Note: Descriptions are shown in the official language in which they were submitted.
_Wo 94l12220 ~ ~13 S 8 8 0 PCT~US93111470
WAT~ SOLI~ NON~MUNOGE:NIC POLYA~E
S C~ OSS-LINNNG AGENT5
'
Field of ~h~ention
The present invention relates to covalent binding of water-soluble
poIyamides to p~teins, polynucleotides and other biologieal subsb~tes to
0: ~onn~ substantially nonimmunogenic water-soluble pmducts. The present
invention~ also relates to~ p~teins, polynucleotides and other biological
: ~ ~ substrates wh~ch are c~oss-li~dced, conjugated, polyme~ed or deco~ed with
water-soluble: polyamides to foI~n substan~ally non~Dmunogel~ic products.
C~oss-~nk~g ~eagcnts~ are used for a ~anety o~ pu~poses, includi~g;
the~ investigation:~ :of ~ e ~a~al anangement ~and fimCtioDs of various
pacmnnolecula~entities,; the~ iden~ficadon of bîndL~g sites (~e~tors) for
hg~ds,:~ lhe:~pn~ on ~of a~finit~ matlices, and:the modification and
20 ~ ~of~dv~se~macl1Dmol~cular s~uc~ures ~ethods ~ l~mology,
Volume 91, pages~580 to~6~9 (1983j3. C~ss-linkers have ~n designed to
~se~ve~:~elec~rostatic; c~ e; to alter ele~static charge;~to d~rease
immu~city; to L~crease and decrease susce~tibili~ to pr~eolysis;~o: ~ :
introduce~fluorescsnt labels, spin labels, ~adiolabels, and elect~n-dense
sùbsli~uorl~s;~ to atlach:~sever~l different t~ipes of c~bohydra~e ~moie~des; toma~y ~enzyme :: ~peCi~lCit,!; and t~ introduce intramolecular and/or
ffite~olecular~ Cl'OS5-1ink~, both to couple already asso~iated ~pecies and to
Join va~ious iprotèins il~ 'order to `co~n~ine the pm~es o~ bdth ~o a single
:mo~lecule;~G. ~. Means and R. F. Peeney, Bioconjugate Chemist~ Volume
3~ page2to12(1990)). :Alargenumberofcross-li~greagentsbavebeen
d~velo~ to serve these and a vanety of other puIposes. Many of these
reagents:are commercially available. : ~
Cross-linldng of proteins and their immobilization, either by
: attachment to an insoluble support or by various other means, has been
3s: ;~ employed to increase the stability of proteins or of cer~ain con~ormational
WO 94/12220 PCT/US93/1147û~ `
2135880
re}ations~ips in proteins; to couple two or more different proteins; to identifyor characterize the nature and extent of cer~n~ protein-protein interactions or
to determis~e distances between reactive ~groups in or between protein
subunits. Proteins may be immobili~ed to facilitate their use and their
s separation from other products. ~::Cross-linking therapeutic proteins or
polype~tides has been shown to decrease immunogenicity and to incre~se the
lifetisne of the cross-linked product in the blood stream.
Irl general, cross-lin~ing agents consist of an orgaILic bAdge be~ween
activated te~. The termini bind to biological macromolecules to fonn a
o link. Vanous organic bridges are recognized in the art, including peptides,
car'oohydra~es ~e.g., dextran, starch, and hydroxyethylstarch), fatty acids,
polyglycolides, polypeptides (e.g., gelatin or coll~gen), poly~lene umits,
and polymers such as poly(vinylalcohol), polyvinylpyrrolidone, and
polyethylene glycol (also llmown as polyoxyethylene).
Commercially available homobifunctio~al and heterbifancdonal
cross-lirldng agents range in size ~:rom about 6 to 16 A. Theis solubili~
vat~s dec~eases with cha~n le~g~. Yet the efficiency of cross~ king is
increased with cha~ le~gth as stenc ~indrance is ~duced.
Peptides composed of ~ to nine amino acid residues a~e
: :: 20 : commoIIly used as GroSS-IiDking age~ts. However, these suffer f~om ~e
follow~g disadvan~ages: the ~hemist~ies used in p~ptide synthesis are
complex"nvolving selec~ive bloc3~g and deblocking of functional groups
d: ~pecific COUpliilg cQnditio~s. Care must be taken ~ot to ~acemize the
amino acid compone~ts. P~ptides must be chose~ ully so that t~ey have
no biological actiYity. F~a31y, ~ey a~e subjeca ~o enzymatic hydrolysis,
which lim~ts their period o~ util~t~, par~cularly during circulation in ~iw~
Synthetic polymers a~e being developed for use as c~oss-lin~ng !
agents. A synthetic polymer ~oss-linker desirably has the following
charactetistics: (1) The polymer must be water-soluble and exhibit a narrow,
30 definite molecular weight distribution. (2) It should provide
attachment/release sites or the possibility of the incolpora$ion of such sites.
(3) Tk.e polymer should be eompatible with the biological environrnental,
~`; i.e., non-toxic, non-an~igenic, and not p~ovocative in any other r~spect. (4)
. ~
' ~ ' '
s wo 94/12220 2135 ~ 8 0 PCT/US93/11470
,
It should be biodegradable or eliminated from the organism a~ter having
fulfilled its function (Duncan and Kopecek, Advances in Polymer Seience,
Volume 97, pages 53 to 101: (1984)).
The conjugation of biologically active polypeptides with water-
soluble polymers such as PEG is well-known. The coupling of biologicaLly
active and pharmaceutically active pe~tides and polyp~ptides to PBG and
similar water-soluble polymers is diselosed by U.S. Patent No. 4,179,377 to
Davis et al. Polype~tides modified with PEG are disclosed as exhibit~g
dramatically re~uced immunogenicity and antigenicity. The PEG conjugates
; loalso exhibit a wide ~nge of solubilities and low to~icity, and have been
shown to remain in the bloodstream conside~ably longer ~an tbe
co~esponding ~ative compounds yet are leadily excreted. The P~iG
: ~ ~ coI~yugates have also bee~ shown not to r~terfere wi~h ~nzymatic activi~r in
the:: bloodstream or ~e conformation of the polyp~tides conjugated theIeto.
