Tm: .Ir-r 5:.-11. or l’lI.|\fl&Im‘._‘I’Jl..(‘Ifil' urn l'lxes:nnri::s-r.-ii. TIII.I1.-\|':i:1'rtI.H
`(‘np_\-right Q I'|r\‘l l-3." Thu \v\'|1luim.-i
`(E Wilkin.-3 (‘r-.
`
`V01. 1513. No. 2
`a"I'1'n.'rd'
`l-I1 U..S.A.
`
`T11]-I ['.‘IIU[.1‘.\'I‘IR(}IC I§FFl*Z(."I‘:-I AND RATl:I'.‘S OF I'IYDROI.YSIS OF
`{'1IXI-‘()R1IATI()NAI.I.Y RIGID .-‘\N:\I.C)Gi‘.-‘s OF ACETYLCHOl.L\'E‘
`
`{'. Y. CI-IIt')I'. .I_ 1’.
`
`l.O_\'(i_ J. 1'}.
`
`('.'.-L\‘f\'{]T\'
`
`\.\'n l’
`
`I)
`
`.\l€.\15'l‘l£0.\'G
`
`rim! I}ii.'i-ion of .-'|uIr‘(i':}‘Eii(:I
`J'}r,m1rf::Ii'n.f of Pi:-:Ii‘r:mC0iugg.r. Coil.-79:.‘ of .-'lfer}I'r'a'Ju'
`i"'iir'iiir'.s'h'_r,r.
`f10.I'i':*gi' 0} Pilinrrnnry. Un1'L;Pr.s'i'.i'_l..r of form. a"mrr:
`I-‘ally. Ioirrz
`
`.-\i-:-r.~}aur:| for iiiilili:-aiinii
`
`f\‘:>\'i.-iiilivr 18.
`
`1962:‘
`
`.\ii.-'i‘ii.-in‘
`
`'[‘|u- L'lIlIliI'II'1‘gl(' --in-c-is nml
`(‘mniu C. Y.. J. 1’. I.:m:. J. G. C.IN?\'().\' nn 1’. JJ. .-\Iui:.~;'r:tnx:;:
`I'.ilI':-3 of l]_\'Il1‘ul_\'.‘."=!:~.-‘- of :-onfornmiion:1|l_\‘ rigid analogs of IIl‘{‘l_\'ll'l|l'Jlll|l’. J.
`l’Ii.-u'ni.'irol_ Exp.
`'|'h:'r. 166: 243-348. 1969. 2—AL-elox_\'
`i'_\'I'll}I:l'[}]I_\.'l
`Il'llIli‘llI_\'l:ll11I11r}!1itll1]
`iuzliilx-.-a E.-\("{‘i\I)
`:|f'£' :-unI'm-marmn:ii1}' rigid zinulugs of :1L'nt_\‘l('h(Jlinu (ACh] will: Ir:iI1.-mill.
`:iI1:l {'i.-zniil
`:-<mf0rI11:I-
`Iiuli.-'. Thu‘
`(+)—lr'im.<—.-\CTl\I had strong ]'l1.'|.15L'.'l.'|I'lI'll('
`:I{‘li\'i|i:'.~i on drug iilnml
`[nra-.-'.-n11':-
`.-ind
`iziiuu-:i-pii: ilr-um pr:=|::Iralions, suggesting ihat
`the trniisniil form of .-\C-“T.\I
`\\':lS :13sor.'i.-1ln:l
`will:
`ii»: miisr':1rinic :H’li\.'i|if‘S. The (+J.(—)—r:1L~'-ACTM w.-1.-i vxpi-zriril
`To have strong
`nimIirnr- :u'tiviIi:'5 owing :0 ii.-=. L-isoizl coiiforninlion.
`l[uwu:v:~r.
