CRYSTALS OF LAOUINIMOD SODIUM AND MPROVED PROCESS FOR TEE
ItIANUFrtCTURE THEREOF
Th~sap pltcatzon clams pnonty of U S Prov~s~onAalp plication No 611785,575, filed March 14,
5 201 3, the entire content of wluch 1s hereby mncorporated by reference herem
Throughout thrs applicatton, vanous publicat~ons are referred to by frst author and year of
publication. Full cttations for these publications are presented In a References section lnmedtately
before the clatms. D~sclosureso f the publtcatlons cited In the References sectlon m theu entireties
are hereby incorporated by reference Into th~sap plication in order to more fully describe the state
10 of the art as of the date of the ~nventiondescribed herein.
Background of the Invention
Laqumunod 1s a compound which has been shown to be etTket~ve ~n the acute expenmental
autounmune encephalomyelltls (aEAE) model (U S Patent No 6,077,851) Its chmcal name IS Nethyl-
N-phenyl-1,2,~1hydrrr4-hydroxy-5~~oro-l-m&yl-2~xoquno~ne-3-car~axn~d1 d~e,t s
L 5 Chmcal Regsky number IS 248281 -84-7
Laquinirnod is a small molecule having the following chemcal structure:
I laquinimod
Laquinimod sodium has htgh oral bioavailability and has been suggested as an oral formulation for
the treatment of Multiple Sclerosis (MS). (Polman, C. et al., (2005) "Treatment with laquinirnod
2 0 reduces development of active MRI lesions in relapsing MS", Neurology. 64:987-991; Sandberg-
Wollheim M, et al. (2005) "48-week open safety study with high-dose oral laquinimod in patients",
Muit Scler. 11:S154). Studies have also shown that laquinimod can reduce development of active
MRI lesions in relapsing MS. (Polman, C. et al., (2005) "Treatment with laquinimod reduces
development of active MRI lesions in relapsing MS", Neurology. 64:987-991).
2 5 In order to prepare laquinimod as a pharmaceutical drug product, manufacturing processes need to
take into constderatton the posstbtltty of the impunties disclosed herein being present m the product.
Early syntheses of laqutntniod produced product contamnated with tmpunttes such as methyl 5-
chloro-4-hydroxy-1- methyl-2-oxo-l,2-dihydroquinoltne-31:arboxylaatne d 5-chloro-il-hy&oxy-l -
methyl-2-0x0-l,2-dthydmquinoltne (K. Jansson et al , (2006) "Synthesis and Reacttv~ty of
5 Laqutnimod, a Quinoline-3-carboxarmde Intranolecular Transfer of the En01 Proton to a Nitrogen
Atom as a Plausible Mechantsm for Ketene Formation, J Org Chem , 71, 1658-1 667) Several
other impunttes have also been disclosed (U S Patent No 6,077,851, U S Patent No 6,875,869,
and U S Patent No 7,881,208) U S Patent Application Publication No 2012/0010239, the
contents of whtch are incorporated by reference tnto this application, cfiscloses the impurity 5-
10 chloro-4-hydroxy-l -methyl-2-0x0-N-phenyl-1,2-dihydroqumotde U S Patent
Application Publicatton No. 2013/0217724, the contents of whch are incorporated by reference
into tlus appltcatlon, dlscloses the impunty iV-ethyl-4,s-dihydroxy--1m ethyl-2-0x0-N-phenyl-l,2-
dihydroqtlmoltne-3-carboxmlde U.S Patent Appllcatton Publication No. 2013100345256, the
contents of whch are mncorporatd by reference into this application, discloses the tmpunty N-
15 ethyl-4-hydroxy-l-methyl-S-(methyl(2,3,4,5,6-pentahydroxyhexyl)amino)-2-oxo-~l-1,2-
dihydroqumnoline-J-carboxmde US Patent No 8,178,127, the contents of which are
incorporated by reference into thls application, dlscloses the impunties 2-chloro-6-(I-ethyl-Nmethyl-
2-oxoindoline-3-carboxamido)benzo~acc td, 1H ,3H-spiro[S-chloro-1 -methylqutnoline-2,4-
dione-3,3'-[l]e(hylmdolm-121-one and 5-chloro-N-ethyl-3-hydroxy-l-methyl-2,4-d1oxo-N-
2 0 phenyl,I,2,3,4-tetr&ydroq~unolme-3-carboxamtde
The processes of synthesis of laquinimod and the preparation of its sodium salt are disciosed in
U.S. Patent No. 6,077,851. An additional process of synthesis of laquinimod acid, but not
laquinimod sodium, having low levels of certain impurities is disclosed in U.S. Patent No.
6,875,869. U.S. Patent No. 7,884,208 teaches a process for the preparation of laquinimod sodium
2 5 which removes certain impurities present aRer the salt formation step, thus resulting in a crystalline
mixture of higher purity as well as a crystalline mixture having large particles, and improved tapped
and bulk density. Pharmaceutical compositions comprising laquinimod sodium are disclosed in
PCT International Application Publication No. WO 2005/074899.
In the preparation of laquinimod sodium disclosed in U.S. Patent No. 6,077,851, laquinimod acid was
3 0 suspended in ethanol, and SM sodium hydroxide solution was added. After stining, the resulting
precipitate was filtered, washed with ethanol, and dried. The method used to make laquinimod sodium
in U.S. Patent No. 6,077,851 is commonly referred to as a slurry-to-sluny salt formation.
In the sluny-to-slurry salt formation method of U.S. Patent No. 6,077,851, the laquinimod sodium is
not dissoived in solution Any solid impurities, if present in the laquinimod sodium suspension, are
therefore not removed by filtration.
U S Patent No. 6,875,869 discloses a process of prepanng the base compound Laqusrumod tn Igh
yeld and low level of rmpuntres, However, the process ur US. Patent No 6,875,869 1s only for
synthesrs of the base compound (lauqmmod acid) and not the salt. As such the slurry-to-slurry salt
5 fomauon process would sull be needed to f ~ r mthe sodlum salt.
U S. Patent No. 7,884,208 teaches an rrnproved process fo~pr epanng laquumod sodium resultmg m
crystals of higher punty as well as crystals havmy tmproved crystallme charactmstlcs, e g , eompnsmg
no more than 2 ppm of a heavy metal and h a wh sgher tapped density In the processes d~sclosedm
Examples 13-17 of U S Patent No 7,884,208, laqmmmod sodium 1s d~ssolvedm water to form an
10 aqueous solution, the aqueous solutlon IS concentrated, and then a water-msctble antl-solvent 1s added
to the concentrated solution to form laqummod sodium crystals. The process of U S Patent No
7,884,208 removes the unpunties afier salt formahon, thus resultmg m laqurnirnod sodlurn of hsgher
purity than the iaquinimod sodium produced directly from the "slurry to slurry" process of U.S. Patent
No. 6,077,851.
Summary of the Iavenaan
The subject invent~onp rovldes a mixture of cfystallme laqumod sod~ump art1c1es, wherein (I) 90%
or more of the total amount by volme of the laqu~mmods odun part~clesh ave a size of 40 mcmm
or less or (u) 50% or more of the total mount by volume of the laquuumod so&um particles have a
5 sue of 15 mcrons or less, and wherem:
a) the mixture has a bulk density of 0.2 @mL to 0.4
b) the mixture has a tapped density of 0.40 gimL to 0.7
c) an amount of heavy metal which is present in the rmxtwe 1s no more than 20 ppm of
heavy metal relative to the amount by wel&t of laqumod sodium;
d) an amount of 5-Chloro-4-hydroxy-I-methylquinolin-2(1H (MCQ) which is
present in the mxture is no more than 0.15% relative to the amount of laqwnimod sodlum as measwed
by HPLC;
e) an amount of 5-chloro-4-hydroxy- 1 -methyl-2-0x0- 1,2-dhyhqumoline-3-
caiboxylic acid (MCQCA) in the mixture is no more than 0.15% relative to the amount of laquinimod
15 sodium as measured by HPLC;
f) an amount of methyl 5 - c h l o m - 4 - h y d r o x y - l - m e t h y l - 2 i l x o - 1 , ~ - 3 -
carboxylate (MCQME) whtch is present m the mixture 1s no more than 0 12% relative to the amount
o f i a q w d d m as mas& by &?LC,
g) an mamoutil of meethyl 5shiod-hydroxy-1 -m&y1-2-0xyr- t ,24hy&qunoiine-3-
carboxylate (MCQME) wluch 1s present in the mixtu~1s no more than 0.10% relative to the amount
o f l a q ~ osdod ium as measured by HPLC;
h) an amount of N-ethylaniline O\IEA) which is present in the mixture is no more than
0.10% relative to the amount of laquinimod sodium as measured by HPLC; or
i) an amount of N-ethyl-4,5-dihydroxy-l-methyl-2-oxo-N-phenyl-1,2-
2 0 dihydroquinoline-3-casboxamide (5-HLAQ) which is present in the mixture is no more than 0.10%
relative to the amount of laquinimod sodium as measured by HPLC.
The subject invention provides a mixture of crystalline laquinimod sodium particles, wherein (i) 90%
or more of the total amount by volume of the laquinimod sodium particles have a size of less than 40
microns, (ii) 50% or more of the total amount by volume of the laquinimod sodium particles have a
ma~ympm suom~ms u q l s sal jo azrs a ahq s a ~ s ~upmdp os
prurrtnnbsl aql jo aumfo.2 Aq junomv @to%ay ljo aiour lo %0t (111) pm 'suolsrm 5 I urryl ssatjo azis
b) cunmntmting the aqueous solution to form a concentrated solution comprising
approdtely 1.7-1.8 mi, of water per gram of laqumod sodiwn;
c) adding acetone to the concmtratd solution of step b); and
d) msolatmg rccrystailized laquinunod sodium.
5 The subject mventlon also provides for a mtxW of crystallme l a q m c d sod~ump arttcles prepared
by the process as described herein, and a pWceuttca1 compostt~onco mprrsing sald rmxture.
The subject mnventmon provldes a rmxture of crystalltne laqwmod sodium partmcles, wherem (I) 90%
or more ofthe total amount by volume of the laqu~nunods o&um particles have a size of 40 mcrons
or less or (u) 50% or more of the total amount by volume of the l a q m o d sodurn particles have a
10 srze of 15 mcmm or less, and wherem
a) an amount of aluminium ~n the mxture is less than 5 ppm relative to the amount by
we~ghot flaqumod sodium;
b) an amount of calcium in the m m e is less than 60 ppm relative to the amount: by
weight of laqu~nimodso dium;
c) an amount of copper in the mixture is less than 1 ppm relative to the amount by weight
of laquinimod sodium; or
& ar, amont of z m UI the mtxture is Ies than 7 ppm reiartve to the mounl by %elgin(
of laqumunod sodrum.
The subject invention dso provides a pharmaceutical composition comprising the mixture as described
2 0 herein and a pharmacwtically acceptable carrier.
The subject invzntion also provides an isolated compound having the structure:
The subject invention also provides aa composition comprising a compound having the structure:
or a salt thereof, wherein the composition IS fiee of laqulmmod or a salt thereof.
The subject mvention also prondes a phmaceutical composltlon compnslng an amount of
laqu~nunod and at least one of 2-chloro-6-(3-(ethyl(phenyl)ammo}-2-hydroxy-N-methyl-3-
5 oxopropanam~do)benzotca c~d( BE-3-HLAQ), 5-CMoro-4-hydroxy-l-methylqumolu~-2(1N)-one
(MCQ), 5-Chloro-4-hydro.cy-l-methyl-2-oxo-l,2-dihy&oq~1notine-3-carbo7taychicd (MCQGA),
Methyl 5-chloro4-hydroxy-l-methyt-2-axo-l,2-dihydroq~noline-3aarhoxyl(aMteC QME), NFthylanlllne
(NEA), and Ethyl 5-chloro-il-hydroxy-I-mdbyl-2-ovo-l,2d-ih ydroqumnol~ne-3-
carbovylate (MCQEE), wherein a) 2-chioro-6-(3-(ethyl(phenyl)amno)-2-hydroxy-N-m~hyl-3-
10 oxopropanmdo)benzo~ca c~d(B H-3-HLAQ) is present in the pharmaceutical composltlon m an
amount not more than lo%, relatlve to the concentratlon of laqumlmod, based on a detemnat~on
by an HPLC method, or b) 5-Chloro-4-hydroxy-1 -methylqumofin-2(1@-one (MCQ) 1s present In
the pharmaceutical composition m an amount not more than lo%, relative to the concentration of
laquumod, based on a detemnat~onb y an HPLC method, or c) 5-Chloro-4-hydroxy-l-methyl-2-
15 0x0-1,2-dthydroquinoline-3-carboxyllc actd (MCQCA) is present tn the pharmaceutical
composltlon in an amount not more than lo%, relatlve to the concentration of laqumlmod, based
en a d&enrunatton by an HPLC method, or 3) :)%ethyl 5-chloro4-hydmxy-1-meth~.1-2-oxo-L,2-
d~hydroqumoltne-3-carboxylate(M CQME) is present m the pharrnaceutlcal composltlon In an
amoult not more than :O0C, relattve to the concmtmt~on of iaqumunod, based on a detemmat~on
20 by an HPLC method, or e) N-Ethylaniime (NEA) IS present m the phamaceut~cacl omposltlon m
an amount not more than lo%, relattve to the concentratlon of laquuumod, based on a d e t e m t i o n
by an HPLC method, or t) Ethyl 5-chloro-4-hydroxy-I-methyl-2-0x0-l,d21-h ydroqumohne-3-
carboxylate (MCQEE) ~s present in the phamtaceuttcat composltlon in an amount not more than
lo%, relatlve to the concentratlon of laqunnmod, based on a determtnat~onb y an HPLC method.
2 5 The subject invention also provides a process for preparing 2-chloro-643-(ethyl@henyl)amino)-2-
hydroxy-N-methyl-3-oxopropanamido)her1zoiacc id (BH-3-HLAQ) comprising the steps of: a)
adding sodium hydroxide solution to a suspension of 5-chloro-N-ethyl-3-hydroxy-1-methyl-2,4-
dioxo-N-phenyl-l,2,3,4-tetrahydroquinoline-3-carboxamiinde w ater, b) stirring the mixture of step
a) followed by addition of hydrochloric acid solution, c) extracting the aqueous solution with ethyl
3 0 acetate, d) washing the organic phase with brine, e) drying the organic phase over sodium sulfate,
f) filtenng the suspension, g) evaporating the filtrate, h) purrfy~ngt he res~dueb y ctystalhzat~on
from lsopropyl alcohol, I) coolmg the suspension followed by filtenng and washrng wlth rsopropyl
alcohol, and j) obtaning and drytng the resultrng whlte solid.
I'he subject ~nvention also provides 2-chloro-6-(3-(etbyI@henyl)amino)-2-bydro~y-N-methyl-3-
5 oxopropanamdo)benzolc acld (RH-3-HLAQ) prepared by the process described above.
Ihe subjeel rnventlon also provides a process for prepanng 5-Chloro-4-hydroxy-1 -methylquinolin-
2(1N)-one (MCQ) compnsmg the steps oP a) heating a mixture of 5-Chloro-4-hydroxy-l-methyl-
2-axo-1,2-d1hydroqu1nolme-3-carboxyl1acc id (MCQCA) and dimethylsulfoxlde, b) cooling the
mxture of step a), and c) tiltang the mixture of step b) and coileetlng the resultmg filtrate.
10 The subject mvention also provrdes 5-Chloro-4-bydruxy-l-methylquinolin-2(1~ne (MCQ)
prepared by the process described above.