15 ~ ~scordingly,~ a number o~ Pl~ conjugates of the~apeutic proteins have ~een
de~reloped~ ex~ibiff~g reduc~d ~u~ogeniGity and a~tigenicity and longer
cle~ance~ ~imes, while~ ~taining a substa~tial portion of the protein's
physiologi~l~ac~ivr~
Affention: bas~ ~also~focused upon the ~ yugation of P~G ~wi~
o~ ~elapeubc drugs. Gnanov et al., "Mac~molecules," 17, pages 945 to: 952
1984):~ observed t~at the ~at~acbrneDt of PEG~to various drugs l~d to
prolonged pharDlacologi:cal a~vi~
U.S~ Patent. No. 5,122,614 to Zalips~ describes the use OI
polyethylene~gycolas-ac~oss-lin~gagent. U.S. PatentNo. 5,053,520to
Bie~ z describes poly~nino acid based coupling agents which a~e not
`: :: w~ter-solu~le. U.S. Patent No. 4,182,695 to Ho~ descnbes protein bound
to poiyamldes. Russian Patent ~plica~on No. SU 1659433 discloses water-
soluble polyamides with luminescent groups in ~e chain. U.S. Patent No.
:5,110,909 to Dellacherie discloses water-soluble macromolecular conjugates
30 of hemoglobin. ~CT ~pplic~tion WO 92/08790 to Cargill discloses the use
of polyamide polymers bonded to a linker group which is bonded to a
protein .
. . . ~ :
Many potentially therapeu~ic proteins have undesirable ~ha~actenstics
-- such as short half life in YiYo, poor solubility, vulne~bil;ty to en~ymatic
WO 94/12220 ; PCT/US93/11470
~,
213S880 -
degradation in ~iv~, or immunogenicity. I?le polyamides of the present
inv~ntion when coupled to such proteins overcome these disadvarltages.
, ~
Summarv of the Invention `~
The present inverl~ion is water-soluble, nonimmunogenic polyamides
having molecuL~ weights of about 300 to about 20,000 grams per mole;
;:~ where the amide repeat units are comprised of: (i) a water-soluble organic
acid subunit having at least one car~oxylate group and fifteen or fewer atoms
sep~g the ~mide ~unctionalities i~ the polyamide; covalently linked as an
o arnide to (ii) a water-soluble organic amine subunit having at least one
p~imary amino group and fifteen or fewer atoms sepa~ating the amide
functionali~es in the polyan~ide.
In other words, the polya~de of the present inveDtion is a water-
soluble, substa~tially nonilDmunogenic polyamide selected f~om the formulas
15 : I, II, andm:
Y-A-X-Y
B^X-Z
~ Y^X^Z
(i) where term~us Y is H or a ca~oxyl coupling group; (i~i) where teIminus
2~ ~: Z is~ r a coupli~g ~oup at~ached to an amine group; ~d (iii) where X is
a~polyanude sel~ed ~rom: (33^A)n, (A^B)n, (AA)~ and b~a~ched polyamides
fo~med by li~g:(B^A)~" (A^B3n or tAA)n to a c~ntIal polyacid, polyamine
:; or~:polyamino acid; a~d~ (ivj where A is a ~ ,~di^acid; B is a ~ ,~diami~e;
: is~ a -c~ ~amino acid; ~ is ~he number of am:ide r~peat units in the
2s pol~e; and (v) where the acid subunits of the amide re~eat unit~ :are (a)
organic acids having fifi:een ~r fewer atoms in the chain and having one or
~more hetèroatom6 O, S, P or N present as substituents onr or atoms in~ thè
chain, or (b) two or more of such organic acids bridged by water^soluble
o rganiG dian~ines; and (vi) where ~e amine subunits of the amide repeat
:30 units are organic, water-soluble amines having at least one pnmaIy amine
~up and haYing f~teen or fewer atoms in the chain and having one or more
heteroatoms O, S, P or N present as substituents on or atoms in the chain;
and (vii) where n is from 2 to about 100.
~WO 94/L2220 213 5 8 8 0 PCT/US93/114~0
:
The present inve~tion includes one or more such polyamides used to
cross^link, conjugate, decorate or polymenze proteins~ antibodies, haptens,
polypeptides, polynucl~tides or ather biological substrates. The cross-
linked, conjugated~ polymeri~ed or decorated product is water-soluble,
5 nonimmunogenic and retains all or a useful portion of the physiolagical
activity ofthe subs~te.
Bnef DescriptionQf the Dlawttl~s
:Figure 1 sbows the polycondensation of ethylene glycol
o bis(methoxycar~onylmethyl ether) a~d 1 ,4-diaminobut2ne.
Pigure 2 shows the lea~ion conditions, product characteristics, and
yield o~ ~ie reactions show~ in Figure 1.
igure :3 sho~s the e~pe imental data, including oxygen bînding
nctio~ ~of ~d;aspisi~ cross-linked hemoglobin polymerized and deco~ed
4 shows~ ~he sLze exclusion chromatog~aphic profiles of
cross-linked hemoglobin polymeriæd and de~oIated with P~S-
Figur~ S shows the reverse phase HPL profiles of di~i~ cross-
d hemoglobin polyme~ized ~d decoIated wi~ PAS-2400.
ure 6 depicts :the componet~ts of polyamide s~thesis.
igure 7 depics th~ syDthesis of BMDAB (a:polyamide componellt).
