIWpCOJ 5C lIJns "PJyllU;)PI Jq PlnOJ SJOlJllj
IC AIICJJljlClIUI UJ,\IS 'Sri S:"ll .lU01IUI!XOllW SC ..... 01
ICUOIIlPPC UOllJ;)rUl)O J\IS ,Jlj\ II: SJSOJJ,JU JO slJod
5C SJSO p 'SAJ>tUOW UI "SnJp J41 01 pJIcpJ \Iqlssod
-JJ ISOW U[ ".IJIlJq SI JUOJlUI:\O\IlU)O SJICSI:\CJ\X,J
SC ..... lI J! lj" UOllClOlJ:I\,JP [CJISOIOJn,JU R SC" .lJ.llII
JO JJUCJJIOI
S')sCJ ISOW Ul Inq "P,JAJ.)SqO SC,,, SIl,JJ JI \ SUldOJU Ul
P;lJUdIUO.) "PSS61
UOllJnp;)J V "(S:86 [ "IC I;) ;)lqcllnZ :S:86 I "IC I,J ,)).Iod
snOU,J\CJlUl \q \1,Jjl'>S P;)\':>llIJI: Jq IICJ ljJllj\\ sjJ,\JI
ICJOI ,'1I1 'SJUIP\JI:JljIUC J41
1I11 .....
"II: I;) SUJq1\,) UOIlCJ\SIUIlUPC
-1>1 :9861 "IC I.l IURlI>t1>1 :qL861 "IC I.) 1JSUlUlj3)
UOIICJIUJJUOJ JlII PUO\Jq d,)JIS SI .)\.InJ ;)SuodsJJ
sucwnll Ul p;)}Jod.lJ U,)Jq ,)\Clj SW t 01 I ,)U01IUCXOI
,Jsop JlIl pUI: (CS861 "[I: IJ SUJqI\') S.lnssn 01 \[J.\15
-!W)O U01\CJ\SlUlIUPC [CJJ41C1IUI)0 SJSCJ ..... JJ \'
-UJI\J ~pulq ihup J41 [IlJI3u[UIlllllJlllj ~I Ul)ILIlJIILI
"U;);)S SR," UO!ss,)JSoJd lJlj}Jn) ou 'SUOlsn)
-dl: J~W;)IS'S jO {l1J IXOI ~UI\IlU II ;)Sop Jljl ICljl SlJCJ
oj;) P;)U!RJp " 1[Clucd UI ".) Iq lssod sc ..... JSCUlClp .l1;)ld
JlII Su!pn[JuI "SUOSCJJ leJJ \;)S JOj P.l!Jl UJJq SClj
-WOJ WOll," UI SlU ;)!lcd IlU UI pJ ,\')lljJC SCM UOISS1W
JUOJIUeXOllW )0 UOllCJlS1UllUPC reuolS;)JOJOl
-;)J ;)1;)ldwOJ pUC 'Suo!IRJqd W OJ 10 uu:d lUCJylU~IS tn0lll~," pJICl;)IOI SC,,, UO!H:JISIUIWpC [clnJ[dcJ\u[
'(dCJJlI.L IcuOl'iI;)JOJOl
";)SOP ;)1I1 J O pJ!1I1-;)UO I noqc SC." Sn1p .lljl JO \I!pq
)0 s).}JdsV JI\;)UI>tOJCIU.lcljd S:"f
-1:1!c,\c J!WJIS,(S "JlnOJ snou,J\CJ lU ! .llj\ (q UOI\C1\ -S!U!WPC .IJljc Ullljl J.l~llj SJWll
OOt
lnoqc 'l (3n'
"\pnIS llCIUS Slljl IUOJJ PJ\U
u,),),,,IJq 1111s J1J" SUOlIC1\UJJUOJ
-;)P Jq 10UUIlJ uOIlJunj J,J\I[ p:ulCdlUI 1I11" SlUJ111!d
ICJnJ[dEJIU! slnolj t(" J.)ljc pUC '3wQ( 01 9 [ W01)
JOj sJlnJ uOllcJI[ddc leJJu.:>Q "\IIJcdcJ Jl[oqll\J1U
OOL [
pUC
OOs: [
"(L861
"IC I,) ljJSn~~1 CWOI[Jlj\05JW
[cnsn ;)1I1 )0 JUnolUC ;)lqCUC\ JWOS UICIJJ \t:41
ICJnJld puc JJJUCJ UC lJ C\O "JJJUI:J ISCJJq P,)S15CISC\
SIlJJ CWOU1JJI:J JClnll;)JOlCdJlI )0 JJUJs,JJd JlII .;q
paSUCJ 5;)sOQ
-JW U! sUOIsnjjJ [cJn.)[d)o lUJW\C;)JI ;)ljl JOj P;)ISJ\
PJU1Cld).,J Jq PlnoJ SJJUJJJlllp ;)S;)ljl IC41 pJ1SJS'ilns
se ..... JUOJIUCXOI1W)0 UOI\CJ\S!UIWPC IClnJ[dClIU[
SJOllJnc JlI.L 'sp\J[ Ulqrul[1Q ICIUJOU 1I11
"SJlpnl5 J;)ljunJ
\I
SlUJIlCd
pip UCljl .\l!JIXOlICJISOIOiCIUJClj JJJ\,)S .)JOW pJJUJ
JOJ P;)PU;)WIUOJ;)J UJJq selj ~wfliw Ot JUOJIUCXOI
-!JJdx.) cWfSlU 810 rl .IJljllJ JUOJlUCXO\IW u,J.\13
-!W jO ')sop ~U!UCIS e puc '.(\lJ! XO I JO 5[J.\JI ;)[qc
sjJ.\.)1 u!qnJl[lq P.lIC\JIJ ljll" SIUJllcd JJJUCJ \SC;)Jq
m
JUOJIUI!\OIl,,\' JO
s")IPUI~O")I~IUJl:lId
1I!JIUIl.)
374
and humans (dt: Dycker ('1 at 1988; Shepherd et al. 1987). Male rats rea:ived mitoxantrone ]mg eit her intravenously or injected into the carotid arlery. I nthe lalter group, brain tissue concen trations were 18 times higher than in the former. Infusions into the hepatic anery for primary hepatocellular carcinoma, and into the internal mammary artery for palients with advanced carcinoma of the breast. have betn tried with good clinical success. Systemic toxicity was reduced In companson with intravenous treatment, bu t pharmacokinetic data were not given.