The subject Invention also pmvides a process for prepanng 5-Chloro-4-hydroxy-I-methyl-2-0x0-
l,2-dihydroquinoline-3-carboxyllacc id (MCQCA) compnsmng the steps of a) heatlng a mxhue of
ethyl 5-cbloro-4-hydroxy-1-methyl-2-0x0-1,2d-~ hydroquinoline-3-cxboxylate( MCQEE) In a
15 solution of hydrochloric acid in acetic aad, h) cooling the mxture of step a), c) diluting the mxture
of step b) with 2-propanol and fudher cooling the d~lutedm rxlure, and d) filtenng off the crystals
resulting from step c)
The subject invention also pmvides 5-Chloro4-hydroxy-l-methyl-2uxo-1,2-dIhydroquinoline-3-
carboryi~ca crd (XftleWA) prepared by the proct3ss desrnbed above
2 0 The subja.t mvmtion also prrjvrds a process for pntjlmilg Methyl 5-chlora-4-hydfoxy--1m &hyl-2-
0x0-1,2-dihydroquinoline-3-carboxylate (MCQME) compnsmg the steps of: a) fonrung sodium
dirnethylmalonate by reaction of dimethylmalonate in dimethlfomrnide with sodium methoxide
solution, b) reacting the intermediate 5-chloro-1-methyl-lh-benzo[D][l,3] oxazine-2,4-dione with
sodium dimethylmalonate to form methyl 5cNoro-4-hydroxy-1-methyl-2-0x0-1,2-
25 dihydroquinoline-3-carboxylate (MCQME) sodium salt, and c) acidifying methyl 5chloro-4-
hydroxy-1 -methyl-2-oxo-l,2-dihydroquinoline-3-carboxylaWte CQME) sodium salt to methyl 5-
chloro-4-hydroxy-l-methyl-2-oxo-1,2-dihydroquino1ine-3arboxyla(MteC QME).
The subject invention also provides Methyl 5-chloro-4-hydroxy-1-methyl-2-oxo-1,2-
dihydroquinoline-3-carboxylate (MCQME) prepared by the process described above.
3 0 The subject invention also provides a process for preparing Ethyl 5-chloro-4-hydroxy-1-methyl-2-
0x0-1,2- dihydroquinoline-3-carboxylate (MCQEE) comprising the steps of: a) adding sodium
hydnde to a solution of j-chioro-1-methyl-lh-bemo[D][1,3]o xazine-2,4-dione and d~ethyl
malonate m d~mahylformamideb, ) heating the mixture of step a) wfule stimng, c) cooling the
solution of step b), d) quenching the reaction mxture of step c), e) ac~difyingth e mixture of step
d), 1) filtmng then drymg the mixture of step e) , and g) crystallizing the crude product of step f)
5 by dissolvmng in ethanol following by slow cooling.
The subject invention also provides Ethyl 5-cNoro-4-hydroxy-l-methyl-2-oxo-l,2-
dihydroqujnoline-3-carboxylate (MCQEE) prepared by the process described above.
The subject invention also provides a process for testing whether a sample of laquimmod contams
an undesirable unpunty which comprises detennilllng whether the sample contains a compound
10 havlng the structure
The subject Invention also provides a process for prepmng a validated pharmaceutical composition
compnsaog iaqwruz~cdc mpnsiny a) obtvmg a batch of laquin~mod.b t dezerrmmtni: the
15 amount of at least one of 5-Chloro-4-hy&oxy-l-methylqumolm-2(1~-on(eM CQ), 5-Chloro-4-
hydroxy-l-metbyl-2uxo-l,2-dihydroquinol1ne-3-carboxylaicci d (MCQCA), Methyl 5-chloro-4-
hydroxy-l-methyl-2-oxo-l,2-dihydroquinoline-3-carboxylaQteX CQME), N-Ethylandine (NEA),
and Ethyl 5-chloro-4-hydroxy-1-methyl-2-0x0-1,2- d~hydroqumoline-3-carboxylat(Me CQEE) m
the batch using by an HPLC method, and c) prepanng the pharmaceutical composition from
2 0 the batch only if
i) the batch is determined to have not more than 0.15% 5-Chloro-4-hydroxy-lmethylquinolin-
2(1H)-one (MCQ), relative to the concentration of laquinimod, or
ii) the batch is detemined to have not more than 0.15% 5-Chloro-4-hydroxy-I -methyl-2-0x0-
1,2-dihydroquinoline-3-carboxylie acid (MCQCA), relative to the concentration of
laquinimod, or
in) the batch 1s detemuned to have not more than 0.15%, 0 12% or 0.1% Methyl fi-chloro-4-
hydroxy-l-methyl-2-oxo-l,2-d1hydroquinolme-3-carboxyl(MatCeQ ME), relatrve to the
concdmtron of laqummod, or
IV) the batch is detenn~nedto have not more than 0.1 % N-Ethylanlline (NEA), relative to the
5 concentration of laquinimod, or
F) the batch is determined to have not more than 0.1% Ethyl 5-chloro4-hydroxy-l-methyl-
2-oxo-1,2- dihydroqurnoline-3-carboxylate (MCQEE), relative to the concentration of
laquinimod.
The subject mventton also proades a process for preparrng a pharmaceutical composition compnslng
LO Iaquinlmod, or for hstnbutmg a validated batch of a phamaceutical composltlon compnsrng
laqummod, compnslng a) obtarung a hatch of laqu~nimodo r of the phmnaceutlcal composalon, b)
perfomng stabrhty testing wlth a sample of the batch, c) detemmng the total mount of at least one
of 5-Chloro-3-hydroxy-l-methylqu~nolmn-2(1N)-on(Me CQ), 5-CNoro-4-hydroxy-l-methyl-l-0~0-
1,2-d1hydroqu1nolme-3-carboxylac1icd (MCQCA), Methyl 5-chloro-l-hydroxy-I -methyl-2-oxo-t,2-
15 d~hydroqmoline-3-carboxylate (MCQME), N-Ethylanlime (NEA), and Ethyl 5-chloro-l-hydroxy-lmethyl-
2-oxo-1,2- d~hydroqurnolme-3-carboxylat(eM CQEE) m the sample of the batch after stability
testing by an HPLC method; and d) vaildatrng the batch for dlstnbution or prepmug the
p b c e u t l c a l composition fmm the batch only ~f the sample of the batch after stability testlng
contalns
20 1) not more than a rota1 of 1)./5% relative to the concentratlon of laqnwod of 5-Chloro-4-
hydroxy-1-methylquinolin-2(1W)-one (,MCQi, or
11) not more than a total of 0.15% relatrve to the concentratlon of laquwod of 5-Chloro-4-
hydroxy-1- rnethyl-2-oxo-1,2-d1hydroqumol1ne-3-carhoaxcyi1d1 (~M CQCA), or
iii) not more than a total of 0.15%, 0.12% or 0.1% relative to the concentration of laquhhod of
Methyl 5-cNoro-4-hydroxy- 1 -methyl-2-oxo-l,2-dihydroquinoline-3-carboxylate
(MCQME), or
iv) not more than a total of 0.1% relative to the concentration of laquinimod of N-Ethylaniline
(NEA), or
v) not more than a total of 0.1% relative to the concentration of laquinimod of Ethyl 5-chloro-4-
3 0 hydroxy-l-methyl-2-oxo-1,2d-i hydroquinoline-3-carboxylate( MCQEE).
The subjcct mvention also provtdes a process for validatmg a batch of a phmaceutical product
contaimng laqwnlmod or a phmaceuticaliy acceptable salt thereof and a pharmaceutlcally
acceptable carner for dlstnbution compnsrng a) subjectmg a sample of the batch to stabtllty testmg,
b) detmnmg the amount of at last one of 2-chloro-6-(3-(ethyl(pheny1janudo)-2-hyd
5 methyl-3-oxopropamdajbe11~oic acsd (BR-3-ZILAQ), 5-CNoro-4-hydroxy-1 -methylquinolm-
2(lIfl-one (MCQ), 5-Chloro-4-hydroxy-1-methyl-2-oxo-1,2-dihydroqu1noi~ne-3tarboxaycliidc
(MCQCA), Methyl 5-chloro4-hydroxy-l-methyl-2-oxo-l,2dlhydroqumnolme-3-cafboxylate
(MCQME), N-Ethylanilme (NEA), and Ethyl S-ehloro-4-hydroxy-1-methyl-24x0-1,2-
drhydroquinolmne-3-carboxylate( MCQEE) m the sample of the batch after stablhty testrng by an
10 HPLC method, and c) validatutg the batch for distribution only ~f the sample of the batch after stability
testmg contains
I) not more than a total of 1.0% relative to the concentratmon of hqtmhod of 2-chloro-6-(3-
(ethyl(phenyl)amino)-2-hydroxy-N-methy1-3-oxopropanamido)bmoic acld (BH-3-HLAQ),
or
15 mi) not mare than a total of 0.5% or 0.1% relatlve to the concentralon of laquinimod of NEthylanilme
(NEA), or
111) not more than a total of 0 5% relatlve to the concentration of Iaqummod of 5-Chloro-4-
hydroxy-1-methylquinolmn-2(1W)-one (MCQ) and 5-Chlora-4-hydroxy-1 -methyl-2-0x0-1,2-
dihydroqumolme-3-carboxyl~acc id (MCQCA) combmed
2 0 The subject mvazlon also pmdm a process for prqanng a packaged pharn~aceut~ccaol mposrtion
compnslng laqulmod or a pharmaceutlcally acceptable salt thereof comrpnslng a) obtainang a
"oath ofpbmaceuticai composltlon compnszng laqurnlmod or a pharmaceutically acceptable salt
thereof, b) perfnmung stabtltty testing with a sample horn the batch, c) detemnlng the amount
of at least one of 2-chloro-6-(3-(ethyl(phenyl)ammo)-2-hydroxy-N-methyl-3-
2 5 oxopropanmxdo)benzoic acid (BH-3-HLAQ), 5-Chloro-4-hydroxy-l -methylqumolm-2(1m-one
(MCQ), 5-CNoro-4-hydroxy-l-methyl-2-oxo-1,2-dlhydroqnoline-3-cboxayclidic ( MCQCA),
Methyl 5-chloro-4-hydroxy-l-methyl-2-oxo-l,2-dlhydroqmolme-3-carboxyla(tMeC QME), NEthylandme
(NEA), and Ethyl 5-chloro-4-hydroxy-1-methyl-2-0x0-1,2d-i hydroqumolmne-3-
carboxylate (MCQEE) m the sample by an HPLC method after stability testmng; and d) packagng
3 0 the pharmaceutical composttlon in only if
i) the content of 2-chloro-6-(3-(ethyi(phenyl)amino)-2-hy-I-3-
oxopropanamido)benzoic acid (BH-3-HLAQ) in the sample is determined to be not more
than 1 .O% to the concentration of laquinimod, or
10 the content of N-Ethylarlmne (NEA) m the sample 1s determined to be not more than 0.5%
or 0 1°/o to the concentration of laqrummod.
in) the comb~nedc ontent ctf S-Chloroii-hydroxy-l-methylqumnol1n-2(11-I)-o(nMe CQ) and 5-
Ckloro-4-hydrowy-I-methyl-2-oxo-l,Z-d1hydroquno1ne-3-carboxyalci~c d(M CQCA) in
the sample is detemned to be not more than a total of 0.5% relatlve to the concentration
of laqurmmod.
The subject uivention also protldes an ~mpunty or a salt thereof for use, as a reference standard to
detect trace amounts of the mpunty In a phmaceut~caclo mpositmon compnslng laquln~modo r a
phmaceut~callyac ceptable salt thereof, wherein the rmpunty 1s selected from the group consist~ng
10 of 2 - c h f o r o - 6 - ( 3 < e t h y l @ h e ~ y 1 ) m 0 ) - 2 - h y d r ~ o cac ~d
(BH-3-IILAQ), 5-Chloro-4-hydroxy-1 -methylqutno11n-2(1 (MCQ), 5-Chloro-4-hydroxy-lmethyl-
2-oxo-l,2-dihydroqurnol1~3-carboxylaiccs d (MCQCA), Methyl 5-chloroil-hydroxy-Imethyl-
2-0x0-1,2-dihydroquinolme-3-carboxylat(eM GQME), N-Ethylamlme (NEA), and Ethyl 5-
chloro-4-hydroxy-I-methyl-2-0x0-1,2d-th ydroqu~nollne-3-carboxylate(M CQEE).
15 The subject Invention also provides a method of determmng the concentration of an impunty m a
pharmaceutical composstion compnsing laquimmod, the method compnsing, a) Prepanng a sample
solut~on from the phannaceuttcal composalon. b) Prepanng a standard solut~on compnslng the
rmpunty, c) Prepanng a resolution solutton compnsmg laqutmmod and the mpunty, d) Prepanng
a buffer solution by dtssolvmg ammomum acetate m water and adjustmng to pH of 7 0 i 0 05 with
20 aqueous amonia or glaclat acettc acid, e) Prepming a diiucnt solution compnstng ibe buffer
solution and acetotutnle, f) Prepanng a blank solutlon compnsmng the mluent solution and aqueous
acetumtaie, gj Injsttng Into the HPLC the rcsolut~un soiutron, the blank soiutron, the standard
solut~ona, nd the sample solutlon, h) Runmng the NPLC usrng ultraviolet absorption at 240 nm and
the d~luensto lutton as the mob~lep hase, 1) Detemmmg the retention tune (RT) and the areas of the
25 peaks of the ~mpuritym the chromatograms of the sample solut~ona, nd j) Perfomg quantltation
of the lmpunty with respect to the correspondsng peaks m the chromatograms of the standard
solutions.
wherein the impurity is 5-Chloro-4-hydroxy-I-methylquinolin-2(1H)-one( MCQ), 5-Chloro-4-
hydroxy-l-methyl-2-oxo-1,2-dihydroquinoline-3-earboxylaiccid (MCQCA), Methyl 5-chloro-4-
3 0 hydroxy-1 -methyl-2-0x0-l,2-dihydroquinoline-c3a-rb oxylate (MCQME), N-Ethyl-4,5-dihydroxy-
I-methyl-2-0x0-N-phenyl-1,2 dihydroquinoline-3-carboxarnide (5-HLAQ) or Ethyl 5-chloro-4-
hydroxy-I-methyl-2-0x0-1,2d-i hydroquinoline-3-carboxylate( MCQEE).
The subject invention also provides a method of determining the concentration of an impurity in a
phmaceutlcal composltron compnsmng laquinmod, the method compnsmg, a) Prnpanng a sample
solutlon fmm the phrmaceuttcal composltlon, b) Prepanng a standard solutton compnslng the
xmpunty, c) Prqartng a resolut~on solutton comprising laqu~mmod and the ~mpuntyd, ) Preparmg
a buffer solutton by dlssolvtng momurn acetate m water, and adjusttng to pH of 7 0 i: 0 05 wlth
5 aqueous w o n l ao r glac~aalc etic ac~de,) Prepmny a blank solutron comprismg the buffer solution
and acetomtnle, f) Injectmg Into the HPLC the resolution solution, the blank solutron, the standard
solution, and the sample solution, g) Running the HI'LC uslng ultrav~oleta bsorption at 240 nm and
a mobile phase of a mxture of the buffer solutton, and acetonrtnle, h) Detcminmg the retention
tune (RT) and the areas of the peaks of the rmpunty In the chmatopams of the sample solutlon,
10 and I) Peiformtng quantltatton of the tmpunty wlth respect to the correspondmg peaks tn the
chromatom of the standard soluttons,
IS The subject rnvenrron also prondea a method of detesmnnng the concentration of an tmpunty m a
phamceuttcal composttion compnsmg laqummod and a pharmaceutically acceptable earner, the
method compnsmg, a) Prepanng a sample solutton fsom the pharmaceuttcal composltlon, b)
Prepanng a standard solutlon compnslng the rmpunty, c) Prepanng a Quantitatton Ltmt (QL)
solution compnslng the ~mpuntyd, ) Prepanng a resolutton stilutton compnsmg laqurmmod and the
2 0 mpunty, e) Fsepanng a buffer solutton by dlssolvlng ammomum d~hydrogenp hosphate m water,
and adjusting to pH o17 0 * 0 10 with aqueom amoma or phosphoric acld, x) Preparing a blank
solution compnslng the buffer solutron and acetomtnle, g) Injecting Into the HPLC the resolutton
foiut~oq the blank solullon, the Q1, folutxon, the danhrd mlutron and :he sample soiutlon, h)
Runnmg the WPLC ustng a ultraviolet absorption at 212 nm and a mobile phase of a mxture of the
25 buffer solutlon, acetomtnle, and methanol, I) Determnmg the retentton ttme (RT) and the areas of
the peaks of the unpunty in the chromatograms of the sample solutton, and j) Perfortrnng
quantttatton of the lmpunty wtth respect to the corresponlng peaks m the chromatograms of the
standard solut~ons,
wherein the impurity is 2-chloro-6-(3-(ethyl(pheny1)amino)-2-hydroxy-N-methyl-3-
30 oxopropanamido)benzoic acid (BH-3-IILAQ) or N-ethyl-4-hydroxy-1-methyl-5-(2,3,4,5,6-
pentahydroxyhexylamino)-2-oxo-N-phenyl-l,2-dihydroquinoline-3-carboxami(MdeE G-LAQ).