;Figu~e 8:d~pic~s t~e polyco~densatio~ of BMDAB with diamine~ to
fonn a polyamide. ~: :
2s ;: ~ : Fig~e 9 depicts the synthesis of p~lyamide actiYated esters~ PAS-
3~7 and~ P~(~
Fl~ure 10 ~de~icts the synthesis of maleimide-capped: polyamide,
desi~ated PAM-4080. ~
Figure l l de~lcts the size exclusion profiles following pclymerization
30:~ ~ of diaspi~in cross-linked~ hemoglobin with PAS-3037.
Figllre 12 depicts size exclusion chromatography following
polymerization of diaspinn cross-linked hemoglobin with PAS-4200.
Figure 13 depicts the reverse phase HP~C pr~files following
polymerization of diaspirin cross-linked hemoglobin with PAS-4200.
~ . ~
WO 94/12220 . PCT/US93111470,_A~
X1358811
Figure 14 depicts the size ei;~oluston chromatog~phy profiles
following polymenzation of diaspilin c~ross-linked hemoglobin with PAM-
4080.
Figure 15 depicts reverse phase HPLC follawing polymeIization of
s diaspilin cross-linked hemoglobin with PAM~080.
Figu~e 16 shows the expenmental data following palyme~zation of
diaspinn cross-linked hemoglobin with P~S-3070.
Figure 17 shows the expelimental data following polymeIization ~f
diaspirin: cross-~nked hemoglobin with PAS-4080.
lo Figure 18 shows the expe~ental data following polymenzation of
diaspinn cross-linked hemoglobln with PAM~080.
,
Detailed De$ç~ip iQn_he In~eDtiQP
~ e ~lyamides: ~ the present invention are substantially non-
15 ~ immunogenic, water-soluble polyamides having molecular weights of about
3 ~ to a~ut 20,000 g~ams psr mole. The amide r~peat units of these
polyamides are composed of a water-soluble organic acid subunit having at
o~e: caiooxylate grwp and fifteen or fewer atoms s~ ing the amide
fill~ctionalities in the:polymer, covalently linked as an ~mide to a water-
20; ~ ` soluble~ o~nc a~ne subu~it haviDg at least one p~naIy amino group ~andfi~l or ~ewer atoms s~parating the amide fimctio~alities in the polymer~
I"nese~polyamides may be~:employed directly or afteE activation, for the
pu~poses of cross-linl~ng, conjuga~ng, polyme~izing and/or deeo~t~g
bidodcal:~substlales~such as ploteins, p~lypeptides, a~tibodies, haptens,
bohyd~ates or p~ ucle~tides to give products which are water-s~uble,
substa~tially nonimm~ogel~c, ~dlwhich re~ all or a use~l~p~ortion of the
substratels physiological act~vity. They may also be used to a~tach subs~ates
to :detecdon agents or so~d mat~ices.
The teml i'non-immunogenic" indicates that the polyamide does not
30 ~ cit a humoral or cell-mediated immune response, either in uw or in vitro.
: The tenn l'water-solublell indicates that the polyamide :has a solubility
i31 w ater that exc~s 500 mg per 100 mL. The term also indicates that the
polyamide does not act as a detergent and does not fonn aggregates such as
~,
micelles in water.
~ ~ .
wo 94/12220 ~ .'13 5 8 81) pcTluss3lll47o
l~e telm "activation" means converting a group of the polyamide
te~ninus to a more reactive coupling g~oup.
The polyamide may be linear or b~anched.
The term "substrate" means the molecule to which the polyamide of
s the present invention is bound. Subs~tes include but are not limited to
proteins such as enzymes, gsowth ~actors, antibodies or blood proteins;
polynueleotides sueh as compleme~ y DNA fragments; steroids and
: hormones, immunoconjugates; caIbohydrates; and conjugates of any of these
: ~ substr~tes. The substIate may also be a solid support ~ bead. Substrates
IO include molecules ~aving therapeutically useful biological activity.
As used herein, a subs~ate is said to be "decorated" when multiple
polyamides a~e bound to the subs~ate by one termi~us of each polyamide
and~ other term~i of the polyamide are not ~ound to a differel~t wbstrate
molecule.:
15 : ~ Ihe water-soluble polyamides of this invention may be pr~pared by
~: :methods~ hiown in the art. ~Cnown methods for the pr~ ation of
poly~nides ane inco~po~ated here by reference as useful methods for
prepalation of tbe polyamides of the preserlt inveDtion. N. Ogata et al.,
Polym Joumal, Volume 5, pages 18~: tl973~ and N. Oga~a and Y. Hosoda,
20 Joun~ Polym Science9 Pobm Lett. Ed., Volume 12, pages:355ff(1974)
des~be the polycondensati~n of dies~ers activated by ~er or !hydroxyl
groups~:with di~ines. N. Ogata et al., Jou2nal Polym Science, P~lym
Cheq~ishy~ Volume 14, pages 783ff ~1976j, N. Ogata et al." Polym
Jou~al, Volum~ ll,: pages 827 to 833 (1979), and ~I. Sa~o, et al.,
mol Chemis~y, Y~lume 182, page 755 ~o 762 (1981) describe the
; polyco~de~sation of a,cti,vated diester~ containing ether, thioether or hydroxyl
ups with diamines. D. Kieley ant T-H. Lin ~ave also described
polyhydroxypolyamides and a p~ocess for making same, U.S. Pa~ent No.
4,833,230. N. Ogata and Y. Hosoda, Joumal Polym Science, Polym
30 ~ Chemis~y Ed., Volume 18, pages 1159 to 1162 (1978) desc~ibe the
synthesis of water-soluble polyamides by polycondensation in solutions of
ethylene glycol dimethoxycarbonylmethyl ether and hexamethylene diamine.
The acid subw~its of the amide repeat units are selected fr~m the
group of orgal~ic acids having fifteen or fewer atoms in the chain and havinc
,~ , .
~:
Wo 94112220 PcTIuss3lll47~ .