`il. had n:'gluz':hIi.-
`I1i:‘0IiI'Ii:.‘
`'lI‘l1\'1l_\'
`run
`I‘:-ng I‘("E‘T.11:-.‘- nhrlnminis nmsr-in, presiamably due to {he l.3—intr‘r:u-[ion of The
`in--1|i_\'l«-nv izruiip of r_\':'ioprop:u1r~ ring with the r:1rhon_\'l znxygen v.'hi:’l1 is l)elie\'eil
`Io he
`|'I'I]lIl1‘I.’rl for nirotinie :1eti\'itics. The po:r:n:r_v ratios of rims:-:irinic :u:tivilies l)et\\'r.-en (+)—
`.u::l f--)—!mm‘~.-\CTM \\'(‘].'i-
`\'er_\' close Po those bvtwct.-n I.(+l- :1n.:I n(''l-:iu:-iyl-_r1-meIl1)'l-
`. lml1lII'. ‘fhv i1i1i:~:r;:ru:iu zuitivitirs of .-\Ch cinii (+l—t‘rflm‘-ACTM on (lei: lllfnlil pre.-‘slire were
`m:irlcz-:ll_\' [um-I'sIi:1l:‘:l by nr-osrigrnine (ll—l'0ld nnrl 23-fold. r:':i|Ir':'1i\‘(‘l_\'l, but [hm nl t'--)-
`r.rm.-~.\L"T')I
`\\':}:§ poorly ['10iI"l'Ill:l[i'Ll
`(3-(old). Thur
`sImIio:= run
`rnzyiimlic li_\‘¢lrol.\'si.-: of
`mms-—.\(.'T.\l
`|l_\'
`llN-U’ll‘lmllI'1{‘S|{!!':'tSlI‘ and eh0lir1r*st.vr:Isr‘
`rI~\'t.\:1|ed that
`the l'{"l.‘|.li\'l" rates of
`|'i_r:l:-nl_\-.-i.-a of {-l-)— .-md (—l—!rrm.5-ACTM by (IN?l}‘l<'l]I’)liI1t'>ilI‘r:l?=f' werv 96 rrml 59"}
`that of
`\f’|i
`\\'i:h iiw i-‘mm:-r-: the h_\'rlml_'L'*_=i~s
`r:1!v=.c l-:_\- r:holim--ster:iur- \\'l"Tl‘ G1 zuui 3-1*‘?
`in relation
`‘in .lI'l"5_\'il'lli|il.Ili’. Tl1l.‘.‘S2'
`I‘L=.~eI.llI£- indirulv that the hiollagir‘.
`.'u‘Ei\'iT_\' of
`E'-i- )—i!mIi.w—_-\CTl\I
`is
`inuln-am.-1lr'al
`l;_\’
`I'H‘1):=ll:.‘,I11in(‘ mun‘ lh:m that of
`[—)-ITI‘l1'?-'4-.\(‘T.\1 l!I‘i'!llIFI'
`illt’
`l0I‘mI‘l'
`is E.
`ll--‘.i'n'I'
`-Ill-:-'1]'.'Iiv fur Ihz» L-l1oline:3ir~r:u.=4-.-_
`
`iiiierisive studies on the In€‘.-l('CUlflI‘
`I}{‘._—'|’I1lII!
`:1c<:L_vlc}1oline
`(ACh),
`the possible
`femiirvs of
`biologi<- iinlaortanee oi" conformational isomerism
`re111:1in.< uncertain. For the elucidation of this
`
`reported have l}l'_’[‘.T‘l
`ihrr omnpnunrls
`pmhlvin,
`ll Sll‘IIUl1lI’:1ll_\' as close to ACh as possible and
`'_’i runinr:n:iiiun:1|ly as rigirl as possible (1\InrtiI1-
`:'-imiih H :il.,
`IF.It3Tl.
`In the present. work 2-
`:ir»r-tnxy l'_\‘(’ll‘11ll'{l])}'i trimethj.'l:m1Inonium indicies
`(.-\f'."l'.\[I were .‘Ei(!li3(‘Tf!Ll
`to meet
`the require-
`I‘.r1t‘ll1.':—‘
`.-'I.'1T("Il
`:ihm'e because these compounds
`Imve :1
`l'}‘£‘lE}[}F{)]!:lIlC ring in place of the choline
`mnir'[\' and :m-
`:~nn.~:iilvrc<l
`to be the .»'in:1llr-.'~:i
`
`i'ieri\'.1ti\'es ca-
`.\Cli
`.-imr.m,r.r
`-TrII:~ltII':‘
`f'l|l‘III|.I'.Il
`iuilalr
`ni mail:-r'r:ii_I1
`:»niifrurni:iIi-mal
`ri,r_rirli:_\‘.
`The tr:u:.~5ni¢|
`:m:l visoicl
`rrrmfnrniers of
`.XCTT.\I
`are $lI(J\\'lI in lir_rI1re l. The (‘holim-rgie Cfi(‘E't:-C and
`the r~}ioIinr-.~'t<=r:::-0 |:_\-ulrrilisis; of ACTH are re-
`
`lii-:'r-ii ml fr:I'|ILil1lii':IIi(JI1 SE_‘pl:‘I11l)£‘I' 15. 1958.
`:'rI|i.\
`\\u|'lc
`\\.'|:~I
`.-='ll]|'|iIii"Tf"l1
`in part
`lw 1'3.
`Pirlulir
`llr'.'Illl1 .*~':-t'\‘i:-i-
`(ir.-ull.-a .\il3—l39fi_. 3-‘B-1~l3l
`::Ii:l
`.\-B-H1110“.
`
`investigation. The rela-
`ported in the present
`tionships of conformational v:.tri:1tinns and the
`role of
`the unsubstituted m<:tli_\'|ene group of
`the eyc1oprop.'1ne ring of
`.v\C'I‘M to nicotinie
`and muscarinic clients are also discussed. The
`potentirttion by neostigrnine of
`the intisearinie
`activities of
`(+)~ and (—l-trans-.-XCTM on
`dog blood pressure is correlated with their rates
`of enzyrnatic hydrolysis h_\' acct}'IrhoIinesier:1se
`(AChI'l] arid ellolinosterase (Chl'-I].