The subject invention also provides a method of determining the concentration of an impurity in a
phamceutical composition comprising laquinimod and a phasmaceutically acceptable canier, the
method compnsmg, a) Prepartng a sample solutron frnm the phmceutlcal campostton, bf
Prqanng a standard solutlon eompnslng the impmty, c) Prepanng a resolutlon solution
compnsmg laqmmmod and the rmpunty, d) Prepmng a butYer solutron by dissolvmng ammomum
acetate tn water, and adjustmg to PH or 7 O * O 05 w~thaq ueous amonla or glacial acetlc acid, e)
5 Prepanng a blank solutlon compristng the buff= solutton and acetomtnle, f) Injecting ruto the
HPLC the resolut~ons olution. the blank solut~ont,h e standard solut~ona nd the sample solution, g)
R m g the HPLC uslng a uitravlalet absorption at 242 nm and the blank solution as the mobile
phase, h) Detmmng the retention t~me(R T) and the areas of the p& of the rmpunty m the
chromatogm of the sample solution, and I) Perfoming quantitation of the Impunty with respeet
10 to the conespondlng peaks In the chromatograms of the standard solutions,
where~nt he unpunty 1s 5-Chloro-4-hydroxy-l-methylqu1nol1n-2(l~+WneC Q), 5-Chloro-4-
hydroxy-l-methyl-2-oxo-l,2d1hydroqinnolme-3-c~boxyalc1idc (MCQCA), Methyl S-chloro-4-
hydroxy-l-methyl-2axo-l,2-d1hydroqumol1ne-3c-a rboxylate (MCQME) or N-Ethyl-4,5-
d~hyclroxy--1m ethyl-2-oxo-N-phenyl-1,d2~ hydsoqumoline-3-carboxamde(5 -HLAQ).
15 The subject invention also prowdes a method of detemnmg the concentration of an unpunty m a
pharmaceutical composition compnsmg I a q u m d and a phaceuttcally acceptable carner, the
method compnsmg, a) Prepanng a sample solutlon from the pharmaceut~cal composrtlon, b)
Prepanng a standard solut~on compnsrng the Impmty, c) Prepanng a resolutlon solut~on
comprlslng laqu~nunod and the impurity, d) Prepanng a blank solutlon compnsmng methanol and
2 0 acetomtnle, e) Prepanng a buffer solutton by dissolvmg ammomum acetate in water, and adjusting
LO pH of 7 6 * 0 05 with aqueous a m m a o r giaclai acetlc ac~d.r ) h j s t ~ n g~ ntath e HPLC the
resolution solut~on, the blank solut~on, and the sample solution, g) Runnmg the HPLC uslng a
ultrwioiet zbsorptton at 2.40 nrn and a mobile phase corripnsmg acetooitnle and the buffer solutllon,
h) Detemnmg the retent~on t~me (RT) and the areas of the peaks of the mpurity rn the
25 chromatograms of the sample solut~ona, nd I) Performmg quantltat~ono f the lmpunty with respect
to the conespondmg peaks In the chromatograms of the standard solutrons,
wherein the impurity is N-Ethylaniline (NEA), 5-Chloro-N-ethyl-3-hydroxy-1-methyl-2,4dioxo-
N-phenyl-1,2,3,4-tetrahydroquinoline-3-cboxde (3-HLAQ) or lH,3H-spiro[5-chloro-lmethylquinoline-
2,4-dione-3,3'-[l]ethylindolin-[2]-one]( SPIRO-LAQ).
Brief Description of Figures
Figure I: Fig. 1 from U.S. Patent No. 7,884,208.
Figure 2: Fig. 2 from U.S. Patent No. 7,584,208.
Figure 3: HPLC Data - Example c h m t o g m of mixture of laquimmod sodium.
5 Figure 4: HPLC Data - Pure chromatogam of Laquinimod Sodium.
Fibwe 5: Mtcroscop~c photograph of typical batch of crude Laqulnrmod Sodluni at a first
magnificat~on level.
Frgure 6: Microscopic photograph of typical batch of crude Laqulmmod Sohum at a second
magmfication level
10 Figure 7' Mrcroscoptc photograph of Batch C at a first magnification level
Figure 8 M~croscopicp hotograph of Batch C at a second magnificatron level
Flgure 9 1 R-NMR S p e c m of MCQ In DMSO
Frgure 10 13C-NMR Spectrum of MCQ m DMSO
Flgure 11 Mass Spectrum of MCQ (ES+ mode)
3 5 Figure 12 Founer Trdnsfom-Infrared Spectrum of MCQ in KBr
Fibme 13 1 W-NMR Spmm of MCWA in DioiKOIf
Figure 14.13C-NMR Spec- of MCQCA in DzO +KOH
Flgure 15 Mass Spectrum of MCQCA (ES+ mode)
Frgure 16. Fomer Transform Infrared Spectrum of MCQCA
2 0 Flgure 17' 1H-NMR Spectrum of MCQEE m CDCI3
Flgure 18 13C-NMR Spectrum of MCQEE in CDCh
Figure 19' Mass Spectrum of MCQEE (ES* mode)
Figure 20: Fourier Transform Infrared Spectrum of MCQEE.
Figure 2 1 : I H-NMR Spectrum of 5-HLAQ 1n DMSO.
Frwe 22. 13C-NMR Spectrum of 5-NLAQ m DMSO.
Figure 23. Mass Spectrum of 5-HLAQ (ES+ mode).
Figure 24: Fourier Transform Infrared Spectrum of 5-HLAQ.
5 Figure 25: 1H-NMR Spectrum of DELAQ in CDCI3.
Figure 26: 13C-NMR Spectmm of DELAQ m CDCI,.
Figure 27: Mass Spectrum of DELAQ (ES+ mode).
Fibwe 28: Fourier Transform Infrared Spectrum of DELAQ m KBr.
Figure 29. IH-NMR Spectrum of 3-HLAQ in CDCtl.
10 R,me 30. 13C-NMR Spectrum of 3-HLAQ ~n CDC12.
Flgure 31: Mass Spectrum of 3-HLAQ (ES+ mode).
Figure 32. Founer Transform Infrased Spectrum of 3-HLAQ
Figure 3 3 1H-NMR Spectnun of SPIRO-LAQ m DMSO
F~gure3 4 I3C-NMR f pectmm of SPlRO-LAQ m DMSO
2 5 Fig~re35 Mass S ~ m omf SP IRO-LAQ (ESt mode)
Figure 36: Founer Transform Infrared Spectnun of SPfRO-LAQ
Flgure 37 IN-NMR of BH-3-HLAQ m DMSO.
Flgure 38: 13C-NMR of BH-3-HLAQ m DMSO.
Figure 39: Mass Spectrum of BH-3-HLAQ (ES+ mode).
2 0 Flgure 40: FT-IR Spectrum of BH-3-HLAQ.
Figure 41: Resolution test chromatogram (Laquinimod Sodium Before Milling (Cryst) and Drug
Substance) - Identification By Retention Time And Deteimination Of Assay And Polar Jinpurities
/ Degradation Products (Polar IDD) By HPLC.
F~gure 41: D~luent chromatogram (Laqumtmad Sodtum Before Mill~ng (Cryst) and Drug
Substance) - Idmt~ficat~oBny Retent~onT ime And Deternunatlon Of Assay And Polar Onpunties
/ Degradat~onP roducts (Polar 1DD)B y HPLC.
Figure 43: Typical chromatogram for assay (Laqulnimod Sodium Before Mtllrng (Cryzt) and Drug
5 Substance) - Idmt~ficationB y Retention T~mAe nd Detemnatlon Of Assay And Polar lmpuntles
/ Degradat~onP roducts (Polar IDD) By FPLC
Figure 44: Typ~caic hromatop~nfo r lmpurittes testing (Laqu~nlmodS odium Before Mlllmg
(Cryst) and Drug Substance) - Identsfication By Retentton Time And Determmatlon Of Assay And
Polar Impuntles i Degradation Products (Polar IUD) By ILPLC.
10 Flgure 45 Resolution test chromatogram (Laqum~modS odium Before Mtll~ng( Cryst) and Drug
Substance) - Detemmtion Of N-Ethylamlme And Nonpolar Impmtres (Non-Polar IDD) By
WPLC
Repre 46: DsIuent chromatogram (Laqu~mmods odium before millmng (Cryst) and Drug Substance)
- Detemnat~onO f N-Ethylan~lineA nd Nonpolar Impurit~es( Non-Polar IUD) By HPLC.
15 Figure 47. Typlcal sample chromatow (Laqulmmod sodtm before m~lling( Cryst) and Drug
Substance) - Determination Of N-Ethylantlme And Nonpolar Impunttes (Non-Polar LDD) by
HPLC
Frgure 48' Resoliitlon Solut:csn 1 (Resoiut~on Test) Chromatogm (hqruntnmd Capsulesf
Ident~ficat~oAnn d Determinatton Of Assay And Polar Impunt~e1s D egradation Products By HPLC.
20 Flyue 49 Resolution Solution 2 Chromtoym (taqunimod Capsules) Identification And
Detemnatton Of Assay And Polar Impunttes / Degradatton Products By HPLC.
Figure 50: Diluent (Blank) chromatogram (Laquinimod Capsules) Identification and
Determination of Assay and Polar Impurities / Degradation Products By HPLC.
Figure 51: Sample chromatogram for Assay (Laquinimod Capsules) Identification and
2 5 Determination of Assay and Polar Impurities i Degradation Products By HPLC.
Figure 52: Sample chromatogram for determination of Impurities / Degradation products -
(Laquinimod Capsules) Identification and Determination of Assay and Polar Impurities /
Degradation Products by HPLC.
Figure 53: Resolution test chromatogram (Laquinimod Capsules) Determination of Non-Polar
Impurities i Degradation Products.
Figure 54: Blank (Diluent) chromatogram (Laquinmod Capsules) Detminatlon of Non-Polar
Impurities / Degradation Products.
Figure 55: Sample chromatopm (Laquinrmod Capsules) Detenninat~ono f Non-Polar impunities /
5 Deyradation Products.
Figure 56: Typical Chromatogram of Resolution Solution (Laquinimod Capsules) - Detm~nation
of MEC-LAQ and BH-3-HLAQ by HPLC.
F~gure 57: Blank (Diluent 2) chromatogam (Laqulumod Capsules) - Determination of MEC-LAQ
and BH-3-FILAQ by HPLC.
10 Figure 58: Chromatogram of QL Solution (Laqurnimod Capsules) - Determination of MEG-LAQ
and BH-3-HLAQ by HPLC.
Figure 59: Chromatogram of Standard Solution (Laqumimod Capsules) - Detemnation of MEGLAQ
and BH-3-HLAQ by HPLC.
Figure 60: Typical Sample Chromatogram (Laqumimod Capsules) - Detemlnation of MEG-LAQ
15 and BH-3-NLAQ by HPLC
Detailed Description of the Invention
Laquinimod 1s a small molecule having the following chemical structure:
Laquinimod
5 It IS an oral immunomodulator which has demonstrated therapeut~ce ffexlt tn varlous expenmental
~nflamatoryiauto~mmame mal models, such as Expenmental Autoimmune Encgtralomyel~t~s
(EAE). an ammal model for Mult~ple Scleros~s (MS), Dextran Sodium Solphate (DSS) Induced
colxt~s for Inflammatory Bowel Dtsease, Non-Obese D~abetic (NOD) mice for Type I D~abetes
(IDDM), Expenmental Auto~mmme Neuntis (EAN) for Gu~llrun-Barre Syndrome, Systeme
10 Lupus Erythcmatosus (SLE), lupus nephnt~s,l upus arthnt~s,C rohn's D~seasea nd Rheumatold
arthritis. The therapeut~c activ~ty of laqutmmod m these models results %om a vmety of
mechmsttc effects, mcludlng reduction of leukocyte mfiltfat~onin to target tlssues by modulat~on
of chemoktne-medlated T-cell adhes~onm, odulation of cytolune balance, down reyllat~ono f MHC
class I1 resultmg m alterat~ono f antigen presentation, and effects on dendntic cells subpopulatlons
15 (PC1 ~ntemal~cnAaip plr-ation kbi:catlon Yo W02011 169746)
ceuticdly accqtabllable salt of l a q u i h d incldcs !ithim sodium, potassilun nragnmium,
calcium, manganese, copper, zinc, aluminum and iron. Salt formulations of taquinimod and the
process for preparing the same are described, e.g., in U.S. Patent Application Publication No.
200510192315 and PCT International Application Publication No. WO 20051074899, which are
2 0 hereby incorporated by reference into this application.
The subject invention provides a mixture of crystalline laquinimod sodium particles, wherein (i) 90%
or more of the total amount by volume of the laquinimod sodium particles have a size of 40 microns
or less or (ii) 50% or more of the total amount by volume of the laquinimod sodium particles have a
size of 15 microns or less. and wherein:
a) the mixture has a bulk density of 0.2 gimL to 0.4 4mL;
b) the mixture has a tapped density of 0.40 g/mL to 0.7 g h L ;

What is Claimed:
1. A mxture of crystalline laqulnmod sodium particles, whereul0) 90% or more of the total
amount by volume of the laqummod sodium particles have a size of 40 mcrons or less or (11)
5OUh or more of the total amount by volume of the l a q w o d s o&m part~ciesh ave a size of
15 microns or less, and wherem.
a. the mixture has a bulk density of 0.2 g/mL to 0.4 &dml;
b, the mixture has a tapped density of 0.40 gimL to 0.7 gimL;
c. an amount of heavy metal which is present In the mxture 1s no more than 20 ppm of
heavy metal relative to the amount by wnght of laqummcd sodrum;
d. an amount of 5-Chlom4-hydroxy-1-methylqumolinn2(1H)-on(eM CQ) which is
present ln the mture IS no more than 0.15% relatlve to the amount of laqulmmi
sodium as measured by EIPLC;
e. an amount of 5-chloro-l-bydroxy- 1 -rnethyl-2-oxo-1,2-dhydroquinoline-3-carboxyl~c
acid (MCQCA) which 1s present in the rmxture is no more than 0.15% relative to the
amount of laqumod sodium as measured by WLC;
f. an amount of methyl 5-ehlom-4-hydroxy-l-methyl-2-oxo-1,2-d1hydroq~nolme-3-
sart.2?xyla?c(M CQME) whch 1s pamt m the mmme 1s na more than 0 12% rclaclve
to the amount of l a q m o d sodmrn as measured by HPLC;
g. an amount of methyl 5-chlom~-hydroxy-l-methyl-2-oxo-1,2-dihydroquLnoline-3-
carboxylate (MCQME) which is present in the mixture is no more than 0.10% relative
to the amount of laquinimod sodium as measured by HPLC;
h. an amount of N-ethylaniline (NEA) which is present in the mixture is no more than
0.10% relative to the amount of laquinimod sodium as measured by HPLC; or
i. an amount of N-ethyl-4,5dihydroxy-1-methyl-2-oxo-N-phenyl-1,2-
dihydroquinoline-3-carboxamide (5-HLAQ) which is present in the mixture is no
more than 0.10% relative to the amount of laquinimod sodium as measued by WLC.
2. The mixture of claim 1, wherein (i) 90% or more of the total amount by volume of the
laquinimod sodium particles have a size of less than 40 microns or (ii) 50% or more of the total
amount by volume of the laquinimod sodium particles have a size of less than 15 microns.