2~.3S8~ ,., , ~
heteroatoms (O, S, P, N) present ~ither as substituents on or atoms in ~e
chain. Alternatively, the acid subunits of $he amide repeat units rnay consist
of two or more such orgianic acids join~ to bAdging water-saluble, organic
diam~nes. The amine subunits of ~ amide repeat UDitS are selected from
5 among the group of orgal~ic aminès having f~n or fewer atorns in the
chain and having heteroatoms ((:~, S, P, N) present as substihlents on or
atoms in the c}liun. Polyamindes of simiLar iand/or dissimilar structllTe may
be linked by a cent~ polyacid, polyamine or polyamino acid to form
b~nched, wtater^soluble polyan~ides.
lOAny of the known coupling chemis~ies may be used to activate
polyamides of this invention to decorate, link, polymenze and/or conjugate
substra~es~ Many examples of such coupling chemistries are give~ in
~:~ nChemistly of Protein Co~jugatio~ and Cross~ kiIlg9~l S. Wong, CRC
Press, Inc. ~1991) which is i~ po~ated by reference herein.
15 ~Such cbcmi~ies include ~eac~ng the polyamides with bi- or poly-
un~;~al protein reagents ~UGh as dialdehydes, N-hydroxysuccinimide
esters, ~function~ acetals, bis-maleimides, bifilnctional imino esters,
iepoxides, and dica~boxylic acid cblondes. The choiee of coupliDg ~:
chemistries will d~pend upon the subs~ate molecule beLng c~ss-l~ked,
~: 20 ~ co~ugated, polymerized and/or decorated. The c~upling chemistIy ~hould
be: selected so that it does not alter the biological or chemieal activi~ of ~he::sub~rate molecule.
Generally, to decoIate a substrate molecule, b~een about 4 and S0
moles~of:polyamide should be used per mole of subs~ate. Iarger substr~te
2s molecules will ~equire a greater propofion of polyamide. To p~ ily
~` ~ : conjugate, cross-linlc or polyme a substIate without decorating ~equires
tho h~owl~ge of a chemist skilled in the ar~ as ~o the chemistries of the
~` coupling agents, the reactive groups on the particular subst~te, the sLze of
t~e subst~te, the size of the polyamide, concent~tion of the subst~te, and
:: 30 gen~ral reaction pa~ameters.
As will be appreciated readily by those of skill in the art, substra~es
such as ~nino acids, pe~tides, proteins, nucleotides, polynucleotides,
::: pharmaceutic agents, and diagnostic agents have functional groups which
may be covalently bound to the pendant fi~nctional groups of the polyamide
.~ WO ~4/12220 PCT/US93111470
t'`~ 213~880
backbone and ~unction~ deAvatives thereof. Those of ordinaly skill in
the art having ~he ben~fit of this disclosure will comprehend the synthetic
approaches that may be employed to covalently join the polyamide and the
substrate. The order of re~c~oll is not important. The pendant functional
s g~oup(s~ of the polyamide may be activated appropnately, if so requi~ed,
and then at~ched to the subs~ate. Lhkewise, the subst~te may be activated
appropnately, if necessa~ d then attached to the polyamide.
For example, amLno, hydroxy, c~oonyl, carboxyl, or thiol
substi~ents ~ commonly found as part of the structure of amino acid,
o peptidb, protein, nucleo~de, polynucleotide, and diagnostic agent
compounds. Moreover, the polyamide may be syn~esized to incoIporate
reactive teImiI~i such as these substituents. The subs~ate may be joL~ed to
~he polyaII~ide by chemis~ies such as those cited below, or by other
chemistries such as those disclosed in Bodans~ly and Bodanszly, "Tbe
15 ~ Piactiee ~ P~tide Syl~thesis," Spnnger-Verlag, ~ew York, (I984);
dblad, nC~hemical Reagents ~or P~otein Modifica~on, " CRC P~ess, Boca
aton, P~o~ida, (1991); Mosbach "Methods in Enzymology, Yolllme XLIV,
Immobili~d ~es," Academic Press, New YoTk, (1976); or Uh1maIm
d Peyman, "An~sense 03igonucleotides: A New T~erapeuti~ Principle,"
Zo~ ~ Chemical ~Reviews, ~olume 90, No. 4, pages 543 to 585 ~June 1990).
For example, ~i~ is ~og~i~ed as a diagnostic probe tllat is
selectively D ained by complexation with a~ridin. Bio~n contains a ca~oxyl
roup that ma~r be acli~,~ated as a succinimidyl ester and at~ached to a
polyamide ~baving a amino te~minus. Either pnor to or ~ollowing cov~en~
bon~i~g to biotin, ~he other te~ us of the polyamide may be covalently
` bonded to a p~p~de, protein or other bio~hemical agent. Under these
: ~ ~ conditions,; the polyamide ser~es as a spacer group that ~ncurIen~y
maint~ns or increases the aqueous solubility of the product. The
bidchemical agent is thereby labeled with a diagnostic probe that is
30 positioned at the end of the polyamide spacer to ~acilitate :interactioD with avidin.
~, ~
Si~iilarly, def~roxamine is a pham~aceutic agent that is used
theIapeutically as an antidote to iron poisoning. The duration of therapeutic
action of de~eroxamine is sho~t, because it is rapidly excret~d via the kidney.
WO 94112220 2 ~ 3 5 8 8 0 PCT/US93/1147~ .
- 10-
It has been recognized that if deferoxamine is conjugated to a larger
molecular weight entity such as a dextIan of albumin, it will be retained in
~he vascular circulation fior longer pe~o~s of time. In accordance with the
present invention, a polyamide ~~ be used as a spacer group that
s concurrently maintains or increases the aqueous solubility of t~e product.
One tenninus of the polyamide may be converted to a carbonyl ~unctional
gr~up and atta~hed ~o ~he ~mino substituent of deferoxamine by reductive
amination, and the other terminus of the polyamide may be converte~ to an
activated ester (e.g. a succinLmidyi ester) and attached to albumin. Thrcugh
0 this conjugation, the duration of vascular circulation OI conjugated
defexaxamine is leng~hened and the agent retains its chelating abilities.