`
`pnr_nr1mfE.0ns.
`'_:m'ssure
`4\«IE'l'ImUF~. Dog Moon!
`Mongrel dogs of 1-ithvr sex.
`\|.'(‘i5.{lIiTIfl
`9 to 13
`kg, \\'("l'l? :‘ll]f‘§l.l}f‘|i?.'f!(l with 15 ing.-"kg of iliiopentnl
`5-'-(li'llIllI'I
`and
`950 mg*'l-cg
`of
`h:':rhii‘:1l
`smiiiini
`aiimiiiistvru-(l
`i.\', The
`T.r:1(‘h(-:1
`\\':1s w;mnn|:1tnrl,
`rind the \':|izi
`in-rv .-'I"I‘lii)m‘:’i.
`'|‘hrm:i:r_|mui
`thr-
`I‘I}'H‘1'iI']lf‘lll:i
`the nloirs
`|\'1"TI"
`:tI'tifir'I.'tll_\' w-nlil.-uni
`with
`n
`Palrnn-r
`H‘:-‘[\EI':lfI’)1'. The riirht
`fi-mnr.-1|
`:1rIr~ri:|l
`[IFl‘:‘~':-'lIrl'
`\\':1.-e ml-:i.-Ilrrul
`\\'ilh rs 5<i:ith.'m1
`pressiirv lr::iis:ium-r {I".23.\.\) and :---mnlr-ii on an
`0fi'n:-r Dynogrrtph fi_vpi_- RS1. All vornprmmls
`\vI-I‘:-
`i1'ljr>:‘i¢-ti
`via
`:\
`}u‘;l.\'I'lli_\'h-m'
`I'Il.ll1i'l|'I‘
`in-
`
`ii.’-13
`
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:21)(cid:20)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:20)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)
`Sun-Amneal-|PR2016-01104- Ex. 1021, p. 1 of 6
`
`EXHIBIT
`
`Ex. 1021
`
`

`
`2-1-1
`
`(_‘llIUL' ET .-u,.
`
`Vol’. I66
`
`
`
`Flu. 1. Thu‘ tr:i::.~:ui:I I.-\l mic! I-isiiicl [I'll r-nnfur1'n:LliuI1:-‘. [sf 2-:l('(3t(JK}' r‘_\'('l:J|JI'up_\'l lrilnttlliyliilnmtmilim
`L-'§.("l").l).
`'|‘l:<- t+ }— .'lfIll
`!—>-1.~:IIlI1l'I":i1-f('ITlIl‘1'
`tr:1r1e-mid or 1'1:-Hrlll
`.-\(‘ l‘.\l I'(m.~:1.II11!I‘ Iwn i|'.'II|.l‘I‘Ill'
`Iillflgur-4
`\\‘l1iI'lI l'.'1.IIlII1l
`Int-. E-§ll|JE'I'll11|:Ilr:-2L'(l.
`
`\\'(-I'(.-
`am]
`fL‘lIlfII':ll V:-in
`lrlil
`into Lln:
`Sr'rt<‘:l
`ml of
`i.-_-':Jl0I1i:.'
`in \\'i1h .'1lI(Jll[
`2
`rapidly W:l.‘.-‘l]i‘:’.l
`\'ul1lmr~ pf llll‘
`ee‘iil11-
`srilim-_ In :ulI
`(':]Sl'.\i
`ll]|' 1n1:1|
`tion.-3 injt-<~t.:'d \\':i.- lcwpt.
`t'I‘.|I1.‘=l.'iI1.L at 3 ml.
`Tlur
`:lu.-e:‘S U:-il'{|
`in
`Iln-
`lniu.'1.-.'-.-'.'1_\'
`\\'m'i~ \':iI‘ir-il
`by -1-fold i1m~r\'.'il.~::
`(1.1 pi:-"iii! and 0,1 #4:. In:
`fnr
`(-l-)-lrr.-re.-:—.-\C'l‘;\l. 30 ,uL( ki.’ uml
`I-ill
`,uL!'l-\';_’.'
`fur
`(—)-i’mn.x'—.\C'l‘.\l
`{lllil U.-I
`,u_i:_.»'k:_'
`flIl(l
`1.5 .14.’ kg
`for ACI1. Tliv lll'l{l'l‘1'
`ca!" ]mlI'I1Il::llfm cal m1i.~‘r-:n'ini:-
`.'1r'ti\‘it)' of
`tl|r‘.%r'
`rrI[11LJr}'l1IIll.~i
`lay
`50
`.t1I:_l-ig of
`Iiw).-‘1is:i11ii1i:
`\\'.-i.-'
`:*[lIIrlI‘l|.
`livforv
`:‘ul:lilioI1
`of
`I100.-'1i;:miru*.