3 The mixture of claim 1 or 2, wherein 10% or more of the total amount by volume of the
laqui&od sodium particles have a srze of 5 microns or less and wherein:
a. the mxture has a tapped denslty of 0.40 yimL to 0.7 @mL: or
b an amount of 5-Chloro-4-hydroxy-l-mahylqumolm-2(tH)-on(eM CQ) which IS
present m the mbxe 1s no more than 0.15% relative to the amount of laqfurumod
sodvum as measured by HPLC.
4. A nuxture of crystallme laqu~rumods ohum particles, wherm (1) 90% or more of the total
amount by volume of the laqmrmod sodvum particles have a slze of less than 40 microns, (it)
50U/oo r Inore of the total amount by volume of the laqurmmod sodium panicles have a slze of
less than 15 microns, and (ni) 10Y0 or more of the total amount by volume of the laqwmod
sod~ump articles have a slze of less than 5 mcrons and whermn.
a. the mixture has a bulk density of 0.2 e/mL to 0.4 g/mL;
b. the mixture has a tapped density of 0.40 e/mL to 0.7 gimL,
c. an amount of heavy metal which is present in the mixture is no more than 20 ppm of
heavy metal relative to the amount by weight of laquinitnod sodium;
d. an amount of 5-Chloro-4-hydroxy-l-methylquinolin-2(1~-o(MneC Q) which 1s
m the m~turri:s no more than 0 15% rei;lt~veto the heom of !aq
sodrum as measured by HPLC;
e. an amount of 5 -chloro4-hydroxy-1 -methyl-2-oxo-l,2-dihydroquinoline-3-carboxylic
acid (MCQCA) which is present in the mixture is no more than 0.15% relative to the
amount of laquinimod sodium as measured by HPLC;
f. an amount of methyl 5-cNoro4hydroxy-l-methyl-2-oxo-l,2-diiydr0quinoliie-3-
carboxylate (MCQME) which is present in the mixture is no more than 0.12% relative
to the amount of IaqWod sodium as measured by HPLC;
g. an amount of methyl 5-chloro4-hydroxy-l-methyl-2-oxo-1,2dihydroquinoline-3-
carboxylate (MCQME) which is present in the mixture is no more than 0.10% relative
to the amount of laquinimod sodium as measured by HPLC;
h. an amount of N-ethylaniline (NEA) which is present in the mixture is no more than
0.10% relative to the amount of laquinimod sodium as measured by HPLC; or
I an o u t of iv-ethyt-4,5-d~hy&oxy-l-methyl;t-oxo--pheny1-i,2-
d~hydroquinolme-3-carboxmde (5-HLAQ) which IS present 1n the mixture is no
more than 0 10% relative to the mount of laqummod sodium as measured by WLC.
5. The mture of any one of claims 1-4 prepared in a single batch comprising 2.5 kg or more of
laqumimod sodium.
6. The mxtwe of any one of clalms 1-5, wheran the laqu~ntmods od~ump articles are detemed
based on an m l l e d sample of the mxture or whereln the size and amount by volume of
Iaquimmod sodurn part~clesa re detemned based on a mlled sample of the mxture.
7. The rmxture of any one of clalms 14, havmg a bulk dens~tyo f 0 2 gimL to 0 4 gimL,a ndlor
h m g a tapped densty of 0 40 g/mL to 0 7 g/mL.
8. The mixture of any one of claims 1-7, whemn:
a. an amount of aluminum in the mixture is less than 5 ppm or less than 2 ppm relative
to the amount by weight of laquinimod sodium:
b. an amount of calcium in the mixture is less than 60 ppm or less than 25 ppm relative
to the amount by weight of laquinimod sodium;
c. an mount of calcmin m the mixture is less than 25 ppm relatlve to the amount by
d. zn musil of z o p p m the mxtm 's las than I ppm or less than 0 5 ppmreiative to
the amount by weight of laqumunod sodium; andior
e. an amount of zinc in the mixture is less than 7 ppm or less than 4 ppm relative to the
amount by weight of laquinimod sodium.
9. The mixture of any one of claims 1-8, wherein an mount of heavy metal in the mixture is no
more than 20 ppm relative to the amount by weight of laquinimod sodium
10. The mixture of any one of claims 1-9, wherein a total amount of polar impurities in the mixture
is w more than 1.00% relative to the amount of laquhimod sodium as measured by HPLC.
1 1. The mixture of any one of claims 1-1 0, wherein:
a. an amount of 5-Chloro-4-hy&oxy-l-methylqumol1n-2(1N)-0ne( NCQ) in the
mixture 1s no more than 0.15% relarrve to the amount of laquinimod sodium as
measured by NPLG;
b. an amount of 5-chloro-l-hydroxy-I -methyi-2-oxo-l,2-d1hyrlrttqumolme-3~carboxyl1~
acid (MCQCA) m the mture 1s no more than 0.15% relat~ve to the amount of
laqumimod sdum as measured by WLC;
c. an amount of methyl 5-chlom-4-hydmxy-l-methyl-2-0~0-1,2&hy~q~olrne-3-
carboxylate (MCQME) m the mxture 1s no more than 0 12% relatwe to the amount of
laqtumod sodium as measured by HPLC;
d. an amount of methyl 5-chloro4-hydroxy-I-methy1-2-0x0-1~2d1hydrclquno1~e3-
carboxylate (MCQME) whch 1s present m the mxture is no more than 0.10% relat~ve
to the amount of laqu~mmods odium as measured by WPLC, &or
e. an amount of ethyl 5-cNom-il-hydroxy-l-methy1-2-oxo-l,2-dihydroqu1nolme-3-
carboxylate (MCQEE) m the mixture 1s no more than 0.10% relative to the amount of
laquimod sodium as measured by WLC.
12. The mxture of any one of claims 1-11 , wherem a total amount of non-polar ~lnpuntiesm the
mlxture is no more than 0.50% relatrve to the amount of laqui~mods odium as measured by
HPLC
13. The mixture of any one of claim 1-1 2, wherein:
a. an amount of N-ethyldine (NEA) ln the IIWL~Wi~s no more than 0 10% relative to
the amount of laquinvnod so&um as measured by HPLC;
b. an amount of N-ethyl-4,5-dihydroxy-l-methyl-2-oxo-N-phenyl-1,2-
dihydroquinoline-3-carboxamide(5 -HLAQ) in the mixture is no more than 0.10%
relative to the amount of laquinimod sodium as measured by HPLC; and/or
c. an amount of 5-chloro-4-hydroxy-1- methyl-2-0x0-N-phenyl-1,2-dihydroquinoline-
3-carboxamide (DELAQ) in the mixture is no more than 0.10% relative to the amount
of laquinimod sodium as measured by HPLC.
14. The mixture of any one of claims 1-13, wherein:
a. an mount of l a q u ~ m dac id in the mixture is no more than 1.00% relative to the
amount of laquimmcd sodium as meamred by WLC;
b. an amount of dimdhyl malomte m the mixture rs no more than 0.10% relative to the
amount of laquinunod sodm as measured by WLC;
c an amount of &&yI donate m the mlxtwe 15 no more than 0.10% relative to the
mount of laqutmmod sod~uma s measured by HPLC;
d. an amount of dimethyl sulfate in the mixture is no more than 1 ppm relative to the
amount by weight of laquinimod sodium;
e. an amount of water m the mixture is no more than 1.5% by weight relative to the
amount of laquinimod sodium as measured by K.F. coulometric titration; andlor
f. an amount of sodium from 5.8% to 6.4% relame to the amount by weight of
laquinimod sodium.
15. The mixture of any one of claims 1-14, wherein:
a. an amount of ethanol in the mixture is no more than 5000 ppm relative to the mount
by weight of laquinimod sodium;
b an mount of n-heptmc In the mxtm 1s nu more than 5000 ppm=i&tve to the asnowt
by wnght of laquinimod sodium;
E. iril amtint ofn-ockne ut the mixture 1s no inore than 2000 ppm reiatlve to the amount
by weight of laquinimod sodium;
d. an amount of n-octane in the mixture is no more than 200 ppm relative to the amount
by weight of laquinimod sodium;
e. an mount of n-octane in the mixture is no more than 20 ppm relative to the amount
by weight of laquinimod sodium;
f. an amount of methanol in the mixture is no more than 3000 ppm relative to the amount
by weight of laquinimod sodium;
g. an amount of acetone in the mixture is no more than 5000 ppm relative to the amount
by weight of laquinimod sodium;
h. an amount of dloxane tn the mixture is no more than 380 ppm relat~veto the amount
by werght of laqumimod sdum; andor
i an amount of &methyl foimmde ln the mxntre is no more than 880 ppm relatlve to
the amount by weight of laqumod sodium
16. A pmceutteal composition compnsmg the mxrure of any one of clams 1-15 and a
phmceutically acceptable camer.
17. The pbaceutlcal cornpositton ofclaun 16. wherein,
a. a total amount of 5-Chloro-4-hydroxy-l-methy1qu~no1in-2(lti)-(oMnCe Q) and 5-
chloro-4-hydroxy-l-methyl-2uxo-1,2-dihydroqulnoline-3-carboxyahccld (MCQCA)
m the pharmaceut~ealc omposition 1s no more than 0.50% relattpe to the amount of
laqunnmod sodium as measured by HPLC;
b. a total amount of polar impurities in the pharmaceutical composition is no more than
2.00% relative to the amount of laquinimod sodium as measured by HPLC;
c. an amount of N-ethylaml'me (NEA) in the pharmaceutical composition IS no more than
0.50% relative to the amount of laquhmod sodium as measured by HPLC;
d. an amount of 5~hlorn-N-ethyl-3-hydroxy-1-methyl-2,4-d~oxo-N-phenyl-l,2,3,4-
1~ydrquinolms-3-carhaxaz~d(3e- 1SLAQ) m he euilcal comqroslt~onis
no more than 0 50% relative to the amount of laqumunod sodium as measural by
!@LC,
e. a total amount of non-polar impurities in the pharmaceutical composition is no more
than 1 .00% relative to the amount of laquinimod sodium as measured by HPLC; andlor
f. an amount of N-ethyl-4-hydroxy-1-methyl-5-(methy1(2,3,4,5,6-
pentahydroxyhexyl)amino)-2-oxo-N-phenyl-l,2-dihydroquinoline-3-carboxamide
(MEG-LAQ) in the pharmaceutical composition is no more than 1.00% relative to the
amount of laquinimod sodium as measured by HPLC.
18. The pharmaceutical composition of claims 16 or 17, wherein:
a. an amount of water in the pharmaceutical composition is no more than 1.50% relative
to the amount of laquinimod sodium as measured by K.F. coulometric titration;
b. an amount of water m the eut~cacl omyos~tionis no more than 0.80% relative
to the amount of l a q ~ msdod ium as mcaasurd by K.F. coulometric titration; andor
&I& composlhon comprises an amount of durn from 5.8% to 6.4%
refatwe to the mount by wag& of lagummod s&m.
19. The phsnmceuticd cornpositton of any onc of clams 16-18. whmeh
a. an amount of ethanol in the p a m t i a l composition is no more than 5000 ppm
reIative to the amount by weight of laquinirnod sodium;
b. an amount of n-heptme in the p h c e u l i c a l composition is no more than 5OOO ppm
relative to the amomt by weight of laquinimod sodium;
c. an amount of n-octiine in the p h c e u t i c a l composition is no more lhan 2000 ppm
relatlve to the amount by weight of laquirnmod sodium;
d. an amount of n-oc?ane in the pharmaceutical composition is no more than 200 ppm
relative to the amount by weight of laquinimod sodium;
e. an amount of u-oc-e in the pharmaceutical composition is no more than 20 ppm
relative to the amount by weight of laquinimod sodium;
f an amount of m e k o l in the p
relatrve to the amount by welght of laquimmod sodium;
y. run munt 3f acetcme m the phmceutlcai composlt~onis no more than 880 ppm
relative to the mount by welght of laquinunod sodium;
h. an amount of dioxane in the pharmaceutical composition is no more than 380 ppm
relative to the amount by weight of laquinimod sodium; andor
i. an amount of dimethyl fonnamide in the pharmaceutical composition is no more than
880 ppm relative to the amount by weight of laquinimod sodium.
20. A method of treating a subject afflicted with, or allevaitaing a symptom of, a form of multiple
sclerosis, lupus nephritis, lupus arthritis, rheumatoid arthritis, a BDNF-related disorder,
Crohn's disease, a GABA-related disorder, a cannabinoid receptor type 1 (CBI) mediated
disorder, or an ocular inflammatory disorder comprising administering to the subject the
truxture of any one of clrums 1-15 or the p h c e u t t d composltlon of any one of claims 16-
20 so as to thereby treat the subject or alleviate the symptom m the subject.
2 1. The mxture of any one of clatm 1 - 15 or the pharmaceubcal composition of any one of clam
16-19 for use m the treatment of, or allevtat~ono f symptoms of, a form of multiple sclerosts,
lupus nephntrs, liipus arthntts, rheumatold athtls, a BDNF-related disorder, Crohn's d~sease,
a GABA-related disorder, a cannabmold receptor type 1 (CB1) mabated dsorder, or an ocular
mfla-tory dtsorder.
22. Use of the mture of any one of clatms 1-1 5 or the pharmaceuttcal composttlon of any one of
cla~ms1 6-19 for the manufacture of a medicment for treatmg, or allmatlng a symptom of, a
form of multlple sclerosis, a GABA-related disorder, a cannabmold receptor type 1 (CBl)
mediated disorder, lupus neplmtis, lupus arthntls, rheumatoid arl.hnt~s, a BDNF-related
disorder, Crohn's dtsease, or an ocular mflammatory d~sorder
23. A process of reerystallization of laquinimod sodium comprising:
a. dissolving an amount of laquinimod sodium in water to form an aqueous solution;
b. eoncentrattng the aqueous solut~on to form a concentrated solution compnsmg
approxtmately 1 7-1.8 mL of water per gram of laqulnunod sodium;
c addmg acetone to the concmtrated solut~ono f step b); and
d. isolating recrystallized laquinimod sodium.
24. The process of claim 23, wherein.
a. the amount of laquinimod sodium in step a) is 2.5 kg or greater;
b. step a) is performed with 10-12 mL of water per gram of laquinimod sodium,
preferably step a) is paformed with approximately 11 mL of water per gram of
laquinimod sodium; andtor
c. step a) is performed by heating the aqueous solution to a temperature of 58-75"C, or
wherein step a) is performed by heating the aqueous solution to a temperature of 60-
73°C.
25. The process of claims 23 or 24, wherein crystallization occurs after step a) and before step
c), preferably wherein crystaliztion:
a, 1s mnduced by rapid stirring during or aAer the concentrating step b);
b. is induced by adddion of a seed crystal cr-ystal during or
c. occurs without addition of a seed crystal.
24. The process of any one of claim 23-25, wherein:
a. step b) ts performed under conditions approprtate to rnduce crystalltzatlon at the
concentration of 1 7-1.8 mL of water per gram of laquinimod sohum; and/or
b. step b) is peTfonned at 28-45°C or at 30110°C.
27. The process of any one of claims 23-26, wherein:
a. step c) is performed with the concentrated solution at 40-55°C or at 45-50°C;
b. step c) is performed with 6-12 mL of acetone per gram of laquinimod sodium,
preferably step c) is performed with approxunately 10 mL of acetone per gram of
laquinimod sokum:
c. step c) is performed over a period of 1-4 hours or over a periodof 1.2-2.5 hours; and/or
d. step c) IS followed by cooling the solution to a temperature no less than -14OC and no
rDre than So&: or wherem step cf is fallow& by m h g the soiutmn to a :
no less than -4OC and no more than 4OC.
28. The process of any one of claims 23-27, wherein the solution is cooled over a period of 3-5
hours or over a period of 3.5-4.5 hours.