All the components of the polyamides of the present invention aré
selected so as ~to p~eserve wa~er-solubility. They a~e water~soluble,
hydwphilic o~er the entire chain length. The le~g~h of ~he polyamide is
chosen~ t~ facilitate inte~action between the substrat~ and the polyan~ide. The
c~oss-iir~ng of a large sub~ will ~equire a longer polyamide sinee it will
minimize~steric interactio~s between two large substrate molecules.
n immunogenic subs~ate should genesally be hig}~y deeo~ated and
should~havere3~tivelylongchainpolyamides.
20 ~ In ~reacting the polyamides of the present invention to biologieallya~ctivo subshates, sueh as enzgmes, care is t~ken to avoid destroying the
; ~ acti~ity;of the subst~ate. One skilled in ~he alt will understand~that va~ying
he degre~ of decora~don and/or ~lymeriza~on will allow one to pr~p a
product~ havi~g a useful biological activiry.
The poly~nides of the present ~ve~ltion ~ not polymers of
` ~ ~ amino acids, so they are not subject to enzymatic hydr~lysis.
In addition, ~hie polyan~ides of the present invention may be` used to
render substrates soluble in organic solvents such as methano], ethanol or
acetolait~yl.
-30 ~ The polyamiàes sf the present invention may be used as
polymenzation agents. In one example descnbed fully below, the ten~ini of
a polyamide have been modified as maleimide groups, suitable for reaction
with thiol substituents of proteins. Bis(maleimide) polyamide was empolyed
to polymen~e human hemoglobin via the cysteine-n93 thiol -residues of that
.. .~ .-
:~
wo 94,~ 213 5 8 8 0 PCT/US93/11470
protein. Similarly, in another exarnple below, the termil~i of a polyamide
were converted to bis(suçcinimidyl) esters or bisaldehydes, suitable for
reaction with amino substit~ents of proteins. Both the bis(suecinimidyl)
.
polyamide and the bisaldehyde poIyamide have been employed to polymeIize
5 human hemoglobin via ~he E -a~ o groups of lysille residues of the protein.
In another embodiment the polyamides of the present invention may
be used to link probes (e.g. fluoresceDt, Iadioactive, etc.3 to a substrate to be
detec~ed.
In the examples that ~ollow, we use the following nonmenclature for
lo our polyamides: since the backbone is a polyamide, the letters PA will
apply; ~he letter desig~ating the coupling group wi~l follow, M for
maleimide and S ~or N-hy~xysuccinimide; a hyphen will sepa~ate the
alphabetic code f~om the approximate molecul~r weigbt. Thus, a polyamide
idelltified as PA~1-3800 is a polyamide bis(maleimide) having a moleeular
1~ weight of abou~ 3800 Daltons.
,
D~SIGN AND SYNT~IESIS O~POLYAMID~S
~; ~ ~ ~e foLlowing cxamples the polyamide condensation pr~ducts are
G baMcterize i in three ways. S~e exclusion chroma~ogIaphic (SEC) analysis
20 ~is connQIeted using a Supersse~A 12 colum~ and 50 m~ phosphate, pH 6.5,
mobile phase delivered at a flow ~ate of 0.4 mL/min. with detec~o~ at 220
nm; ~ is analysis conf~ns that polymerized pr~ducts were fo~ed and
p~mits a~proximation of molecular weights and the Iange of molecular
weight~ o f the componeDts in the prodllct m~xtu~e. Thin-layer
2s chlomatographic (II,C) analysis pen~its se~a~ation and charactenzatio~ ~f
end-group ~ctionality of the components in the product mrxt~re. The
St~UGh~re af each component is assigned on thé basis of relative mig~ation
(Rf) and reactivity toward ninhydxin spray reagent. Under the TLC
condi~ions, poly~mides with diester end~ ups have the largest Rfj followed
30 by components with mons-ester - mono-amine end-groups, and di-amine
end-gr~ups, res~ectively. Only components having an amine end-group are
reactive toward ninhydrin. The structure sf the mono- and di-esters is
co}~L~med by base-catalyzed hydrolysis and TLC of the resulting products;
~- - un~er these conditions esters are hydrolyzed to acids and the Rf of the
, ~
Wo 94/12220 2'~.358~ PCTJUS93/114
material decreases. Finally, the molecularSweight is estimated by amino
end-group analysis using fluorescamLne.~`Précisely and accurately weighed
polyamide samples are dissolved in ~ thanol/phosphate buffer, derivatized
by adding fluorescamLne dissolved~n acetone, and then analyzed by flow
s injection with a ~IPLt: system equipped with a fluorescence detector.
Equivalent weights are detem~ined by ~omparison of responses for sta~dard
solutions of diaminohexane/PEG/ethyl acetate in methanoltphosphate bu~er.
Equivalent weights are converted to molecular weights based on the average
number of amines per molecule.
o Alte~natively, the ~ spectrum un be used to estimate the
molecular weights of the poly~des, as follows: the first step is to divide
the structure of t~e polyamide into e~d groups and r~peating units. Then the
mol~cular weight of each par~ is calculated. Next one id~ntifies u~ique
compon~ts L~ each pa~t and correlates the co~esponding NMR p~sona~lGe
with that component. Polyamides have a rwmber of well-resolYgd
resonances ~at ca~ be colTelated with ~pecific function~l ~ups~ For
example, the two pairs of two hydrogens on the succinate group in PAS-
4200 ~,ve rise to (triplet) resonances at about 2.53 and 2.92 ppm having
ls of 2.197 and 2.605 uni~s, respectiv~ly. Similarly, the intemal
20 ~ meffi lene groups o~ ~e butanedian~e ~esidue give ~ise to a broad
sonaoce at 1.5 p~m baving an integ~ated area of 16.034 units.