`lllf‘
`.-:.'1mr-
`ilu.-‘i~
`ll‘\'l'l:-i
`:1.-'
`:-‘.t:1lr'1l
`:'1l)(n'r:
`\ri-rv 1i.~;ml_
`.\fir:'
`I|t'H.‘Sll!.’.l]!lT1f‘, 0.0lJh‘
`iuslfkiz
`:lI1:l 0.032 ,LI5.'._-"lip:
`raf
`("l-)-trrrns-.\C‘.TM, 3 ,c-Ia.’-lig
`:m:l
`32
`,r..tg.£
`lip.‘
`nil
`f—)—h':m.-4-_\(I'l‘l\l.
`illlll
`".015
`;.¢g:_x'kg and 0.061 ,u1."l\'l!
`rail
`.\{'li
`\\':'1‘:~ l1>=[‘Il.
`'l‘}1i~
`.In.~:r:s
`:i|i-l
`\rm‘i~
`I':1n-
`a:'lmini.~_:Ii'.'iIinn.<
`uI'
`lll'1I}.'!§
`rlomixn-«I. and all
`rnlizl
`:1
`:-Hln-I‘i.'i.
`I'm'
`p.'|:'.'1ll(rl—
`lino |1in.'1.~'.-e;i_\‘
`\\'r-rr~ m--I.
`!"r‘rJ_u‘} n:'.fu.v ri--larhrn.-:'ia:'.~'
`rm-fn.-—'
`:llrtliI]JIllT1l.~‘
`IIIII.‘-I‘l1‘
`
`.=nI:.~'t‘r’r' _m':-prri'r:!i‘0n. Tho
`\l.'l.‘~ I1lJl:illlI'll fimn Hxrairt
`
`lyv Burn (1952). Tlirn mu.-:-lr~
`;r:i;n'm;.< us (1:-.<:-riiu-rl
`was ll|.I'i‘fillll"fl
`:iT
`lmlln l'T]Il‘3
`:iml
`\\'.-is
`e<I|]ir-I'fII!=i~tl
`will:
`fmiz RiIIL'.:~I".-:
`.~=nli:|i--n
`f_\':i(‘|.
`£5.-13: K(‘l.
`0.30;
`f‘:1(Tlu_-'2l[-_-0.
`l'|.'2C{: X.-Ill(‘l');, £1.35:
`Lilli:-o.=-'4-.
`
`this was ox_\‘g:»I1utNl with 9572: 0:-
`;:,’|it:-r):
`(].'Il
`5‘.'F- CO-.- at mom lII‘l'Il]1I'I'ElllIl'E‘_ 'l‘In~ !'.'1l.(‘ of lluw
`of slip:-rl'1i.~;inn Iliiinl was 3 to -1 ml,-’n1i11 and was
`m:iinl:iini=d lJ_\'
`:1
`I-lnltvr inntm pinup (l_\'pI" RD
`-15). Drug solutitmu \\'('I'[‘
`injt-i-1:-nl
`into tho str(~am
`oi s11pi~t'f1::=i0ii
`llllltl
`in \‘0l111m'.< of nut niom tlmi
`0.1 ml. Tliv I‘l.‘l‘lI[=:i
`II111St.!lt- was |rl.'u':‘d .'LI. an initial
`ll‘II:-§l()I1 of
`1
`1:. TltE'
`ti-Iirainn (iv\':-lu|u'd In‘ cun-
`II'.'1r'ti0II ui Hm m11S('lI- w.-1.:
`II’I{‘i'lE‘iIlI‘l'.‘I.'l
`in grams on
`:1
`t'31.-illmm lI'.'I|lSfl'llE'f‘l‘ (tygw GT-03) and recorded
`on un 0lTnm- TJ_\'nnp:1'.'1pl1
`I‘l‘I'(l['ll{'l.'
`(lypv RS).
`WI ‘ T’! ‘
`.'*4::lIl1i:me-i of
`l.l1i'
`r-niliptallliui.-'
`;vrvp:i|‘i‘(l
`in
`rlistillvd
`\t".1lt‘l'. Tlw :~m1:-i~n1r.-itinnrs
`of
`drugs
`II:5{?ll
`in Ilur
`l:in:1:~'.~':L_\'
`\\1-:'--
`.-']I:l:~ml
`l'l_\'
`-1-fold in-
`I‘r‘T‘\'.'ilSZ 0.1 pg and 1.6 pg:
`fan‘ AC1},
`-10 ,btS.‘.' am!
`15-0 Hi!
`I'm‘
`(4.')~frrm.<—.-\("l‘.\I. N0 H; and S00 .u1.’.'
`for (v )—£rnn.<—."l.CTi\I and 100 fill.’ mid 401]
`,:.:_2: for
`f-l ).f—)-.-".='.-.~'-.-\('.'l‘l\I,
`Tin‘
`.'1IlT11iT1l.-ifI‘.'JIlIJl1=.-'
`of
`i1r1:;.'s nlul {lf‘|S1'S \\1‘r(.- 'I‘:lI'l(lUll]i7.l‘{l.