3.6 Discussion of Phannacokinetic Data in Huma ns
All of the studies reviewed above demonstrated a vel')' short t 'hl.l (range 4. 1 to 10.7 minutes). Serum
mitoxantro ne concentrations 31 the end of infusion were between several hundred ,.glL and I mg/l and declined to below 10 ~81L. which was in the ra nge of the assay sensi tivity of several methods, within 8 to 24 hours. T hus, mitoxantrone rapid ly leaves the plasma compartment, and binds to endo thelial surfaces and penetrates into the formed elemen ts of blood (Albens et al. 1983, 1985a, b, 1989; Savaraj et al. 1982b). T he d is tribution phase (tYl.lo2) is given at between 0.3 and 3.1 hours. Many of the reported half-life values o f the elimination phase (tYl.lo}) a re between 10 and 40 hours, but 3 authors reponed much longer values (between 7 and 12 days). It is possible that in the present autho rs' own study the adm inistration of cyclophospha mide might have altered the e li mination ofmitoxantrone, altho ugh in a study in leukaemia patien ts wi th si ngle agent treatment, the same values were achieved. In addition, the authors' concept is supported by pharmacokinetic studies in animals and the data from autopsy studies with high tissue coocen tralions a variably long time after dosing. Variabilities in the estimated pharmaco. kinetic parameters in these studies may also be due to the differences in data poin ts beyond 24 hours, weight ing a nd assay sensi tivi ty. Concentra tions at day 21 after administration were between 290 and 1000 n8lL as measured by RIA ; none o f the HPLC
methods ISsenSItI ve enough to measure mLloxantrone concentratIons in the lower range. The auth· ors' combination of the measurement of mitox· an trone concentra tions in plasma and unne mIght help to overcome these problems panly, Since the solid phase extractIon method permItted the load· ing o f large volumes of urine and resulted In a sen· sitivity of 0.2 I181L murine. In summary. the foll owing consensus may be suggested: mItoxantrone ex hibits arapid inlllal dIS' tribution phase followed by a relatively slow eltm· ination phase. The results suggest a deep tIssue companment with a slow release. The very large volume of distribution shown by all studies sug· gests that much of the drug is sequestered in the tissues. All tissue measurements showed that mItoxantrone persists m human tissues for prolonged pe. riods. However, concentrations of drug in tIssues in which a tumour was located generally did not correlate with concentration of drug In the tumour itself, although exceptions to this were the lIver and brain (Stewart et al. 1986). The calculated mea n renal clearance values were between 0.9 and 2.7 L/ h/ m2 and accounted for about 4% of the dose in 48 hours. The observation in humans that 28% of the dose is excreted in the faeces within 5 days. as well as the high rate of biliary excretion in animals. supports the concept that bile may be the major route for the ehmmation of mitoxantrone.