The~e ~e two succinate residues in ~e ~nd g~ups of the polyamide
derivatiYe: ~erefo~, the l~soDance at about 2.53 ppm and the one at 2.92
- .
~; ~` ppm each results from four hydrogens. The aver~ge area P~ponse ~ (2.197
~;,
2s ~ 2.605)/2 or2.401 unitsperfourhydrogens o~ each succinate. Similar~,
the two internal methylPne groups of the butanediamine residue m the
r~e~tir;g ùi~it con~ain four hydrogens. The obseIvation th~k the integrated
area of the latter resonance (16.034 units) is larger than that of the f~ur-
hydrogen ~esponse for either type of succinate hydrogen indicates that there
are muldple butanediamine residues within the repe~t units in the polymer.
We ean estimate the value of the multiple by ratioing the integrated areas:
16.iO34/2.401 or approximately 7. Thus, there are sevcn repeat groups iD
the polyamide. The molecular weight of the polyamide is the sum of the
-; molecular weights of each of the end groups (416.44 and 198.14,
,
~Wo g~/~22~ PCT~93/~1~70 '
~135880
respectively) and the multiple seven times the molecular weight of the re~eat
unit (7 x 504.57 or 3532). The sum is 4146.57 or about 4200 Da. This
value was also obtained inde~endently by end-group az~alysis of the
polyamide bisamLne precursor of PAS-4200 using fluorescamine.
s ~
SYnthesis of PAS-2400
Example 1 (a~ .
Polvcondensatipn of Ethylene ~1YCO1 biS(methOXYCar~OnY1methY1L~d
0 Ethylene~glycol bis(methoxycarbonylmethyl~ ether (E~:E3), which has
an ether group ~ to each ester group, was condensed with 1,4-
diaminobutane (:DAB) to p~uce polyamides. See Figure 1. Two poly-
condensa~ioll me~hods were us~: the solution method and t~e melt met~od.
In genelal, :the polycondensations were completed as follows. For
the ~, lED~ ~d D~ in the desired molar ratio we~e
dissolved: in met~anol, and the solution was heated at 3ûC for seventy two
hours or at 6~~ for twe~ty ~our hours. The solve~t was ev~porated and the
esidue~ was trea~d with acetone and re~eatedly evapo~ated to remoYe
: residual methanol. Tnturation of the ~esidue with acetone afforded a solid.
o ~ ~ In the melt method, a mixtur~ of ED~i and DAB was heated at 120C u~der
Yacuum wi~ magnetic sti~g to rem~ve me~hanoL A~ one to ~wo houn
the mixture ~vas dissolved m me~hanol. Tne sohltion was eivaporated ~o
~ ~ .
ess:and the residue was ~ ated with aceto~e to give polyani~de
product. ~ ~
Analysis ~f the reac~on mixtu~es by SEC confilmed tha~ polymerized
products were fo~m~l. TLC analysis (stationaIy phase: silica gel; eluellt: 2-
propanol I ~H40H / ~ 1 l 7:1:2, by volume~ of the pIoduct show~ three
spots having Rf valueis of 0. 1, 0.4, and 0.7, re ;pectively. ~e stmc~ureis of
: :the co~Tes~onding polyamides were assigned on the basis of reactiv;ty toward
30 ni~hydrin and base as x ,cll-diaminopolyamide (designated I in the figure),
amino-~-es~eIpolyamide ~designated II), and oc ,~-diesteIpolyamide
(designated III), ~espectively (~;igure 1). In addition, product ~ is
ninhydrin-negative while products I and ~ are ninhydlin-positive, indicatiDg
,~ . .
:~ .
WO 94l12220 PcT~uss3/1147~
2~ 3 5 a~
14-
the products has at least one primary amine g~up. Finally, products II and
m can be hydrolyzed with dilute aqu~,obs NaO~I, whereas I cannot,
indicating products II and III contaill at lç~st one ester g~oup.
The yield of these products d~jSends on the molar ratio of DAB to
s EDE. A molar ratio of one gives I as the major product. A molar ratio of
DAB/EDE greater than one gives polyamide II as the major product. In
contrast, m became the major product with a molar ratio of DAB/ED~ of
: less than one.
The results of polycondensation of ED~. and DAB are summanzed in
o Figure 2. The experimental data indicate that -amino, ~-ester polyamide
II having a molecular weight ~ of about 2,400 Dalton is best produced
by the solution method at 30C. ,~-Diamino-polyamide I havi~g a MW~
in the :range of 1,300 to 1,500 Dalton could be prepared either by the
solution or ~e melt method employing a DAB/liDE malar ~atio of 1.3 to
5 1.5.~ ~ ,c~Diester-polyamides m were ob~ained in good yield by ~he melt
method with ~equi~olar ~DE and DAB. Because DAB is a volatile
compound, ~AB is gIadually removed from the rea~on mixt~re when the
: melt:method is utilized~ leavillg ~DE in large excess. Collsequently m is
obtained as the major product.
o~ ~ amplel~
ConversiQnQ~ lvan~idem to_an actvate~l~li:~.
Crude diester m (Figure 1), obtai~ed by condensation of EDB and
DAB9 was hydrolyzed with dilute sodium hydroxide to the co~esponding di-
acid.~ ~ After hydrolysis, thç reaction mixture was treated with AG50W-X8
25 res~ (Bio~d) to remoYe sodium ion and by-products I and ~. The di-acid
- was obtaîned in a pure state as judged by TLC. The di-acid was trea~ed with
dicyc!ohexylcarbodi~ll~ide (DCC) and N-hydroxysuccinimide (NHS) ii~
chloroform to comert it to the co~e~onding polyamide bis(N-
hydruxysuccinimide este~) (designated PAS-2400).
Example 2
PolvmeIization of Hemo~lobin with PAS-240Q.
A typical polymerization of diaspirin cross-linked hemoglobin
3~ (designated DCI~Ib) with PAS-2400 was completed as follows. DCl~Hb
i~ ~
.i ,
Wo 94/122~0 213 5 8 8 0 PCT~S93/11470
. .