`pin.-3
`(iiiiiiv.-1
`(Elmira-,u:'g
`ii’:-mn
`;1JTc]JfIFflHr}?M_
`lay I!
`l)lr:\\' on
`\\'i-igliitii: 300 tn 500 L!
`\\‘[‘l‘[' slIm1:r':l
`lllr‘
`nl‘
`lllf'
`ilmim.
`lu-ml.
`'|‘lu-
`trlrmin.-tl pm-timi
`\\':l-‘S
`iiserl. An
`ap]1mxi|nari~l.\‘ 3 mi
`in
`lT‘T1|.{lll_
`inilinl
`lvn:-eirnl of
`]
`5,:
`\r:i.-‘ pl:1m~rl an !|1:'
`li.~:.~'11:'.
`Till‘ mnllimls of injm-lion nf fll'IT:.!:~= and rt-mriliul:
`
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:21)(cid:20)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:21)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)
`Sun-Amneal-IPR2016-01104- Ex. 1021, p. 2 of 6
`
`

`
`196.9
`
`PHARMACOLOGY OF ACTH
`
`245
`
`of the contractions were as described in the last
`section. The ilea were superiused with Krebs—bicar-
`honate solution (NaCl, 0.93: KC-1, 0.351; CaCl:'
`2HsD, 0.373; KHaP0i,
`0.1.63; Mgs0., 0.18;
`Na]-ICOa, 2.00; glucose, 1.80 gjliter). The con-
`centrations of
`drugs
`used
`in the
`bioamay
`were spaced by 4-fold intervals: Oml as and
`0.004 cg for ACh and 10 as and 40 pg for (+).
`(—)-c£s—ACTM. The
`administration of drugs
`and doses were randomised. The relative po-
`tencies and 95% confidence limits of (+)— and
`(-}-trons-ACTM were cited from Armstrong at
`at. (1968).
`Radiometer titration method jar chotinesterose
`activity. The acetic acid formed during the hy-
`drolysis of esters was titrated with 0.05 N NaOH
`on a Radiometer titrator type T'IT1c and titri-
`graph type SBR2c. One milliliter of p-otamium
`hydrogen phthalate (0.3-676 5/21]] ml; 1 ml = 9
`..::mol of NaOH) was titrated with 0.05 N NaOH
`under N3 gas. From this titration curve,
`the
`ordinate of
`the titrigraph chart was calibrated
`directly in micrornoles of NaOH used, which is
`equivalent to micromoles of acetic acid liberated
`from esters by cholinesterssu. The values were
`expressed in micromoles per hour per unit of
`enzyme. The enzyme used was prepared 1.0 U/ml
`in Krebs-bicarbonate solution without NaHCOu
`(7.5 X 10" M NaCl, 7.5 X 10" M KCl and 41 X
`10" M MECL'5H10). The substrate solutions
`were prepared with the same solution in a con-
`centration of 5.6 X 10" M. The total volume of
`the reactants was 1 ml. The reaction vessel con-
`tained 0.8 ml of
`the enzyme at pH 7.0. The
`substrate (02 ml) was added through the sample
`hole, and the reaction mixture was titrated at
`pH 7.0 at 38°C for 10 min. The air in the reaction
`vessel was replaced by N: gas.
`Drugs used. The drugs used in this study were
`ACh
`bromide,
`atropine
`sulfate,
`neostigmine
`methyl sulfate and d-tubocurarine chloride. The
`
`(—)-trons-ACTM and (+5.
`(+)-trans-ACTM,
`(—)-cis-ACTM iodides were
`synthesised
`by
`Armstrong et al. (1963). All doses of drugs used
`refer to the salt form. AChE and ChE were ob-
`tained irom Nutritional Biochemicals Corpora-
`tion with specific activities of 1000 U/mg of pro-
`tein and 4 U/mg of protein, respectively.
`Statistical analysis. The relative potencies and
`the degree of potentiation of the compounds were
`calculated from a four-point parallel-line bio-
`assay as described by Finney (1055). Paired ob-
`servations were evaluated with Student’s t
`test
`(Snedecor, 1956}. A probability value of
`.05 or
`leas was considered to be significant.
`
`Rescue. The mosccrinic activities of ACTH
`on dog blood pressure. The response elicited by
`ACTM was a fall
`in blood premure that was
`immediate in onset and of brief duration. The
`relative potencies and the 95% confidence limits
`of (+)- and (—)-trans-AC'I'.M are shown in
`table 1. The (+)-trans-ACTM was 4.7 times
`more potent
`than ACh, whereas
`(--)-trans-
`ACTM was only ’/45 as potent as ACh. Direct
`comparison of
`(+]-
`and {—)-tram-AC'I'M
`showed that the former was 192 times more
`active than the latter (the 95% confidence limits
`were 105-385). The depressor efiects of (+)- and
`(-—)-trans-ACTM and ACh were completely
`blocked by 2 mg/kg of atropine sulfate.