29. The process of any one of claims 23-28, wherein
a. step d) further comprises washing the recrystallized laquinimod sodium with 1-4 mL
of acetone per gram of crude laquinimod sodium used in step a), preferably step d)
further comprises washing the recrystallized laquinimod sodium with
approximately 3 mL of acetone per pm of crude laquinimod sodium used in step
a);
b. step d) further comprises drylng the recrystallized laquinimod sodium for no less than
one hour at 304°C under a vacuum of no more than 50 mmHg; and/or
c the rsotated rsvsbllued laymimod sodlm m step d) IS a mxhrre of crystall~ne
laqulmod sodium partlcles havtug a particle slae distnbullon such that (I) 90% or
more of the total amount by volume ofthe laqmmod sodium particles have a size of
40 mcrons or less, (11) 50% or more of the total amount by volume of the laquttumod
sdum partlcles have a size of 15 microns or less, and (nr) 10% or more ofthe total
amount by volume of the laqumtmod sodturn partxcles have a sEe of less than 5
mcrons or less
30. A mxture of crystalline laquinimod sodium particles prepared by any one of clam 23-29.
31 The mxtue of claim 30, wheran (1) 90% or more of the to& amount by volume of the
laqulmmod sodlum particles have a size of 40 rmcrons or less, (11) 50% or more of the total
amount by volume of the laqummod sodturn particles have a s~zeof 15 mcrons or less, and
(1u) 10% or more of the total amount by volume of the laquinunod sodrum particles have a sue
of 5 mcrons or less
32. The mixture of claim 30 or 31, wherein:
a. the mixlute has a buk density of 0.2 gimL to 0.4 g/mL;
b. the mixture has a tapped density of 0.40 g/mt to 0.7 g/&,
c an mount of alumram m the mrxtwe 1s !ess t k 5 ppm relative to the amoilnt by
waght of laqu~rumodso dium;
d. an amount of &rum ue the auxzwe is less than 60 ppm retar~ve to rhe mount by
weight of laquimmod sodium;
e. an amount of copper in the mixture is less than 1 ppmrelative to the amount by weight
of laquinitnod sodium;
f. an amount of zinc in the mixture is less than 7 ppm relative to the amount by weight
of laquinimod sodium;
g. an amount of heavy metal in the mixture is no more than 20 ppm relative to the amount
by weight of laquinimod sodium;
h. a total amount of polar impurities in the mixture is no more than 1.00% relative to the
amount of laquinimod sodium as measured by HPLC;
an amonnt of 5-Chloro4-hydroxy-I-methyiqu1nol1n~2(S~-(oMneC Q) m the
rrnxture 1s no more than 0.15% relatlve to the amom of laqumnnod sohum as
measured by HPLC,
an amonnt of 5-cNom4-hydmxy-I -methyl-2-oxo-1,2-dihydn,qumoline-3-~arboxy1ic
actd JMCQCA) m the mixture is no more than 0.15% relative to the amount of
laqwnunod sdum as measured by HPLC;
an amount of methyl 5-chloro-4-hydmxy-1-methyl-2-0~0-1,2--3-
carboxylate (MGQME) in the rmxture is no more than 0.12% relative to the amount of
laqumimod sodium as measured by WLC;
an amount of methyl 5-chloro3-hy&oxy-l-methyl-2-oxo-l,2-dihy~q~oline-3-
carboxylate [MCQME) in the mixture is no more than 0.10% relative to the amount of
laquirumod sodium as measured by HPLC;
an amount of ethyl 5-cNom3-hy&oxy-l-methyl-2-oxo-l,2-dihy&oquinoline-3-
carboxylate (MCQEE) in the rmxture is no more than 0.10% relative to the amount of
laqumimod sodlum as measured by WLC;
a total amount of non-polar impurities in the mixture is no more than 0.50% relative to
the amount of laquinimod sodium as measured by HPLC;
an amount of Ndylanrhe PEA) in the mxture is no more than 0.10% relatlve to
the amount of laqunumod sodm as measured by HPLC;
an amount of laquinimod acid in the mixture is no more than 1. @relatiIve% to the
amount of laquinimod sodium as measured by HPLC;
an amount of dimethy1 malonate in the mixture is no more than 0.10% relative to the
amount of laquinhnod sodium as measured by HPLC;
an amount of diethyl malonate in the mixture is no more than 0.10% relative to the
amount of laquinimod sodium as measured by HPLC;
an amount of dimethyl sulfate in the mixture is no more than 1 ppm relative to the
amount by weight of laquinimod sodimq
an amount of water in the mixture is no more than 1.5% by weight relative to the
amount of laquinimod sodium as measured by K.F. coulometric titration;
u. the mixture comprises an amount. of so&m tiom 5.8% to 6.4% relat~veu t the amount
by weight of laqmmcd scd~um;
v. an amount of ethanol in the mixture IS no more than 5000 pprn relative to the amount
by weight of laquimmod sodium;
w. an amount of n-heptane in the mixture is no more than 5000 ppm relative to the amount
by weight of laquimmod sodium;
x. an amount of n-octane in the mixture 1s no more than 2000 ppm relat~reto the amount
by wmght of laqu~nimodso d~um,
y. an amount of n-octane tn the mixture is no more than 200 ppm relative to the amount
by weight of laquinimod sodium;
z. an amount of nuctane in the mixture is no more than 20 ppm relative to the amount
by weight of laquinimod sodium;
aa. an amount of methanol in the mixture is no more than 3000 ppm relative to the amount
by weight of laquinimod sodium;
bb. an amount of acetone in the mixture is no more than 5000 ppm relative to the amount
by weight of laquinimod sodium;
cc. an amount of dioxane tn the mxture IS no more than 380 ppm relative to the amount
dd. an amount of dimethyl fonnamide in the mixture is no more than 880 pprn relative to
the amount by weight of laquinimod sodium.
33. The mixture of claim 30 or 3 1, wherein:
a. the mixture has a bulk density of 0.2 g/mL to 0.4 g/mL,
b. the mixture has a tapped density of 0.40 gimL to 0.7 dmL;
c. an amount of aluminum in the mixture is less than 5 ppm relative to the amount by
weight of laquinimod sodium;
d. an amount of calcium in the mixture is less than 60 ppm relative to the amount by
weight of laquinimcd sodium;
an amount of copper in the mxme 1s Ies than 1 ppm relattve to the amount by wetght
of 1.Suu11mod sodium;
an amount of zinc in the mixture IS less than 7 ppm relative to the amount by weight
of laquuumod sod~um;
an anlowit of heavy metal In the uuxture is no more than 20 ppm relative to the amount
by welght of laquinimod sdum:
a total mount of polar unpunties m the mixture is no more than 1.00% relative to the
amount of laqwntmod sodium as measured by HPLC;
an mount of 5-Chloro-4-hydroxy-I-methylquinolin-2(1N-one(M CQ) in the
nuxture 1s no more than 0 15% relative to the amount of laqumod sodlum as
nleasured by KPLC;
an amount of 5-chloro-4-hyhxy-I-mehyl-2-0x0,2--1d ihydroq~o1ineoq3-cartx,xyhc
acid (MCQCA) m the mixture is no more than 0.15% relative to the amount of
laquinimod sodrum as measured by WLC;
an mount of methyl 5-chloro-il-hydroxy-l-methyl-2-oxo-l,2dihydroquinoline-3-
carboxylate (MCQME) m the mmture is no more than 0.12% relative to the amount of
liyuinimod sodlum as measured by HPLC;
an amount of methyl 5-ehloro-4-hydroxy-l-methyl-2-oxo-l,2dihy&oquinolme-3-
h x y l a t e JILZCQME) m the mxbre is no more t,hm 0 Im6 relaz~veto the amount of
laquuumod sod~urnas measured by HPLC;
an amount of ethyl 5-cNoro-4-hydroxy-l-methyl-2-oxo-l,2dihy~-
carboxylate (MCQEE) in the mixture is no more than 0.10% relative to the amount of
laquinimod sodium as measwed by HPLC;
a total amount of non-polar impurities in the mixture is no more than 0.50% relative to
the amount of laquinimod sodium as measured by HPLC;
an amount of N-ethylaniline (NEA) in the mixture is no more than 0.10% relative to
the amount of laquinimod sodium as measwed by HPLC;
an amount of laquinirnod acid in the mixture is no more than 1.00% relative to the
amount of laquinimod sodium as measured by HPLC;
q. an amount of dimethyl nzalonate in the mixture is no more than 0.100/0 relative to the
mount of laqmmod ?,odium as measured by HPLC,
r. an amount of dlcthyl malonate in the mxturr: is no more than 0.10% reiacwe to the
amount of laqmmd sdum as measured by WPLC,
s. an amount of dimethyl sulfate m the mutture IS no more than 1 ppm relative to the
amount by weight of laqunnnod sod~um;
t. an amount of water In the mixture is no more than 1.5% by weight relative to the
amount of laquinimod sodium as measured by K.F. coulometric titratioq
u. the mixture comprises an amount of sodium from 5.8% to 6.4% relative to the amount
by weight of laquinimod sodium;
v. an amount of ethanol m the nnxture IS no more than 5000 ppm relative to the amount
by wtnght of laquuumod sodium;
w. an amount of n-heptane in the mixture is no more than 5000 ppm relative to the amount
by weight of laquinimod sodium;
x. an amount of n-octane in the mixture is no more than 2000 ppm, 200 ppm or 20 ppm
relative to the amount by weight of laquinimod sodium;
y. an amount of methanol m the mixture is no more than 3000 ppm relative to the amount
by wa&t of laqlu-d ~~,
z. an amount of acetone in the mixture is no more than 5000 ppm relative to the amount
by weight of laquinimod sodium;
aa. an amount of dioxane in the mixture is no more than 380 ppm relative to the mount
by weight of laquinimod sodium, and
bb. an amount of dimethyl formamide in the mixture is no more than 880 ppm relative to
the amount by weight of laquinimod sodium.
34. A pharmaceutical composition comprising the mixture of any one of claims 30-33 and a
pharmaceutically acceptable carrier.
35. The pharmaceutical composition of claim 34, wherein-.
a total mount of 5-Chloro-4-hydroxy-1- methylquinolm-2(1II)-*7n(eM CQ) and 5-
chlom-it-hykoxy-1- methyl-2-oxo-l,2-drhydmqrunol~ne-3-~arboxyalardc ( MCQCA)
In the phmceutrcal eomposrtion 1s no more than 0.50% relative to the amount of
Iaqu~wodso dtum as measured by HPLC,
a total amount of polar mpunt~esin the phmceutrcat composition is no more than
2.00% relative to the mount oflaqGwod sodium as measured by HPLC;
an amount of N-ethylmlme (NEA) m the pharmaceutical composition is no more than
0.50% relative to the amount of laquhmod sodium as measured by HPLC;
a total miount of non-polar impurities in the p-ceutical eompositton is no more
tban 1.00% relative to the amount of l a q m o d sodium as measured by HPLC;
an amount of water m the phamamt~caclo mposltion is no more than 0.80% relative
to the amount of Iaqummod sodrum as measured by K F coulometnc titration;
the pharmaceutical composition comprises an amount of sodium c om 5.8% to 6.4%
relative to the amount by weight of laquinimod sodium;
an amount of ethanol in the phamaceut~cal composition is no more than 5000 ppm
relative to the amount by weight of laquinimod sodium;
an mom! of n-hqme m the p
relative to the amount by weight of l a q m o d sodium;
an mount of n-octane in the pharmaceutical composition is no more than 2000 ppm
relative to the mount by weight of laquinimod sodium;
an amount of n-oetme in the pharmaceutical composition is no more than 200 ppm
relative to the amount by weight of laquinimod sodium;
an amount of n-octane in the pharmaceutical composition is no more than 20 ppm
relative to the amount by weight of laquinimod sodium;
an amount of methanol in the pharmaceutical composition is no more than 380 ppm
relative to the amount by weight of laquinimod sodium;
an amount of acetone in the pharmaceutical composition is no more than 880 ppm
relative to the amount by weight of laquinimod sodium;
n. an amount of droxane m the p h m ~ u t i cdom pos1tlon IS no more t h3 50 ppm
relative to the amount by wet& of laquinunod sodrum, or
a. an amount of &methyl formamde m the phamceut~caclo mposttlon 1s no more than
880 ppm relame to the amount by welght of laquuumod sodium.
36. The pl~at~tlaceuticwalr nposition of claim 34, comprising:
a. a total amount of 5Chloro-4-hydroxy-1-methylquinolm-Z(1EI)-on(Me CQ) and 5-
ckloroil-hydroxy-1- methyl-2-oxo-l,2dthydroq~~1oIme-3-~arboaxcy~ld(1M c CQCA)
m the phmceutical wmposltion is no more than 0 50% relative to the amount of
laqummod sodium as measured by HPLC,
b. a total amount of polar impurities m the pharmaceutical composition is no more than
2.00% relatlve to the amount of laqumunod sodium as measured by HPLC;
c. an amount ofN-ethylaniline in the pharmaceutical composition is no more th
0.50% relative to the amount of laquinimod sodium as measured by HPLC;
d a total amount of non-polar impuntles ~n the pharmaceubcal composition 1s no more
than 1 00% relative to the amount of laqutmod sod~uma s measured by HPLC,
e. an amount of water in the pha,rmaceut~cacl ompos~tionis no more than 0.80% retatwe
to the amumt of 1aqi;nnrmod &turn as nmm& by K.F couiornetne trtration;
mt~cawi rnpnsition comses an mount of' sodxum from 5.8% to 6.4%
relative to the amount by wnght of laquinmod s o d i q
g. an amount of ethanol in the pharmaceutical composition is no more than 5000 ppm
relative to the amount by weight of laquinimod sodium;
h. an amount of n-heptane in the pharmaceutical composition is no more than 5000 ppm
relative to the amount by weight of laquinimod sohum;
i. an amount of n-octane in the pharmaceutical composition is no more than 2000 ppm,
200 ppm or 20 ppm relative to the mount by weight of laquinimod sodium;an amount
of methanol in the pharmaceutical composition is no more than 380 ppm relative to the
amount by weight of laquithod sodium;
J. an mount of acetone in the phaceuticaf compositton is no more than 880 ppm
relative to the mount by weight of laqmunod sodium;
k. an amount of dioxane in the pharmaeeutleal composition IS no more than 380 ppm
relative to the amount by weight of laqununod sodium; and
I an amount of dlmethyl f o m d e tn the pharmaceutical composttlon is no more than
880 ppm relahve to the amaunt by we~gbot f laqummod sdum.
37. A mture of crystallme laqummod sodium particles, wherem (I) 90% or more of the total
amount by volume ofthe laquniunod sodium part~clesh ave a size of 40 mcrons or less or (11)
50% or more of the total amount by volume ofthe laqummod so&um parttcles have a slze of
15 microns or less, and wherein
a. an amount of aluminium in the mixture 1s less than 5 ppm relative to the amount by
weight of laqutnimod sodium;
b. an amount of calcium in the mixture is less than 60 ppm relative to the amaunt by
weight of laquinimod sodium;
c, an amount of copper in the mixture is less than 1 ppm relative to the amount by weight
of laquinimod sodiwn; or
d. as, momi of m cm the mxturt: rs less than 7 ppm rekit~veto the amount by wnght
of laquinimod sod~m.
38. A pharmaceutical composition comprising the mixture of claim 37 and a pharmaceutically
acceptable carrier.
39. An isolated compound having the structure:
' 'H I , or a salt &ereof.
40. A composition comprising a compound having the structure:
or a salt thereof.; wherein the composition is free of laquinilnod or a salt thereof.