,~ - 15 -
was prepared aceordLng to ~he method described in U.S. Patent No.
,~ 5,128,452. A solution of DCLl~Ib in 0.1 M ~S of about pH 7 to 8 was
.~ deoxygenated by successive ~acuum / nitrogen cycles ~or one and a half
~ours at room temperat~lre.. P~S-2400 was dissolved in deoxygenated
s water, and the solution was added immediately to the DCLHb solution. Tbe
reac~ion n~ix~re was sti~red at ~oom tempera~ure under nitrogen9 and the
,
reaction was moDitored by size exclusion chImoatography usi~g TSK-
. (;4000SW b~d and $SK~G3000SW b~and columns coMected in series with
mobile phase consis~ing of 2-propranol/SOm~ phosphate buffer, pH 6.5
~ ~o (1:9, V/V)j delivered at a flow rate of 1 mLlminute de~ection at 280 nm.
.~ The latter method demonstrated that the polymerization was completed in
, ~ less than ~hirty minu~es and that polyme~ tion was ac~mpaI~ied by
I; d~on. The soluti~n was cooled to 5C and ~ solution of 1 M NAC (:N-
: ~ acetyl-L-c~rstoine~ (mola~ Iatio of NAC/Hb about 5:1) was added. The
lS solu~ion was: sti~ed at 5C under nitrogen ove~night and then dialyzed
ag~inst lact~ted Ringer's solution to give the final produc~. ~ixpe~imental
:''`! ;; ~ data are summ~ in Figures 3, 4, and 5. Note: ID the figures NHS-PA 6
,i ~ is an alter~ate designation ~or PAS-2400.
i~ ~ e da~ indicate ~e followirlg. I~e yield of oligomer is i~creased
!;1 ~ 20: ~with ~inc~easQ~g ~ s of PAS-2400 to DCI~Ib. SE~ :elution dmes of
., ~ D~I~ mollomer dec~ease with increasing molar ra~ios of PAS-2400,
indic ting that PAS-2400 decolates DCLEIb. Polymenza~don is fast; it was
~ ~ complete L~ less than:thi~ minutes. How~ver, ~mpe~dtive hydro~sis of
p;~ ~ the polymenzation agen~ is also fast. As ~e solution p~I is increased, higher
,J',f~ ~ yields of high molecuL~ weight polymers are ob~ed. For example, five
;:~ equivalents of PAS-240Q give 7%, 17%, anid gel, respectively, of high
~` ~ ~ : moleallas weight polym~rs at vah~es of p~I of 7.0, i7.5, a~d 8.0,
~ ~ respectiYely.
50 values: and n values of DCLHb polymenzed with PAS-2400 are
: 30 in the ~ ge of 29 to 33 mm Hg and 1.8 to 2.1, respectively. Pso is the
-oxygen partial pressure at which hemoglobin is half satu~ted w~e the "n"
- value is a measure of the cooperatiYity of oxygen binding. The P~o of
human hemoglobin in red blood cells is- about 28. Thus, the excellent
oxygeD-binding functi~n of DCLHb is maintained in these polymers. RP-
,.,. ~ ~ -
,
W094/12ZZ0 2~,3~ PCT/D593/1147,
- 16-
HPLC analysis ~Figure S) indicates`than both ~- and o~- chains are
modified. However9 ,~-chains app~èntiy are more extensively modified than
3 are ~-ehains.
Thus, PAS-2400 can be used to produce decoraled, polymerized
s DCLHb. The short reaction time (thirty minutes) is favorable for large-~cale
synthesis. Two to Pour equivalents of PAS-2400 at pH 7.0 are suitable for
polymerizatiorl. The hemoglobin maintains its biologic~ activity, i.e.
oxygen bindil~g and, as described below is no~unogenic.
10 Example 3
Methods for the Svnthesis of Longer Polvamides.
Longer polyamides are obtained if the lengths of the component acid
and amine are i~creased, i.e., polymerization with adipic acid (six ca~ons)
or 1 ,6-hex~nedi~e (six carbons) yi~lds longer polymers than does
~; ~ 15 ~ polymonzatio~ with succinic add (four carJoons) or 1,4-butanediamine (~ur
`~ ca~ons):. ~awever, in~eases in chain leDg~h using hydrocarbon components
',: ;~ : would reduce ~e aque~us solubility of the protein.
With this in mind, we synthesized polyamides ~rom ffiester EDE and
~ch of two longer diami~ies: ethylene glycol bis(3-aminopropyl) ether
20 ~ (EGBEi; MW 176) and diethylene glycol bis(3-aminopropyl) et~er (l:)GBE;
;~ 220). See Pi~re 6~ Ihe SEC re~e~tion times of each of the
. ~ polyamidès: suggested these pr~ducts had higher molecu~ weights, but ~e
'!` ~ :products were waxy and ~ad low melting points. Purification of ~ sllch
products by c~ystallization is ~xtremely difficult.
To mir~mize these shortcomings we combined three concepts to
~: . select approp~iate activated esters for the synthesis of longer polyamides.
! First, we identifi~d ~mpanents that are di-acids }i~ving B-ether lin~s; ~ese
di-acids are easily converted to activated diesters. Our initial di-acid o:f
hoice~ was diglycolic acid~ Second, we converted one end of this di-acid to
30~ an amide by r~ng ~wo equivalents of di-acid with one equivalent of
diamine; this genera~ed a neW and longer di-acid that we can use as a
component for longer polyamides. Our first di-acid of choice was 1,4-
:~
.. ~ ~; ~ (carboxyméthoxyacetamido) butane, whi~h we used as the activated me~hyl
,. ~ diester BMDAB (MW 348). Insertion of the methylene (hydrocarbon~
;~ ~
WO 94/1222~ - PCT/US93/11470
i
- 17-
groups reduced the flexibility of the molecule sufficiently to render it a
crystalline solid and retention of the ether link preserved the solubili~r in
water~ Third, we inc~eased the length of the diamine cornpoDent L~ a way
that will maintain water solubility; thus, we used ethylene glycol bis(3-
5 an~inopropyl) ether (~3GBE) and diethylene glycol bis(3~aminopropyl) ether(DGBE) as the diamine components in polyamide synt~esis.