`The muacorinic activities of ACTH on guinea-
`pig ileum. Table 1 shows the relative muscarinic
`activities of {+)- and (—)-trans-AC'1‘M and
`(+),(—)-cis-ACTM on guinea-pig ilea. The
`(+},(—)-C58-AC'i‘M was 5‘io.aoo as active as
`ACh. There was no significant difference in
`muscarinic activity on guinea-pig ileum between
`ACh and (+)-trons—AC’I‘M. The (-—)-trofl.s-
`
`The relative muacarinic activities of (+)- and (-)-trons-ACTH‘ and (+),{-}-cis-ACTH with respect
`to acstytcholine (A(.'h}
`
`TABLE 1
`
` Dog Blood Prim
`
`No of
`
`Relative
`potency
`
`95% Confidence
`limits
`
`a\Ch
`(+)-trans-ACTM
`(— )-trons—ACT'M
`(+),(—)-cis-ACTM
`
`1
`4 .70
`0.02%
`
`10
`10
`
`3 .21-9.79
`0.021-0.025
`
`Game:-Pi; Ileum
`
`N_c. at
`
`Relative
`|1I‘l0en¢-1!’
`
`95% Confidence
`limits
`
`1
`1.13*
`0.0022‘
`0.00010
`
`10
`10
`6
`
`0.81-1.46
`0.00l9—0 .0025
`0.00004-0.00029
`
`' ACTM, 2-acetoxy cyclopropyl trimethylammonium iodide.
`" Cited from Armstrong at at.
`(1908).
`
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:21)(cid:20)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:22)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)
`Sun-Amneal-|PR2016-01104- Ex. 1021, p. 3 of 6
`
`

`
`246
`
`CHIOU ET AL.
`
`Vol. 166’
`
`ACTM was about 1/ion as active as ACh (Arm-
`strong et at, 1968) .
`The nicotiriic activities of ACTH or: frog
`rectus abdominis muscle. The efiect of ACTM
`
`on the frog rectus abdominis muscle was con-
`traction, which was abolished by 5 X 10" M
`d-tubocurarine chloride. As indicated in table 2,
`ACh was 77 times and 357 times more active
`
`than (+}- and (—)-trans-ACTM, respectively.
`A direct comparison of
`the (+}- and (—J-
`trons-ACTM was made, and the (+)-trans-
`ACTM was found to be 4.6 times more active
`
`(the 95% confidence limits were 3.3-6.5). The
`(+],(—)—cis—ACTM was about 1165:: as active
`asACh.
`
`Potentiotiort of muscorinic activities of ACTH
`by rteostigmine on dog blood pressure. As indi-
`cated in table 3,
`the muscarinic activities of
`AC}: and (+)-trans-ACTM were potentiated
`
`TABLE 2
`
`The relative mlcotimic activity of (+)- and (—)-
`trans-ACTH“
`and
`(+},(—)-cis-ACTM with
`respect
`to acetyickoline (A011)
`cm frog rectits
`abdomirtis muscle
`
`Cnmpoud
`
`No. of Relative
`Animals Potenqr 9s% dum
`
`ACI1
`(+)-trans-ACTM 10
`(— J-trans-ACTM
`10
`(-I-), (—)-cis-
`5
`ACTM
`
`1
`0.013
`0 .0028
`0.0042
`
`41-fold and 23-fold by 50 pg/kg of neostigmine,
`indicating that both compounds were good
`substrates for cholinesterases. The activity of
`(--)-trcms—AC'I'M was potentiated 3-fold only,
`suggesting that it was a poor substrate for
`cholinesterases.
`
`The enzymic hydrolysis of trcms—ACTM by
`chotinesterases. The relative rates of hydrolysis
`of ACh and trons-ACTM at the substrate con-
`centration of 5.6 X 10" M are shown in table 4.
`The results
`indicated that both ACh and
`
`for
`(+)-trans-ACTM were good substrates
`AChE, whereas (—)-trons-ACTM was a poorer
`substrate than (+)-trons-ACTM for AChE.
`The rate of hydrolysis was measured at substrate
`concentrations of 1.8 X 10" M, 1 X 10'' M,
`5.6 X 10" M, 3.2 X 10" M, 1.8 X 10" M and
`1 X 10“ M. The substrate concentration-
`
`activity curves obtained were bell-shaped with
`optimum rates of hydrolysis at a substrate con-
`
`TABLE 3
`
`The degree of pots-ntiotion of muscori-mic activities
`of (-l-)- and (— }-trons-ACTH“ and ocetytclmtine
`(ACII) by neostigmine° on dog blood pressure
`N .
`1'
`E Pggfifgggn C E:C:
`
`Compound
`
`0.008~0.021
`0 .0018-0 .0046
`0.0039-0.0047
`
`AC1:
`(+ )-trons -ACTM
`(—)-trans-ACTM
`
`22400
`41 -fold
`11-79
`23-1’old
`2.8-fold 1.6-5.4
`
`7
`7
`
`0 ACTM, 2-acetoxy cyclopropyl
`monium iodide.