4, Elimination, Metabolism, Metabolites 4. 1 Elimi nation and Metabolism Investigations with bi le--duct cannula ted rats. rabbi ts, dogs and monkeys, and isolated perfused rat liver (Batra et al. 1986: Chiccarelll ct al. 1984; Ehninger et al. 1984; Lu el al. 1984: Richard et al. 1989). showed that biliary excret io n is the major excretory pathway for mitoxantrone and ItS me· tabolites. In rats 56% of the radioacti vi ty of 14C·labelled mitoxantrone was excreted in 120 hours via the bile, and in to faeces of non-cannu1ated rats; an avo erage of 35% o f administered radioactivity was
found In fa{'c{'s dunng Ih{' firSI 48 hours aft{'r dosIng (Balra ct al. 1986: Chl{'carclli el al. 198-1 ). StudIes '>11th mo n lo.{'~s (Batra {'lOll. 1986) and dogs (Lu el al. 198-1) also sho'>lcd that 47.3 and 50.5%. respectJlel~. ofdrug-rclated radlOaClI\II~ '>las ellmmOIled Iia Ihe blk III\h1l1 110 hours. I n humans. Ihe mean recoler~ of radlOaCllllt~ In faeces oler 5 da~s '>las 18.3000. Indleal1l1g that. as '>11th animals. blliar~ delollflcation IS Ihe major excretor) palh'>Ia~ In humans (o\lberts CI al. 1985al. Urlnar~ excrellOn of radioacllll\Y was found to be comparalllciy 10'>1 In all SpeCiCS cxamlned. I n rabbits. kss Ihan 6 10 8% of Ihe dosl' '>I as excreted during a 6-hour obsenalion period; 10 rals and dogs. 6.7 or 4.9% of radlOaclil'lI~ has been found In Ihe same collectIOn IImc, Increasing to 13.6% of Ihe admlnislered dose in rats when the lime of colleClion '>las e.\lended to 120 hours, Following a single Intralenous dose of l~C-label!ed mllOlanIro ne. renal e\cr..:l1on of radlOaClllll~ In cancer palients lIas similar 10 Ihal 10 animals. Thest' dt'lermlnallOns of bll1ar~ or rl'nal c\cre· lion '>Icrc PI-'rfoTrlll'd b~ mcasurt'nll'nt of th..: 101;11 radloactll II~ of blk and unne. Some Inl cSllgators hal{' abo c\anllncd IhL' collected bod~ flUids b~ H PLC monlloTlng lighl absorpllon al 658nm. 10 delermlne Ihe fracllon of radioacil I H~ belonging 10 the unchanged drug. The resulls are summarised In lable I for animal studies and lable II for studies 10 humans. The signlficanl differences demon· strale thaI up 10 511\1 (dogs). n.5 10 25% (rabbits) and -10000 (rOllS) of Ihl' IOlal ncn:ted radlOactllll~ mu~1 be allnbUlcd to melaholllcs of mllo\antrone (Alberts el a1. 19S5b: Balra et al. 1986: Chiccarelll el a1. 198-1: Lu et OIL 198-1: ~·l acph..:rson e\ a1. 198-1: Salaraj el al. 198101). Three mon.' polar melabolites hale been delected In an Isolated perfused rat Iller model after separation yia H PLC (Ehnlnger el a1. 198-1). H PLC Inlesllgallons of unne. bile. and plasma of pal1ents showed up 10 -I melaboll t('s (Alberts el aI, 1985a: Chiccarelll el al. 1986: Czej ka & Georgopoulos 1988: Ehnlnger 1'1 al. 1985a.b: Lu et al. 1984: Macpherson 1'1 a1. 1984: Pa~el et a1. 1988: Sm~th 1'1 a1. 1986: Van Belle el a1. 1985. 1986) '>I hich were also obsc-rved 10 Ihe unne and bile of rals (Al'ramlS 1981: Chlccarcill el al. 198-1: Wolf el
r (Rich. al. 1986). dogs (Lu e l al. 1984) a nd abbits ard et al. 1989). However. the amount of the melaboilles delected by lighl abwrpl ion b) no means equalled Ihe radIoactIvity nOl bound 10 the pare nt compound (EhnlOger et al. 1985b). -I.:! Structural .\nal)sis of ~lelaboliles