41 A pharmaceutical composition comprising an amount of laquimmod and at least one of 2-
cbloro-6-(3-(~hyljphenyl)armno)-2-hydroxy-N-methyl-3oxopropanamdo)beoic acld
(BH-3-HLAQ), 5-Chloro-4-hydroxy-1-methylquinolinn2(1N)-0ne( MCQ), 5-Chloro-4-
hydroxy-1-methyl-2-oxo-1,2-dihydroqu~nol1ne-3-c~boxyalcildc (MCQCA), Methyl 5-
chloro4-hydroxy-l-methyi-2-oxo-l,2-d~hydroquinoline-3-~arbo~yl(aMtCe QME), NEthylanilme
(NEA), and Ethyl 5-chlor0-4-hydroxy-l-methyl-2-oxo-1,d2~- hydroquinolmne-
3-carboxylate (MCQEE), wherein
a) 2-chloro-6-(3-(ethyl(phenyl)mino)-2-hy~l-3-
oxopropanmdo)benzoic ac~d (BH-3-HLAQ) is present m the pharmaceutical
composition m an amount not more than 1096, relat~ve to the concentratton of
laquimmod, based on a detemnat~onb y an HPLC method, or
b) 5-Chloro-4-hydroxy-1-methylqumolm2((~M CQ) is present in the
phamaceutical comp~sltiori m an amount not more thaa 10%, relative to the
concentration of laqmmmod, based on a detematlon by an HPLC method, or
c) 5-Chloroit-hydroxy-l-methyl-2-oxo-l,2-dihydroquinoline-3-carboxylic acid
(MCQCA) is present in the pharmaceutical cornposition in an amount not more than
lo%, relative to the concentration of laquinimod, based on a determination by an
HPLC method, or
d) Methyl 5-chloro-4-hydroxy-1 -methyl-2-oxo-l,2-dihydroquinoline-3-carboxy1ate
(MCQME) is present in the pharmaceutical composition in an amount not more than
lo%, relative to the concentration of laquinimod, based on a determination by an
HPLC method, or
e) N-Ethylaniline (NEA) is present in the pharmaceutical composition in an amount
not more than lo%, relative to the concentration of laquinimod, based on a
determination by an HPLC method, or
1) Ethyl 5-chloroil-hydroxy-1-methyl-2-0x0-1.2- d1hydroquinolmne-3-carbOxy1ate
(MGQEB) 1s present m the pharmaceutlcal composition man mount not more than
lo%, relatlve to the concentratlon of laqummod, based on a determinat~on by an
HPLC method.
42. The phmceutlcal composition of claim 41, wherein
a) 5-Chloro-4-hydroxy-I-methylquznolinn2(t~-onJMe CQ) IS present in the
phamraceutlcai composrtion in an amount not more than 0.15%, relative to the
concentratton of laqmimod, based on a detemnation by an WPLC method, or
b) 5-Chloro-l-hydroxy-l-methyl-2-oxo-l,2-dihydroqu1noline-3-carboxyl1c ac~d
(MCQGA) is present m the phannaceutrcal composition ~n an amount not more than
0 IS%, relatlve to the concentratron of laqulmmod, based on a detemnation by an
HPLC method, or
c) Methyl 5-chloro-4-hydroxy-l-methyl-2-oxo-l,2-dihydroqumoline-3-carboxylate
(MCQME) is present ln the pharmaceutlcal composition ln an amount not more than
0 15%, relatrve to the concentratlon of laqulmmod, based on a determmation by an
HPLC method, or
d) N-Ethylanrline (NEA) 1s present in the pharmaceutrcal composition m an amount
not nlare than 0 I%, relative to the concmtratiun of iaqutn~mod, based on a
detmnation by an HPLC method, or
e) Ethyl 5-cMoro-4-hydroxy-l-methy1-2-oxo-1,2d-ih yhqumoline-3-carboxylate
(MCQEE) is present in the pharmaceutical composition in an amount not more than
0.1 %, relative to the concentration of laquinimod, based on a determination by an
HPLC method.
43. The pharmaceutical composition of claim 42, wherein methyl 5-ehloro-4-hydroxy-lmethyl-
2-oxo-1,2-dihydroquinoline-3-carhoxylate (MCQME) is present in the
pharmaceutical composition in an amount not more than 0.12%, relative to the concentration
of laquinimod, based on a determination by an HPLC method; and/or wherein methyl 5-
chloro-4-hydroxy-l-methyl-2-oxo-l,2-dihydroquinoline-3-carboxylate (MCQME) is
present in the pharmaceutical composition in an amount not more than 0. I%, relative to the
concentration of laquinimod, based on a determination by an HPLC method.
44. The pharmaceutical composition of any one of claims 41 -43, wherein
a) 5-Ch10ro-Q-hydraxy-1-mcthylquinolin-2(lm~ne(M CQ) is present m the
phmaceuttcal composition m an amount less than 0.05%, relative to the
concentratlon of laquuumod, based on a d e t m a t t o n by an BPLC method, or
b) 5-Chloro4-hydroxy-I -mcthyl-2-oxo-l,2-d1hydroquinoltne-3-carboxylic ac~d
(MCQCA) 1s present in the pharmaceutical composltlon m an amount less than
0 I%, relative to the concentratlon of laquimmod, based on a detm~nationb y an
WLC method. or
c) Methyl 5-chloro4-hydroxy-1-methyl-2-oxo-l,2-drhydroquinolme-3~arboxy1ate
(MCQME) is present in the phmnaceutlcal composition m an amount less than
0 05%, relative to the concentratton of laqu3nrrnod, based on a detmnatton by an
HPLC method, or
d) N-Ethylanilm WEA) rs present in the pharmaccutlcal composition man less than
0 06O4, relative to the concentratton of laqumtrnod, based on a determmation by an
HPLC method, or
e) Ethyl 5-chioro-4-hydroxy-I-methyl-2-0x0-l,2d-~ hydroqu~nolm-3carboxylate
(MCQEE) is present in the pharmaceuttcal composltton m an amount less than
0 05%, relattve to the concentratton of laquimmod, based on a detemnation by an
WLG method
45 The pharmaceutical composition of clatm 44, wherein S-Chloro-4-hydroxy-l-methyi-2-
axo-l,2-drhydroyurrtoi1ne-3-~ahxyllc acid (MCQCA) 1s prcsent m the phameur~cal
composition m an amount less than 0 05%, relattve to the concentration of laqutnunod,
based on a dctmnatlon by an HPLC method.
46. The pharmaceutical composition of claims 44 or 45, wherein
a) S-Chloro-4-hydroxy-l-methy1quinolin-2(1H)-one (MCQ) is present in the
pharmaceutical composition in an amount less than 0.02%, relative to the
concentration of laquinimod, based on a determination by an HPLC method, or
b) S-Chloro-4-hydroxy-l-methyl-2-oxo-l,2-dihydroquinoline-3-carboxylic acid
(MCQCA) is present in the pharmaceutical composition in an amount less than
0.03%, relative to the concentration of laquinimod, based on a determination by an
HPLC method, or
c) Methyl 5-cNoro-4-hydroxy-l-methyl-2-oxo-l,2-dihydroqu1noline-3-carboxylale
(MCQME) is present m the phmaceutlcal composit~on In an mount less than
0.02%, relative to the concentratlon of laqutnunod, based on a determmat~onb y an
HPLC method, or
d) N-Ethylan~hne (NEA) is present in the pharmaceutical compositlon In an less than
0.02%, relative to the concentratlon of laqtummod, based on a detmnation by an
HPLC method, or
(MCQEE) is present in the pharmaceutical compasit~on in an arnount less than
0.02%, relative to the concentration of laquinimod, based on a determination by an
NPLC method.
47. The phamaceutical composttion of clalm 46, wherein 5-Chloro-il-hydroxy-l-methyl-2-
oxo-1,Z-d1hydroqu1n011ne-3-carboxyltc acid (MGQCA) 1s present In the pharmaceutical
compositlon In an amount less than 0 02%, relatlve to the concentration of laquinunod,
based on a detenmnatton by an HPLC method.
48. The pharmaceutical composition of claim 42, wherein
a) 5-Chloro-4-hydroxy-l-methylqumolin-2(1H)-one (MCQ) is present in the
pbaceuticai, compstbon rx an arnount greater thm O 02%. and not more t a k
0.1 5%, relative to the concentration of laqwmod, based on a determmation by an
f-me mdhod, or
b) 5-Chloro-4-hy&oxy-l-methyl-2-oxo-l,2-diby&oquinoline-3-carboxylic acid
(MCQCA) is present in the pharmaceutical composition in an amount greater than
0.02%, and not more than 0. IS%, relative to the concentration of laquinimod, based
on a determination by an HPLC method, or
c) Methyl 5-cNoro-4-hydroxy-l-methyl-2-oxo-l,2-dihy&oqu~oline-3-carboxylate
(MCQME) is present in the pharmaceutical composition in an amount greater than
0.02%, and not more than 0.15%, relative to the concentration of laquinimod, based
on a detemrination by an HPLC method, or
d) N-Ethylaniline (NEA) is present in the pharmaceutical composition in an greater
than 0.02%, and not more than 0.1%, relative to the concentration of laquinimod,
based on a determination by an KPLC method, or
e) Ethyl 5-chloro-rl-hydroxy-1-mechyl-2-0x0-1,2d-i hydroquinol~ne-3-carboxylate
(MCQEE) is pzsmt m the pharmaceut~cd compositron tn an amount greater than
0 02%, ,md not more than 0 I%, relat~veto the concentratton of laquimmod, based
on a detemunat~onb y an HPLC method
49. The phmcmtical composition of claim 48, wherein
a. methyl 5-chlora4-hydmxy- t -methyl-2-oxo-1,2-dihy~oqu~nol~ne-3-c~boxy~ate
[MCQME) is present rn the pharmaceutical compositron m an amount not more than
0 12%. relative to the conczntrat~ono f laqutrnmod, based on a deterrmnatlon by an
IPLC method, or wherem methyl 5-chloro-4-hydroxy-1-methyl-2-oxo-1,2-
dihydroqmolrne-3-carboxylate (MCQME) 1s present m the pharmaceuttcal
compos~tlon in an amount not more than 0 I%, relative to the concentratlorr of
laqumimod, based on a detemunatron by an HPLC method, and/or
b. 5-Chloro4-hydmxy-I-methyl-2-oxo-l,2-d1hydroqumnol~ne-3-carboxyl1cac id
(MCQCA) IS present in the phmceut~caclo mposttion m an amount greater than
0 0376, relattve to the concentration of laqumtmod, based on a detennatlon by an
HPLC method.
50. The pharmaceutical composition of of claims 48 or 49, wherein
a} 5-CRIoru-4-hyciroxy-1 -merhyiqurnolan-2( ~~~one (MGQ) rs presmf m the
pharmaceutical composit~on in an amount greater than 0.05%, and not more than
0 15%, relative to the concmtwtlon of laqutrumod. based on a ddernunatlon by an
HPLC method, or
b) 5-Chloro-4-hydroxy-l-methyl-2-oxo-l,2dihy&oquinoline-3-earboxylic acid
(MCQCA) is present in the pharmaceutical composition in an amount greater than
0.05%, or greater than 0.10%, and not more than 0.15%, relative to the
concentt-dtion of laquinimod, based on a determination by an HPLC method, or
c) Methyl 5-c~oro-4-hydroxy-l-methyl-2-oxo-l,2dihydroquinoline-3-carboxylate
(MCQME) is present in the pharmaceutical composition in an amount @eater than
0.05%, and less than O.15%, relative to the concentration of laquinimod, based on a
determination by an HPLC method, or
d) N-Ethylanillne (NEA) IS present m the phmceutlcal composition in an greater
than 0 06%, and not more than 0.1094, relattve to the concentration of iaqurnimod,
based on a d e t w a t t o n by an IGLC method, or
e) Ethyl 5-chloro-4-hyhxy-I-methyl-2-0x0-1,2d-i hy&oquinolme-3-earboxylate
(MCQEE) IS present in the phmceut~caclo mposttlon In an amount greater than
0.05%, and not more than 0.10%, relative to the concentration of laqunnmod, based
on a detemtmat~onb y an HPLC method.
51 The pharmaceutical composttlon of clam 50, wherem methyl 5-chloro-4-hydroxy-lmethyl-
2-oxo-l,2-dihydroqumolmne-3-carboxylate (MCQME) is present m the
pharmaceutical composition tn an amount not more than 0 12%, relative to the concentration
of laqunimod, based on a determrnatlon by an HPLC method, or IS present m the
phannaceutlcal composition in an amount not more than 0 1 %, relative to the concentration
of laqutmrnod, based on a dctmnation by an HPLC method
52. The pharmaceutical composition of claims 48 or 49, wherein
a) 5-Chloro-4-hy&oxy-l-methylquinol~n-2(1~-one (MCQ) is present in the
pharmaceutical composition in an amount greater than 0.02%, and less than 0.05%,
relative to the concentration of laqu~nimodb, ased on a determination by an I-LPLC
method, or
b) 5-Chloro4-hy&oxy-1-methyl-2-oxo-l,2-d~hydroqmnolme-3-carboxyl1c ac~d
(MCWA) 1s present m the phamcmt~cacl ornposit~onm an amount greater than
0 02%, or greater than 0 03%, and less than 0.1%, relatlve to the concentrattlon of
laquinimod, based on a determmat~onb y an HPLC method, or
c) Methyl 5-chloroil-hydroxy-I-methyl-2-oxo-l,2-dihy&oquinoline-3-carboxylate
(MCQME) is present in the pharmaceutical composition in an amount greater than
0.02%, and less than 0.05%, relative to the concentration of laquinimod, based on a
determination by an HPLC method, or
d) N-Ethylaniline (NEA) is present in the pharmaceutical composition in an greater
than 0.02%, and less than 0.06%, relative to the concentration of laquinimod, based
on a determination by an HPLC method, or
e) Ethyl 5-chloro-4-hydroxy-1-methyl-2-oxo-1,2-di hydroquinoline-3-carboxylate
(MCQEE) is present in the pharmaceutical composition in an amount greater than
0.02%, and less than 0.05%, relative to the concmtmtion of laquinimod, based on a
detmnation by an HPLC method.
53. The phamcmtical composltlon of claim 52, wherem 5-CNoro-rl-hydroxy-l-methyl-2-
oxo-1,24ihydroqu~noI~ne-3-carboxy1aicc~ d(M CQCA) IS present sn the p b c e u t l c a l
composition In an amount less than 0 05%, relatsve to the concentratlon of laqulmmod,
based on a detmnatlon by an HPLC method.
54. The phmaceutical composition of claim 41, further comprising at least one
pharmaceutically acceptabie carrier.
55. The pharmaceutical composition of claim 54, wherein
a) 2-chloro-6-(3-(ethyl@henyl)amno)-2-hydrwxy-H-methyl-3-
oxopropanamldo)benzo~ca cid (BH-3-HLAQ) IS present in the pharmaceutical
composltlon In an amount not more than 1 0%, relatlve to the concentratson of
laquinlmod, based on a detemnatlon by an HPLC method, or
b) N-Ethylantline (NEA) IS present In the phamaceut~cal composstlon m an amount
not more than 0.5%, relative to the concentratlon of laqummod, based on a
deternunatlon by an HPLC method, or
c) the combmed amount of 5-Cbloro-4-hydrory-t-met:hyIqumoI1n-@2~fC Q)
and 5 - C h l o r o 4 - h y d r o x y - l - m e t h y l - 2 - o x o - 1 , 2 ~ o x y l c acid
(MCQCA) present m the phaceutscal campositlon 1s not more than 0 5%.
relative to the concentratlon of laqu~tumodb, ased on a deternunation by an HPLC
method.
56. The pharmaceutical composition of claim 55, wherein N-Ethylaniline (NEA) is present in
the pharmaceutical composition in an amount not more than 0.1%, relative to the
concentration of laquinimod, based on a determination by an HPLC method.
57. The pharmaceutical composition of claims 55 or 56, wherein
a) 2-chloro-6-(3-(ethyl@henyl)amino)-2-hy~-3-
oxopropanamido)benzoic acid (BH-3-HLAQ) is present in the pharmaceutical
composition in an amount less than 0.05%, relative to the concentration of
laquinimod, based on a determination by an HPLC method, or
b) 5-Chloro-4-hydroxy-l-methylqurnolin-2(1I'l)-one (MCQ) 1s present rn the
phamceutrcal composrtlon In an amount less than 0 05%, relative to the
concentration of laqmnimod. based on a detemunatlon by an HP1,C method, or
c) 5-Chloro-4-hydroxy-I-methyl-2-oxo-l,2-drhydroqui1~oline-3-carboxyl1acc rd
(MCQCA) is present in the phmceutical composition in an amount less than
0.5%, relative to the concentration of laquinimod, based on a determination by an
HPLC method, or
d) N-Ethylanliine (NEA) 1s present m the pharmaccutrcal composttlon m an amount
less than 0.1%, relative to the concentration of laquinrmod, based on a deternunation
by an HPLC method, or
c) the combined amount of 5-Chloro-4-hydroxy-1 -methylquinolmn-2(1H)-one (MCQ)
and 5-Chloro-4-hydroxy-l-mthyl-2-oxo-l,2-diby&oqutnolmne-3-carboxyllacc rd
(MCQCA) present in the pharmaceutical composrtlon is less than 0.1%, relatrve to
the concentratlon of laqmmod, based on a detemnation by an HPLC method.