`
`trimethylam-
`
`‘ ACTM, 2-acetoxy cyclopropyl. trimethylarm
`monium iodide.
`
`‘ 50 ;.:g/kg of neostigmine methyl sulfate.
`
`TABLE 4
`
`The rates of hydrolysis of {+)- and (—)—t-rims-ACTH‘ amt acetytchoii-no (A Oh) by ACJ:E° and 0ItE'
`ACIIE
`CIIE
`
`Substrate
`
`N _
`f
`i
`-
`..‘.’..:'. "‘t.:.:. ".’.’%'.'i?'>“’
`Jm0U-ir/U 9!
`
`AC]:
`(+)-trans—ACTM
`(‘l-‘Tans-ACTM
`
`5
`5
`5
`
`9.2 :1: 0.5
`8.8 -_-h 0.4
`6.2 :l: 0.9
`
`.
`Relative rate of
`.......""“:'.‘*'-.33,
`
`%
`99 8 :l: 5 4
`96 0 :l: 4 4‘
`58 9 :l: 7 9‘
`
`A
`.
`‘£3.35 “t:.:‘.“;‘§° J’
`
`Relative rate of
`,,,,::;:=-33,
`
`fill“/if/U 9}
`33.8 .-.l: 0.6
`20.5 :l: 0.2
`11.3 i 0.3
`
`%
`100.3 :I: 1.8
`60.8 :I: 0.7‘
`33.5 :I: 0.3‘
`
`5
`5
`5
`
`‘ ACTM, 2-acetoxy cyclopropyl trimethylammonium iodide at 5.62 X 10" M.
`3 ACIJE, acetylcholinesterase, 1 U/ml.
`= ChE, cholinesterase, 1 U/ml.
`‘ P > .05 compared with aeetylcholine.
`‘ P < .05 compared with acetylcholine.
`
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:21)(cid:20)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:23)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)
`Sun-Amneal-|PR2016-01104- Ex. 1021, p. 4 of 6
`
`

`
`I969
`
`PHARMACOLOGY OF AGPM
`
`247
`
`centrstion of 5.6 X 10" M for ACb as well as
`
`for (+)- and (—)-trans-ACTM, indicating that
`a high substrate concentration of
`(+}— and
`(—)-trons-ACTM inhibits AChE. The relative
`rates of hydrolysis of
`(+)- and [—]-trona-
`ACTM by ChE were 61 and 34% of that of
`ACI1. The
`substrate
`concentration-activity
`curves showed no inhibition of ChE by high
`substrate concentrations.
`DISCUSSION. It is reasonable to assume that
`the flexible ACh molecule has difierent con-
`
`formations and thus is capable of fitting to the
`different types of ACh receptors. It has been
`suggested that the cisoid form of ACh is asso-
`ciated with its nicotinic activity and the transoid
`form with muscarinic activity (Schueler, 1956;
`Archer et at, 1962; Smissman at at, 1966).
`Based on this hypothesis, it would be expected
`that
`the transoid form of ACTM (fig.
`IA)
`would elicit mainly muscarinic responses and
`the cisoid form (fig. 1B) mainly nicotinic re-
`sponses. The results indicate that this is true
`for muscarinic responses (table 1} but not for
`nicotinic responses {table 2). Therefore, some
`factors other than c1's-trons isomerism must be
`
`involved in determining nicotinic activity.
`Structurally, ACTM is
`similar
`to acetyl
`methylcholine and resembles a hybrid of acetyl-
`a-methylcholine
`(A-ac-MCh)
`and
`acetyl-
`,B-methylcholine
`(A-,8-MCh), both of which
`have been synthesized and studied by Simonart
`(1932) and Major and Bennett. (1935). The pre-
`dominant muscarinic activity of A-fit-MCh is
`presumably due to the 1,3-interaction of the
`,6‘-methyl group with the carbonyl oxygen,
`which is
`required for
`the nicotinic activity
`(Selcul and Holland, 1961a,b; Sekul et al., 1963 ;
`Coleman et al., 1965; Triggle, 1965), whereas
`the predominant nicotinic activity of A-a-MCh
`is probably due to the 1,3-interaction of
`the
`ca-methyl group with the ether oxygen which
`is
`required for the muscarinic activity (Ing
`at at, 1952; Waser, 1961; Beckett at
`£11.,
`1961; Triggle, 1965). As shown in figure 1, the
`methylene group of the cyclopropane ring of
`ACTM would interact with the carbonyl oxy-
`gen but not with the ether oxygen. Therefore,
`the methylene group abolishes the nicotinic
`activity of ACTM. Accordingly, ACTM is
`structurally similar to A-,8-MC}: but not A-eh
`MCh. The studies on cholinergic effects of
`ACTM in the present work support this con-
`
`clusion because (+3-trans-AC'I'M has strong
`muacarinic activity (table 1) but very weak
`nicotinic activity (table 2). In other words,
`(+)-trons-AC'I‘M has predominant muscarinic
`activity (table I} owing to its transoid con-
`formation, which is favorable for proper fitting
`with the muscarinic receptor. In addition, the
`1,3-interaction of
`the methylene group of
`trans-ACTM with the carbonyl oxygen elimi-
`nates the nicotinic activity. The sis-ACTM is
`not a muscarinic stimulant due to its cisoid
`conformation (table 1), nor is it a nicotinic
`stimulant due to the 1,3-interaction of
`the
`methylene group with the carbonyl oxygen
`(table 2). The opposite hypothesis suggesting
`that the transoicl form of ACh favors nicotinic
`activities and the cisoid form rnuscarinic ac-
`
`tivities (Jellineck, 1957; Canepa at al., 1966]
`is unlikely because in the present study (+}-
`trons-ACTM had strong muscarinic activities.