Fell a\tempts have been made 10 e1ucida le Ihe metabolism of mlloxantronc. and on I) 1 laboratories hale reponed efforts 10 Ihe elucidation of the chemical slructure of melaboliles deleCled by sel'eral IIll'Cstlgators (Chiccarell i e l a1. 1986: Ehninger el al. 198-1. 1985a.b: Macpherson el al. 1984: Richard el al. 1989: SmYlh el OIL 1986: Van B elle et al. 1986: Wolfet oIl. 1986). The main reasons for Ihis gap In struct ural Io.no'>lledge seem to be Ihe veT) low occurrence of uri naT) melabohles and diflieullics III finding chromatographic condlllOns. allowmg their preparatllc separallOn. The anlhraqulnonoid mOlel~. coupled with Ihe high polan I) of the molecule. causes leT} low 1'01alllll~. malo.lOg gas chromalographlC separalions IOlpQsslble and complicatlOg mass spectromelric inlesligallons. Mass spenromclric delection of lhe molecule Ion o f mitoxantrone 10 unnar) cxtraCIS IS I ef) dlflicult and requires negatlle Ion deso rptIOn chcmlcallOntsa tlOn or Ihcrmospray IOnisa tion (Blanz 1989). The strong basic aminO grou p ofl he Side chams necessnales aCidiC chromatograph ic condlllons 10 nrcumlcnt 10rl l(' in teraCtions wil h uncapped Sllanol groups of reI crsed-phase Silica gel. Less acidic cond1\1ons can be applied bu t require Ihe usc ofhlSh sail conccnlral10ns, Ihercb~ limni ng Ihelr appll('ablllt~ to prepara\lle separallon. So far Ihere ha\e been no repons of effons to overcome these separallon problems b} uSing macrophasc re\ersed-phase pol~mers lackmg the potenl1al for 10ntC IO leraCllons. All de\eloped chromatographic mel hods In\olle strong changes In pH. so Ihal chemical denalunng of pH.senslllle metaboliles. especiall~ of conjugates. mUSI be e\JX'clcd. In general. the 10'>1 degree of solubllit~ of Ih(' parent drug and metabolites after ennchment IS surpnSlOg. and represe nls
Om Pharmacoklrl('/ 18 ($) 1990
J76
a major problem in elucidation of the chemical struclure. The low solubility could be related to chemical denaturing during i50lation or to ionic or hydrophobic interactions with components ofbiological origin, especially proteins. which denature during enrichment procedures. The identification of metabolites as drug-related material has been achieved either by application of a radioactive now detector monitoring the radioactivity of the eluate after adminiSU'ation of 14e. labelled mito;\antrone (Richard el al. 1989). or by monitoring the typical light absorption of the ch romophoric system al 658nm . Two metabolites of mitoxantrone isolated from the urine ofpalienls have been characterised as the mono- and dicarboxyiic acid derivatives resulting from the oxidation of the terminal methylene groups of the side chains (Chiccarelli et al. 1986). The structural el u-
T.~
I. BtIi'ry.Nl urinary '>lCtIIIOn
""""
....
R........
.... U....
""
Urino
"C 1%"
...
28.5
A. I.
""'"
...-'Y'
U_
• b c d
Unchanged
Oruo
"""
"'"
19.5 4.5".2
SO.5"
3.7
~.
0'rldioactMty loIIowIng intrlvenout
'"
•••
...
cidations have been performed by negative Ion chemical ionisation mass spectrometry and comparison of mass spectrometric fragmentation and HPLC retention times of isolated metabolites and synthesised referentt substantts. The synthesis of 5.g·bIS-(I-IJcH(2-«2·hydroxyethyl)amino)ethyl)amino )-I.4...·rl\ D ...