58 The pharmaceutical composltlon of clam 57, wherein 5-Chloro-4-hydroxy-l-methyl-2-
0x0-l,2-drhydroqumolme-3-carboxyllca cid (MCQCA) IS present m the pharmaceutical
cornpositron m an amount less than 0 1% , relat~veto the concentration of laqurmmod, based
on a detertmnatlon by an HPLC method
59. The pharmaceutical composition of claims 57 or 58, wherein
a) 2-chloro-6-(3-(ethy1Cpheny1)amino)-2-hy~-3-
oxopropanamido)benzoic ac~d (BH-3-HLAQ) is present in the pharmaceutical
composrtion in an amount less than 0.02%, relative to the concentration of
laquinimod, based on a determination by an HPLC method, or
b) 5-Chloro-4-hydroxy-I-methylquinolin-2(1H)-one (MCQ) is present in the
pharmaceutical composition in an amount less than 0.02%, relative to the
concentration of laquinimod, based on a determination by an HPLC method, or
c) 5-Chloro-l-hy&oxy-l-methyl-2-oxo-l,2-dihy&oquinoline-3-carboxylie acid
(MCQCA) is present in the pharmaceutical composition in an amount less than
0.03%, relative to the concentration of laquinimod, based on a determination by an
HPLC method, or
d) Methyl 5-chlaro-4-hydmxy-l-methyl-2-oxo-l,2-d1hydroquinol1ne-3-carboxylate
(MCQME) 1s present in the phmceutrcal composrtlon in an amount less than
0 02'36, relative to the concentmtlon of laquuumod, based on a detem~nationb y an
HPLC method, or
e) N-Ethylaniline (NEA) is present m the pharmaceutical composition in an less than
0 03%, relattve to the concentration of laquinimod, based on a detemrnatlon by an
HPLC method, or
f Ethyl 5-chloro4-hydroxy-l-merhyl-2-oxo-l,2- dhydroqumaline-3-carboxylate
(MCQEE) 1s present in the gharmaceuttcai composition In an amount less than
0 02%, relative to the concentration of laquimmod, based on a d e t m a t i o n by an
HPLC method, or
j the combtned amount of 5-Chloro-4-hydroxyl-methylqumlin-2(1N)-one (MCQ)
and 5 - C h l o r o 4 - h y d r o x y - l - m e t k y 1 - 2 - o x o - 1 , 2 ~ o x y 1 cac ld
(MCQCA) present in the p ~ c e u t i c aclo mposit~on1 s less than 0.03%, relative to
the concentration of taqwmmod, based on a detemnation by an HPLG method
60 The pharmaceutical composition of clalm 59, whercln 5-Chloro-4-hydroxy-l-methyl-2-
oxo-1,2-d1hydroquinoline-3-carboxyl1acc id (h4CQCA) 1s present tn the pharmaceutical
composition m an amount less than 0 02% relative to the concentration of laquinlmod,
Sased on a detemnut~onb y an EPLC method
61. The pbmaceuticai ~om~~1sflo1f0 cn1 am 54, wheran
a) 2chloro-6-(3-(ethyl(phmyl)amino)-2-hydroxy-N-methyl-3-
oxopropanamido)benzoic acid (BH-3-EILAQ) is present in the pharmaceutical
composition in an amount greater than 0.02%, and not more than 1.0%, relative to
the concentration of laquinimod, based on a determination by an HPLC method, or
b) N-Ethylaniline (NEA) is present in the pharmaceutical composition in an greater
than 0.03%, and not more than 0.5%, relative to the concentration of laquinimod,
based on a determination by an HPLC method, or
c) the combined amount of 5-Chloro-4-hydroxy-1-methylquinolin-2(1EI)-on(eM CQ)
and 5-Chloro-4-hydroxy-l-methyl-2uxo-l,2-dihyoquoline-3-choxyliacci d
(MCQCA) present in the pharmaceutical composition is greater than 0.03% and
not more than 0 5%, relative to the concentration of laqumn~mod, based on a
determination by an HPLC method.
62 The pharmaceutical composition of cla~m6 1, where~nN -Ethylaniline (NEA) is present m
the phmnaceut~cal compositlon in an amount not more than 0 176, relative to the
concentration of laquimmod, based on a detemnatron by an HPLC method
63. The pharmaceutical composition of clam 54, wherem
a) Zahloro-6-(3-(ethyl(phenyl)amno)-2-hydroxy-N-methyl-3-
oxopropanmtdo)benzoic acld (BH-3-HLAQ) is present m the pharmaceutical
composition In an amount greater than 0 05%, and not more than 1 0%, relatlve to
the concentration of laquinlmod, based. on a deternunation by an BPLC method. or
h) N-Ethylaniline WEA) 18 present in the pbarmaceutrcal composition in an greater
than 0 I%, and not more than 0.5%, relative to the concentralion of laquimod,
based on a determination by an HPLC method, or
c) the combmed amount of 5-Chloro-4-hydroxy-1 -methylquinolin-2(1H)-one (MCQ)
and 5-CNoro-4-hydroxy-l-methyl-2-oxo-1,2-d~hydr~umolme-3-carboaxcy~1di c
(MCQCA) present in the pharmaceutical compositlon is greater than 0.1%, and not
more than 0 5%, relative to the concentratron of laquimmod, based on a
detmnation 5y an WI C me&&
64, The phannaceutical composition of claim 54, wherein
a) 2-chloro-6-(3-(cthyl(phenyi)amino)-2-hy~l-3-
oxopropanarnido)benzoic acid (BH-3-HLAQ) is present in the pharmaceutical
composition in an amount greater than 0.02%, and less than 0.05%, relative to the
concentration of laqninimod, based on a determination by an HPLC method, or
b) 5-Chloro-4-hydroxy-I-methylqninolin-2(lH)-one (MCQ) is present in the
pharmaceutical composition in an amount greater than 0.02%, and less than 0.05%,
relative to the concentration of laquinimod, based on a determination by an HPLC
method, or
c) 5-Chloro-4-hydroxy-l-methyi-2-oxo-l,2-dihydroquinoline-3carboxylic acid
(MCQCA) is present in the pharmaceutical composition in an amount greater than
0.0296, and less than 0.5%, relative to the concentration of laqu~mmodb, ased on a
detm~nationby an HPLC method, or
d) Methyl 5-chloro-4-hydraxy-t-methyl-2-oxo-l,2-d~hydroqurndine-3-carboxylate
(MCQME) 1s present m the pharmaceutical composlt~oni n an amount greater than
O 02%, and less than 0 05%. relative to the concentratlon of laqu~tumodb, ased on a
detemunatlon by an HPLC method, or
e) N-Ethylamline (NEA) rs present tn the pharmaceutical composition m an greater
than 0.03%, and less than 0.1%. relative to the concentration of laquimmod, based
on a detematlon by an WLC method, or
I) Ethyl 5-chloro4-hydroxy-l-methyl-2uxo-1,2-d hydroquinohe-3carboxylate
(MCQEE) 1s present tn the pharmaceutlcal composltlon In an amount greater than
0.02%, and less than 0 OS%, relative to the concentratlon of laqu:n:mod, based on a
detm~nat~boyn a n HPLC method, or
g) the combmed amount of 5-Chloro-4-hydroxy-l-mcthylqumo1m-2(1Hj-on(eM CQ)
and S-Chloro4-hydroxy-l-methyl-2~xo-l,2-d~hydroqurnol1ne-3-carboaxcyidI ic
(MCQCA) present in the pharmaceutical composition IS greater than 0.03%, and
less than O I%, relative to the concentratlon of laquimmod, based on a deterrnmation
by an HPLC method.
65 The pharmaceutlcal composltlon of clam 64, wherein 5-Chloro-4-hydroxy-l-methyl-Zoxo-
l,2-d~hydroqurnoitnc-3-c~xyitacc ld (MCQCA) is pmsent Irn the pkmcmticd
composrtlon m an amount greater than 0 03%, relative to the concentratton of laqummod,
based on a detemnatlon by an HPLC method, or 1s present m the pharmaceutlcal
cornpositron In an amount less than O I%, relatlve to the concentratlon of laquimmod, based
on a deternunation by an HPLC method
66. The pharmaceutical composition of any one of claims 41-65, comprising laquinimod sodium
salt.
67. The pharmaceutical composition of any one of claims 54-66, in a oral unit dosage form, or
in the form of a capsule, a tablet, or a liquid suspension.
68. The pharmaceutical composition of claims 67, wherein the oral unit dosage form comprises
more than 0.3 mg laquinimod, more than 0.5 mg laquinimod or more than 0.6 mg laquinimod.
69. A process for preparing 2-chloro-6-(3-(ethyl(phenyl)mi11o)-2-hy~yl-3-
oxopropanamdo)benzo~cac id (BW-3-HLAQ) comprising the steps of:
a) adding sodium hydroxide solutlon to a suspens~ono f 5-cNoro-N-ethyl-3-hydroxy-
I-methyl-2.4-dioxo-N-phenyl1-, 2,3,4-tetrahydroqu1noline-3-carboxam1Idne w ater,
b) stirring the mixture of step a) followed by addition of hydrochloric acld solution,
c) extracting the aqueous solution with ethyl acetate,
d) washing the organic phase with brine,
e) drylng the organic phase over sodium sulfate,
t) filtering the suspension,
g) evaporating the filtrate,
h) purifyrng the residue by crystallization from isopmpyl alcohol,
i) cooling the suspension followed by filtering and washing with isopropyl alcohol,
and
j) obtaining and drylng the resulting white solid.
70 2 - c h l o r o - 6 - ( 3 - ( e t h y l ( p h e n y l ) a m m o ) - 2 - h y ~ i cac id
jBF-i-3-HLMf prwared by rhe process of c h ~ m59
71. A process for preparing 5-Chloro-4-hydroxy-l-methylquinolin-2(1~e (MCQ)
comprising the steps of:
a) heating a mixture of 5-Chloro-4-hydroxy-1 -methyl-2-oxo-l,2-dihydroquinoline-3-
carboxylic acid (MCQCA) and dimethylsulfoxide,
b) cooling the mixture of step a), and
c) filtering the mixture of step b) and collecting the resulting filtrate.
72. 5-Chloro-4-hydroxy-1-methylquinolin-2(1H)-on(eM CQ) prepared by the process of claim
71.
73. A process for preparing 5-Ch1oro-4-hydroxy-i-methyl-2-oxo-1,2-d1hydroquino1ine-3-
carboxyl~ca cld (MCQCA) comprising the steps o t
a) heatlng a rnixhrre of ethyl 5-chloro-4-hydroxy-I-methyl-2-oxo-1,2-
dthydroqumnoltne-3-carboxylate (MCQEE) tn a solutlon of hydrochloric acid m
acetlc aad,
b) cooling the mixture of step a).
c) diluting the mxtwe of step b) with 2-propanol and further cooling the &luted
mlxture, and
d) filtering off the crystals resulting from step c).
74. 5 - C h l o r o - 4 - h y d r o x y - 1 - m e t h y l - 2 - 0 x 0 - 1 , ~ o x y l i c acid (MCQCA)
prepared by the process of claim 73.
75. A process for preparing Methyl 5-chloro-4-hydroxy-1 -methyl-2-0x0-1,2-dihydroquinoline-
3-carboxylate (MCQME) comprising the steps of:
a) forming sodium dimethylrnalonate by reaction of dimethylmalonate in
dimethlformatnide with sodium methoxide solution,
br reacting the tnremedzate -F-chloro-i-~hy!-Ih-bmo[Dj[t.3ajx az1ne-2,4-dtone
wlth sodium d~methylmalonate to form methyl 5-chloro-4-hydroxy-l-methyl-2-
o~i)-l,24ihydroqulnoIine-3;i&oxyae (MCQME) sodium ~atta,n d
c) acidi@ng methyl 5-chloro-4-hydroxy-l-methyl-2-oxo-1,2-dihydroquinoline-3-
carboxylate (MCQME) sodium salt to methyl 5-chloro-4-hydroxy-1 -methyl-2-0x0-
1,2-dihydroquinoline-3-carboxylat(eM CQME).
76. Methyl 5-chloro-4-hydroxy-1 -methyi-2-oxo-l,2-dihydroquinoline-3-carboxylate
(MCQME) prepared by the process of claim 75.
77. A process for preparing Ethyl 5-chloro-4-hydroxy-1-methyl-2-0x0-1,d2i-h ydroquinoline-
3-carboxylate (MCQEE) comprising the steps of:
a) adding sodium hydride to a solution of 5-chloro-1-methyl-lh-benzo[D][1,3]
oxazine-2,4-dione and diethyl malonate in dimethylformamide,
b) heating the mixture of step a) while stirring,
c) cooling the solution of step bf,
d) quenclung the reaction mixture of step c],
e) acidifying the mixture of step d),
9 filtering then drying the mixture of step e) , and
g) crystallizing the crude product of step t) by dissolving in ethanol following by slow
cooling.
78. Ethyl 5-chloroil-hydroxy-I-methyl-2-oxo-1,d2~-h ydroquinoline-3-carboxylat(eM CQEE)
prepared by the process of claim 77.
79. A process fir testmg whether a sample of laquimod contains an undesirable lmpunty
wh~chco mprises detmnmg whether the sample contams a compound havlng the structure.
80. A process for preparing a validated pharmaceutical composition comprising laquinimod
comprising:
3) obtaining a batch of laquinimod;
b) determining the amount of at least one of 5-Chloro-4-hydroxy-l-methylquinolin-
2(1H)-one (MCQ), 5-Chloro-4-hydroxy-l-methyl-2-oxo-1.2-dihydroquinoline-3-
carboxylic acid (MCQCA), Methyl 5-chloro-4-hydroxy-l-methyl-2-oxo-l,2-
dihydroquinoline-3-carboxylate (MCQME), N-Ethylaniline (NEA), and Ethyl 5-
chloro-4-hydroxy-1 -methyl-2-oxo-1,2- dihydroquinoline-3-carboxylate (MCQEE)
in the batch using by an HF'LC method; and
c) preparing the pharmaceutical composition from the batch only if
i) the batch is deterrmned to have not more than 0.15% 5-Chloro-4-hydroxy-
I-methylqu~nolin-2(lH)-one (MCQJ, relative to the concentration of
laqulmmod, or
u) the batch IS determined to have not more than 0 15% 5-Chloro-4-hydroxy-
I -methyl-2-oxo-l,2-dihydroquinoline-3-carboxyl1c acid (MCQCA),
relative to the concentration of laqmmmod, or
111) the batch is determmned to have not more than 0 IS%, O 12% or 0.1%
Methyl 5-chloro-4-hydroxy-1 -methyl-2-axo-1,2-dlbydroqu1noline-3-
carhoxylate WCQME), relative to the concentration of laquimmod, or
iv) the batch is d e t d e d to have not more than 0.1% N-Ethylaniline (NEA),
relative to the concentration of laquinimod, or
v) the batch is determined to have not more than 0.194 Ethyl 5-chloro-4-
hydroxy-1-methyl-2-0x0-1,2- dihydroquinoline-3-carboxylate (MCQEE),
relative to the concentration of laquinimod.
81 The process of claim 80, wherein m step c) the phamaceut~cacl ompositron 1s prepared &om
the batch only ~f the hatch is detmned to have not more than 0.12% Methyl 5-chloro-4-
hydroxy-l-methyl-2-oxo-1,2-dihy&oquinoline-3arboxylate (MCQME), relative to the
cormct*nrrailon of laqu~nlfnodc,r whaeln ur srtp c) the phamaceur~sacio mpns:t~oni s gqared
from the batch only if the hatch is determined to have not more than 0.1% Methyl 5-chloro-4-
hydroxjr-i-m~hyII2~xo-i,?-d~hy&oqumolme-3~arboxyIlMatCeQ MF], relat~ve to the
concentration of laquimmod.