`It is interesting to note that there is about a
`250-fold difference in the muacarinic activities
`between L(+)- and o(—)-A-[3-MCh on guinea-
`pig ileum and cat blood pressure
`(Beckett
`et at., 1961; Beckett ct at, 1963). It has been
`suggested that the difierence in activity is due
`to the B-methyl group in n(—)-A-,3-MCh,
`which prevents its proper interaction with the
`muscarinic receptor. This hypothesis is further
`supported in the present work, as there is a
`similar difference in muscarinic activities be-
`tween (+)- and (—)-trons-ACTM (192-fold
`and 330-fold differences in activities on dog
`blood pressure and guinea-pig ileum,
`respec-
`tively).
`The studies on the enzymic hydrolysis of
`ACTM by AChE and ChE reveal that (+)-
`trons-ACTM is a good substrate for AChE
`since it is hydrolysed by AChE as fast as ACh.
`(--}-Trans-ACTM is a poorer substrate for
`AChE since its relative rate of hydrolysis is
`59% that of ACh. The relative rates of hy-
`drolysis of (+)- and (—)-trons-AC'1‘M by ChE
`are 61 and 34% that of ACh (table 4). These
`results explain the observation that the muses.-
`rinic activities of ACh and (+}- and (—}-trans-
`ACTM are potentiated by neostigmine 41-fold,
`23-fold and 3-fold, respectively (table 3). In
`other words,
`the muscarinic activity of (+)-
`trons-ACTM is potentiated by neostigmine
`more than that of
`(—)-trons-ACTM because
`the former is a better substrate for the cho-
`
`(cid:54)(cid:88)(cid:81)(cid:16)(cid:36)(cid:80)(cid:81)(cid:72)(cid:68)(cid:79)(cid:16)(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:25)(cid:16)(cid:19)(cid:20)(cid:20)(cid:19)(cid:23)(cid:16)(cid:3)(cid:40)(cid:91)(cid:17)(cid:3)(cid:20)(cid:19)(cid:21)(cid:20)(cid:15)(cid:3)(cid:83)(cid:17)(cid:3)(cid:24)(cid:3)(cid:82)(cid:73)(cid:3)(cid:25)
`Sun-Amneal-|PR2016-01104- Ex. 1021, p. 5 of 6
`
`

`
`248
`
`CEIOU ET AL.
`
`Vol. 166
`
`linesterases than the latter. Since the intact
`animals are much more complex than the iso-
`lated enzyme preparations, one would not ex-
`pect
`that the degrees of potentiation of bio-
`logic activities of
`the drugs by neostigmine
`measured in vivo and the difierence in magni-
`tude of
`the relative rates of enzymatic hy-
`drolysis of these compounds measured in vitro
`would be the same. It should be true, however,
`that the relative rates of hydrolysis of these
`eaters by cholinesterases and the degrees of
`potentiation of
`the muscarinic activities of
`these compounds by neostigmine are in the
`same order and that is the case in this study:
`AC1: ? (+)-trams-AC'I‘M > (—}—trans-ACTM.
`CoNci.usIoNs. The activities of 2-acetoicy
`cyclopropyl
`trimethylaminoiiium
`iodide
`(ACTM) have been studied in a number of
`pharmacologic and ensymologic systems. The
`tram-ACTM had strong muscarinic activities
`but weal: nicotinic activities. The cis-ACTM
`
`had negligible muscarinic and nicotinic activi-
`ties. The possible reasons of
`these findings
`have been discussed. The potency ratios of
`muscarinic activities between (+)— and (—)-
`tram-ACTM were fairly close to those between
`1.(+}- and Ii{—)—acetyl-,3-inethylcholine. The
`relative rates of hydrolysis of ACTM and
`ACh by cholinesterases and the degrees of po-
`tentiation of their biologic efiects had the same
`order: ACh 2 (+1-trans-ACTM > (—l—t'roiis—
`ACTM.
`
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