P~n~
Pharma,·ol~~
318
Cf/ll. Phur",uml../IIf"/ M (.~) /WO
.n,sm of k llon and p/'La~Hltl," of no"nl~ In In\1'1I_
'cnous and 'nlf1lpmIOM1lII~py In Collman (Ed ) Thr (Uf"'i'lL n.lus Dfno'·.nlro~. pp I So2 I. John W,k,. Ne ..... Yon.
""
"Ibens OS,
Pr", Viol. Bo...·(kn GT. M lK'kel C. o.llon \\IS
Mech-
anIsm of Kllon and p/'Larmaeoklnclln of no~an!roM ,n ,n....
' '1'Il0\l1 .nd IMlra~ntonoell C'hcmolht1'1lp) 1S-21. 1989 Alben. OS. ~n, YM. u'ch S.
t1\(,1'1Ip)
Ad"1l(n In ("!lttr
0."" TI'. \\ood_rd DL DIspm,tlOn of m,'ountronoe ,n pallen,s, ("nttr Treatment Re"~"'I 10 (B); 21-27, 198) Albtns Pt'na YM. u .." s. TP. Woodward OL 0.,.. POSItIOn of mllOUnlrOM In nllKff pfllJCnl1. C'!Kff RncaKh
0.",
os.
45' 1179.18&Too:ftdmp 4) 345. 1984 Chlebo ..'Sk, RT. Ton, M. Bulca ..1t L \!o oodwud DL. Mno~. Intron .. In h.. p.:alle dysfuIKIlon: faclon Ifll""I\(InIIO\I("II) Ind n:spon5f. Abslract PrQ(ft(j,np of lho: Am"'lC1In SocI .. I) of C'*MlC'DIOncolos) 5: 46. 198b CIIoi KE. Sm~ulc- JA. llan) OS. McGralh SC. 11lI1) KM CI al HIJh·ptrformanC\' hqu,d ehrom.IOSl'1lpi'll(" 1)101) for m,IO~' In lroM In pluma UIJ"I clcctroc~.c-mlCllllkl«hon JO' \ \Hand hrCdl' In klll-n'm, ,.'11> f1f1ll,h Journ,1 "t Itd.'mawl',!!'~' "-" 1 ul J",Ilum"r urug, amrlam",n, Jnd 01" lO.am,oO,·(OU'JOlr,)O,·1 anu Ih,'lr ab,,,,, lU ."n.!,'n...' oud.·" alld, f>r, ...·....-..Jln~' 011he '~.",nJI \.-a.!,·m, ,,( .... I.'",., ~1 hIli:. /\1.(111 Iarl\n~''''''''1 I T.apn", I \klan.,.,J 11.11. l'lIc.aU,on, ", J n,," ~nlliumuur ~nL I.J-d,h,d.",,·~ ~·II, .. :[~_ II ~-h>d"" ... (h, llam "'o[~lh' 'lam,"o-~ In.J n. h.a,cnedHln,' ""h n""le .. ;KId, 1I,0....... ml(';ll Pharm~_ ok'!!' 11, ~11·~.w'. I'I~I ,..har:uh 1.1) ',,,~lll. ~ Inhlblhon of ~u"an.,,'n ,"ml.l\~"'.l ml n~mll "I'ld pc'O"dlloon b, mOlO,~nl"'''''' ~rId 3m,'IanITO~, 1"0 nc.-" ~n!h~n~ j-HIII~~ I\lH5 Lalhano , ... lU'labk -\(i, Polla.d (\, \I"n.· \1 ,,'I,'J,,'n J_ CI al P~"'l'klla afu" 'mr.llh,...·al nmuIJn""n.· I In.~1 ~ 1.1nu.-lc" a"d, "uul ~~ -'11~8·"OJS, 1985 \ u ... ~'3r.lJ' l,)Q IT I'ha.maeol""nl dl~po~'"0n 01 '''_ dlh~. l\lb5 ">'J~ RI "'hJrJ,h I D \ ,tountronc I propen,'" tOI fr,-': rad· Kal (."m3I1lln an.! 101'1