82. A process for preparing a pharmaceutical composition comprising laquinimod, or for
distributing a validated batch of a pharmaceutical composition comprising laquinimod,
comprising
a) obtaining a batch of laquinimod or of the pharmaceutical composition;
b) performing stability testing with a sample of the hatch;
c) detemhing the total amount of at least one of 5-Chloro-4-hydroxy-1-methylquinolin-
2(lH)-one (MCQ), 5-Chloro-4-hydroxy-1-methyl-2-0x0-1,2---3-
carboxylic acid (MCQCA), Methyl 5-chloro-4-hydroxy-1-methyl-2-0x0-1,2-
dihydroquinoline-3-carboxylate (MCQME), N-Ethylaniline WA), and Ethyl 5-
chloro-4-hydroxy-I-methyl-2-0x0-1,d2-~ hydroqutnoUne-3-carhxylate(M CQEE) in
the sample of the batch after stabtlity tesimg by an LIPLC method; and
d) validattng the batch for dlstnbution or preparing the pharmaceuttcal compos~tionE rom
the batch only ~f the sample of the batch after stability testtng contams
1) not morc than a total of 0.15% relatlve to the concenhtton of laquimod of
5-Chloro-4-hydroxy-l-mcthylquinoI1n-2(1~-(M0nCe Q), or
11) not more than a total of 0.15% relative to the conm*tion of laquhmod of
5-Chloro~-hydroxy-I-methyl-2-oxo-l,,2-dihydroquinoline-3-carboxyl~c
acld (MCQCA), or
111) not more than a total of 0 If %, 0.12% or 0.1 % rclatlvc to the concentratton of
laqmmod of Methyl 5-chloro-4-hydroxy-1-methyl-2-0x0-1,2-
dlhydroqwnoline-3-carboxylate (MCQME), or
iv) not more than a total of 0.1% relative to the concentration of laquinunod of NEthylaniline
(NEA), or
v) not morc than a total of 0.1% relative to the concentration of laqummod of
Ethyl 5-chloro-4-hydroxy-1-methyl-7-0x0-1,2- dihydroquinolme-3-
earboxyiate (MCQEE)
83. The process of claim 82, further comprising step c) distributing the batch if in step d) the batch
is validared for distnbuiion.
84. The process of claims 82 or 83, wherein in step d) the batch is validated for distribution or the
pharmaceutical composition is prepared from the batch only if the sample of the batch after
stability testing contains not morc than 0.12% Methyl 5-chloro-4-hydroxy-1-methyl-2-0x0-
1,2-dihydroquinoline-3-carboxylat(eM CQME) relative to the concentration of laquinimod, or
wherein in step d) the batch is validated for distribution or the pharmaceutical composition is
prepared Gom the batch only if the sample of the batch aRer stability testing contains not more
than 0.1% Methyl 5-chloro-4-hydroxy-1-mcthyl-2-oxo-l,2-dihydroquinoline-3~arboxylate
(MCQME), relative to the concentration of laquinimod.
85. A process for validating a batch of a pharmaceutical product containing laquinimod or a
pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier for
distribution comprising
a) subjecting a sample of the batch to stability testing;
b) deremuning the amount of at least one of 2-chloro-6-(3-(ethylfphmyl)armno)-2-
hydroxy-N-methyl-3~xopropanmdo)bellzoic actd (BH-3-HLAQ), 5-Chloro-4-
hyhxy-1-methylqumnolm-2(lfo-one (MCQ), 5-Chloro4-hydroxy-1-methyl-2-0x0-
1,2dihydroqumnoline-3-carboxylmc acid (MCQCA), Methyl 5-chloro-4-hydroxy-lrncthyl-?-
oxo-l,2-d1hydroquinol~ne-3-carboxylate (MCQME), N-Ethylamlme
WA), and Ethyl 5-chloro4-bydrclxy-l-melhyl-2-oxo-1,2d-r hydroquinoline-3-
caiboxylate (MCQEE) m the sample of the batch aAer stabllay testing by an HPLC
method; and
c) validating the batch for dtstrrbution only if the sample of the batch after stability
testing contains
i) not more than a total of 1.0% relative to the concentration of laquinimod of 2-
c h l o r o - 6 - ( 3 - ( e t h y l j p h e n y 1 ) m n o ) - 2 - h y ~ l - 3 -
oxopropanarmdo)bellzoic acid (BH-3-HLAQ), or
it) not more than a total of 0.5% or 0.1% relat~ve to the concentration of
laquimod of N-Ethylamline (NEA), or
111) not more than a total of 0.5% relative to the concenttabon of laqulnunod of 5-
Ckitam-l-hy&oxy-l-mahyiqumoiur-2~1N)-~1n(eM CQ) and 5Xhlofo-4-
hy&oxy-l-methyl-2-oxo-1,2-dihydroquinol1ne-3-cboxylaicmc d (MCQCA)
cnmbmed.
86, Tne process of claim 85 wherein in step c) the batch is validated for distribution only if the
sample of the batch after stability testing not more than a total of the 0.1 % of N-Ethylaniline
(NXA) relative to the concentration of laquinimod.
87. A process for preparing a packaged pharmaceutical composition comprising laquinimod or
a phannaceutically acceptable salt thereof comprising:
a) obtaining a batch of pharmaceutical composition comprising laquinimod or a
phannaceutically acceptable salt thereof;
b) performing stability testing with a sample kom the batch;
c) determining the amount of at least one of 2-chloro-6-(3-(ethyl(pheny1)amino)-2-
hydroxy-N-methyl-3-oxopropanamido)benzoica cid (BH-3-HLAQ), 5-Chloro-4-
hydroxy-l-methylquinol~n-2(1H-one (MCQ), 5-Chloro-il-hy&oxy-l-methyl-2-
oxo-l,2-d1hydroqumolmne-32arboxylic ac~d (MCQCA), Methyl S-chloro-4-
hydroxy-1 -methyl-2-oxo- 1 ,Zd1hydroqu1noIine-3-carboxylate (MCQME), NEthylanll~
nc (NEA), and Ethyl 5-chloro-4-hydroxy-l-metbyl-2-oxo-1,2-
d1hy&oqunol1ne-3aarboxylate (MCQEE) in the sample by an HPLC method after
stabihty testing, and
d) packagng the pharmaceutical composition in only if
i) the content of 2-chloro-6-(3-(ethyl(phenyl)~no)-2-hydroxy-N-methyl-3-
oxopropanamdo)bemo~ca c~d(B H-3-HLAQ) in the sample is detemned
to be not more than 1 .O% to the concentration of laqumimod, or
ii) the content of N-Ethylaniline PEA) in the sample is determined to he not
more than 0.5% or 0.1% to the concentration of laquinimod.
iii) the combined content of 5-Chloro-4-hydroxy-l-methylquinolin-2(lJ+one
(MCQ) and 5-Chloro-4-hydroxy-l-methyl-2-oxo-1,2-dihy&oq~oline-3-
carboxylic acid (MCQCA) m the sample is determined to be not more than
a total of 0.5% relative to the concentration of laquinimod.
88 The process of cla~m8 7, wherein in step d) the pharmaceutical composition 1s packaged only
lt the content of N-Ethylamline (NEA) in the sanipie is detmned to be not more than 0 i04
to the concentration of laquinimod.
89. The process of any one of claims 69-88, wherein the laquinimod is laquinimod sodium salt.
90. An impurity or a salt thereof for use, as a reference standard to detect trace amounts of the
impurity in a pharmaceutical composition comprising laquinimod or a pharmaceutically
acceptable salt thereof, wherein the impurity is selected from the group consisting of 2-
chloro-6-(3-(ethyl(phmyl)~no)-2-hydro~y-N-methyl-3-oxoprop~~do)benzoacicid
(BH-3-HLAQ), 5-Chloro-4-hydroxy-l-methylquinolin-2(1l+om (MCQ), 5-Chloro-4-
hydroxy-l-methyl-2-oxo-1,2-dihydroquinoline-3-cboxyliacc id (MCQCA), Methyl 5-
chtoro-4-hy&oxy-l-methyl-2-oxo-l,2dihydroquinoline-3-carboxylate (MCQME), NEthylaniline
WA), and Ethyl 5-chloro-4-hydroxy-l-methyl-2-oxo-1,d2i-h ydroquinoline-
3carboxylate (MCQEE).
91. A method of determining the concentration of an impurity in a pharmaceutical composition
comprising laquinimod, the method comprising,
a) Preparing a sample solution from the phamaceutical composition,
b) Prepanng a standard solut~onc ompnslng the tmpunty
c) Prepmng a resolution solution compnstng laqutmmod and the impunty,
d) Preparing a buffer solutmon by dissolving ammonsum acetate in water and adjusting
to pH of 7 0 ic 0.05 with aqueous ammonia or glacial acetlc ac~d,
e) Preparing a diluent solution comprising the buffer solution and acetonitrile,
0 Prepanng a blank solution compnsmg the diluent solut~ona nd aqueous acetonitrile,
g) Injecting into the HPLC the resolution solution, the blank solution, the standard
solut~ona, nd the sample solution,
h) Running the HPLC uslng ultravtolet absorption at 240 nm and the diluent solution
as the moblle phase,
1) Detemlmng the retention time (RT) and the areas of the peaks of the smpunty in
the chromatograms of the sample solution, and
j) Performing quantltatlon of the impmty with respect to the correspond~ngp eaks In
the chromtomms of the standard solut~ons,
wherem the impunty IS 5-Chloro4-hydroxy-l-methylquin011n-2(lu)e (MCQ), 5-
Chloro4-hydroxy-l-m&1hyi-2-axo-l,2-drhy&~ummlme-3-carboxy1a~c:ed (MCCfCA),
Methyl 5-chloro-4-hydrory-l-methyl-2-oxo-l,2-dthydroqumolme-3- carboxylate
(MCQME), N-Ethyl-4,5-dihydroxy-l-methyl-2-oxo-N-p-1 dihydroqumolme-3-
carboxamde (5-HLAQ) or Ethyl 5-ehloro-4-hydroxy-l-methyl-2-oxo-l,2-
dihydroqumnolme-3-carboxylate( MCQEE)
92. A method of determining the concentration of an impurity in a pharmaceutical composition
comprising laquinimod, the method comprising,
a) Preparing a sample solution Erom the pharmaceutical composition,
b) Preparing a standard solution comprising the impurity
c) Preparing a resolution solution comprising laquinimod and the impurity,
d) Prepanng a buffer solution by dissolving momum acetate in water, and adjusting
to pH of 7.0 i: 0 05 wlth aqueous ammonia or glac~aal cetic acid,
e) Prepanng a blank solution compnsing the buffer solution and acetonitnle,
F) Injecting Into the HPLC the resolution solution, the blank solution, the standard
solution, and the sample solution,
g) Runnmg the HPLC using ultraviolet absorpt~on at 240 nm and a mobile phase of a
mixture of the buffer soiunon, and acetomtnle.
h) Detemmng the retention time (KT) and the areas of the peaks of the impurity m
the chromatograms of the sample solution,
I) Performing quantitatlon of the impunty wlth respect to the correspondmg peaks 1n
the chromatograms of the standard solutions,
wherein the tmpunty is N-Ethylarulme (NEA), lH,3H-sptro[5-chloro-1-methylquinolmne-
2,4-dtone-3,3'-[1]ethylindolin-[2]-one] (SPIRO-LAQ), or 5-CNoro-N-ethyl-3-hydroxy-lmethyl-
2,4-dioxo-N-phenyl-1,2,3,4-t&&ydroqwnoline-3-carboxamde (3-HLAQ).
93 A method of detemunlng the concentration of an impunty in a pharmaceutical composition
compnsing laquimmod and a phamaceutically acceptable carrier, the method compnsmg,
a) Preparing a sample solution from the pharmaceutical composition,
bj Prepanng a standard ror;oiurion compnsing the tmpunry
c) Preparing a Quxtitation Limit (QL) solution comprising the impurity,
d) Preparing a resolution solution comprising laquinimod and the impurity,
e) Preparing a buffer solution by dissolving ammonium dihydrogen phosphate in
water, and adjusting to pH of 7.0 * 0.10 with aqueous ammonia or phosphoric acid,
f ) Preparing a blank solution comprising the buffer solution and acetonitrile,
g) Injecting into the IHPLC the resolution solution, the blank solution, the QL solution,
the standard solution and the sample solution,
h) Running the HPLC using a ultraviolet absorption at 212 nm and a mobile phase of
a mixture of the buffer solution, acetonitrile, and methanol,
i) Detemning the retention tune (RT) and the areas of the peaks of the impurity in
the chromatograms of the sample solution, and
J) Performing quantitation of the impurity w~thre spect to the corresponding peaks in
the chromatoyams of the standard solutions,
wherem the t~npunty IS 2-chloro-6-(3-(ethyt(pheny1jamno)-2-hydhyl-3-
oxopropanamidojbenzo~c acld (BH-3-HLAQ) or N-ethyl-4-hydroxy-l-methyl-5-
(2,3,4,5,6-pentaJlydroxyhexylam1no)-2-oxo-N-phenyl-1,2-dihydroqumoline-3-
carboxamde (MEG-LAQ).
94 A method of determmmg the concmtmt~ono f an lmpunty ma phamaceut~cacl omposition
eompnsmg laqu~mmoda nd a phmceutlcally acceptable earner, the method comprising,
a) Prepanng a sample solut~onf rom the pharmaceutical composition,
b) Prepanng a standard solution comprising the lmpurtty
c) Preparing a resolution solution comprising laquinimod and the impurity,
d) Preparing a buffer solution by dissolving ammonium acetate in water, and adjusting
to PH or 7.0 + 0.05 with aqueous ammonia or glacial acetic acid,
e) Prq&ng a blank solution cowrising the buffer solution and aedonitrile,
f) Injecting into the HPLC the resolution solution, the blank solution, the standard
solutron and the sample soiut~on,
g) Running the HPLC using a ultraviolet absorption at 242 nm and the blank solution
as the mobile phase,
h) Determining the retention time (RT) and the areas of the peaks of the impurity in
the chromatograms of the sample solution, and
i) Performing quantitation of the impurity with respect to the corresponding peaks in
the chromatograms of the standard solutions,
wherein the impurity is 5-Chloro-4-hydroxy-l-methylquinolin-2(1H)-one @ICQ), 5-
Chtoro4-hydroxy-l-methyl-2-oxo-1,2-dihydroquinole-3-carboxyliaec id (MCQCA),
Methyl 5-chloro4-hydroxy-l-methyl-2-oxo-l,2-dihydroquinoline-3- carboxylate
(MCQME) or N-Ethyl4,S-dihydroxy-1-methyl-2-0x0-N-phenyl-d1i,h2y droquinoline-3-
carboxamide (5-HLAQ).
95. A method ofdetemnmg the concentration of an mpunty in a pharmaceut~cacl omposltlon
compnsing laqutmmod and a phaceuttcally acceptable carrier, the method compnsmg,
a) Prepanng a sample solution from the pharmaceutical composition,
b) Preparing a standard solution eompnsmg the impurity
C) Prepanng a resolution solution compnslng laquinimod and the impunty,
d) Preparing a blank solution compnsing methanol and acetomtrile,
e) Prepmug a buffer solutton by dzssolving ammomum acetate In water, and adjusting
to pH of 7 0 + 0.05 w~thaq ueous ammoma or glac~aal cetlc acld,
f) Injecting into the HPLC the resolution solution, the blank solution, and the sample
solution,
g) R w g the HPLC uszng a ultravlalet absorption at 240 nm and a mobile phase
comprising acetonitnle and the buffer solution,
h) Determining the retention time (RT) and the areas of the peaks of the impurity in
the chromatogams of the sample solut~ona, nd
i) Peffonmng quantitation of the impurity with respect to the corresponding peaks in
the cFrmma:a~a_mos f the standard soiutior~,
wherein the impurity is N-Ethylaniline (NEA), 5-Chloro-N-ethyl-3-hydroxy-1-methyl-2,4-
dioxo-N-phenyl-1,2,3,4-tetrahydroquinolide (3-HLAQ) or lH,3H-spiro[5-
chloro-l -methylquinoline-2,4dione-3,3'-[1]ethylindolin-[2]-on(eS]P IRO-LAQ).