;l ~he activated diester building block, B~DAI~, was obtained ~ two
steps (Figure 7). DA13 (1,4-di~obutane) was allowed to ~eact with two
equivalents of glycolic anhydride in N,N~imethyl~onnamide (D~;) to give
o ~ ~ost quantitative yield of B(:DAB [1,4-bis(carboxymethoxyacetamido)
butane~. The latter was esterified in methanol in the plesence of aqueous
: ~Cl or XCl in dioxane solution. The advantage of using ~ICl in soludon is
the ease of ca~ying out the ~ction, e~pecially in a large scale syn~hesis,
and the observation ~at an exac~ amount of HCl can be emplvyed to av~id
the fo~mah~ clf by-products. Gaseous HCl was t~i~, b~t a by-p~uct was
` ~ de~ected in the p~dYa mi~.
` ~ In :;contlast to ~e polyconde~sation of ~D~ and DAB by the solution
.~ ~ method, which gives the a-methylester-~amîn~olyamide as the major
~ ~ product when a mol~ ~tio of ~)E to DAB of 1 was used, the
!~ ~ 20: ~polycondensation of equunolar quanti~s of BMDAB and EC;BE or DGBE
or~ of molar Iatios of BMDAB to DGBE of g~eater than 1 ~Fi~re 8) gave
~ ::~ containing substantial amsunts of ~ee prod~cts: an cY-ester-~
p, ~ amine~(reactive~to ninhydlin; hydrolyzed by base); an a,~e (~ctive
!'' '~ t~;riilhydrin); ~and a diester (unreactive to ni~hydrin). IJnfoItunatoly, the
presence of large amountis of other products made the purifilcatio~ ~ a
desired produc~ tedious. HoweYer, we ~ound that the use of an ex~ss of
diamine (e.g., a molar ratio of diamine to BMD~B of 1.3) gave the a,~
: ~ bisamine polyamide as the major product eontaining only ve~y small` ~ mounts of the molloamine by-prQduct. This latter proc~ure is there~re
: 30 prefe~cd tv produce the polyamide backbone of the polyme~ ion ~eagents.
~ '
;:
Wo 94/~220 2 ~ 3 S ~ ~ Q i PCT~s93lll47o~-
- 18 -
. .
Example 4 .`'
(:onversion to Activated P.ol~t~ieIization A~ents: Polvamide bis(N-
hvdroxvsuccinimide) ester.
The first attempt to synthesi~e polyarnide bis(N-hydroxysuccinimide)
s ester (designated PA5-3070) was a three step synthesis from BMDAB and
EGBE (Figure 9~. First, EGBEl and BMDAB in a molar ratio of 1.3 to 1.0
were condensed by the solution method using methanol as solvent at 65C
for 24 hours to give a slightly orange solution. The product could be
decoloIized by adding decolorizing charcoal (Norit~ A) to ~he solution,
o filterLng, and evaporatixlg to dryness. A white product (:E;igure 9, 2a~
having a MW of 2700, ~vas isolated by crystallizatioll fr~m methanol-
acetone. The product was not stable and tumed yellow dunng storage.
S~nd, conversion of '~he white product to the colTesponding bis(2-carboxy-
~: ~ ethylcarbo~yl) polyamide ~E;igure 9, 3a) was completed by reactioll of (2a)
15 with SUI~DiC anllydride i~ DMF (a small amoll~t of methanol was added to
:: enhance to the solubility of 2a). The reaction produced a yellow product
mixture containing bis(2~ boxy-ethylcaIbonyl)polyamide (3a) as the major
p~oduct and two minor by-products: the methyl ester of (2a) and an un-
~0WII by-product containing a free amino group as indic~ted by TI,C.
There~ore, the m~ was treated with sodium hydr~xide to coslvert the
methyl ester to bii(2-carboxyethylc~onyl)polyamide, (3a) and theII stiITed
with: cation exchange resin (AGSOW-X8) to absorb polyamide amine by-
product. Af~r removal of the res~n by fi1tration, ~he filt~ate, which
c ontained a single product as i~dicated by II,C, was concen~}ated. Pure
1 2s p~oduct bis(2-carboxyethyl~bonyl)polyamide (3a) ~vas obtained by
c~ystaliz~tion from methanol/acetone. Third, conversion ~ the pure
: ` product `to ~he activate~ d~ster (4a) (désignated` PAS-3070) was
accomplished by treatment with ~-hydroxysuccinimide in the presence of
dicyclohexylcarbodiimide (DCC) in DMF. The polyam;de bîs-succil~imide
ester was soluble in water but the coupling groups were slowly hydrolyzed~
Therefore when dîssolved in water the actîvated polymeIi~ation agent was
~-~ used without delay.
~: The synthesîs descrîbed above has several drawbacks~ For example,
~:~ the isolated white pr~duct, (2a) is not stable; it îs oxîdLzed during storage to
.
~WC~ 94112220 21 3 ~ 8 8 ~ ; PCT/US93/11470
- 19-
an unknown yellow product which could not be removed ~eadily by
cIystallization~ Furth~rmoré, recrystallization of bis(2-carboxyethyl-
carbonyl) polyamide (3a) ~n methanol/acetone converts some of the di-acid
to t~e co~Tes~onding methyl ester(s). To avoid these d~wbacks, our
5 .pre~eIIed synthetic strategy is as follows. First, st~ps 1 and 2 (Figure 9)
were canied out as an integ~ted process in which product 3 in Figure 9,
obt~ed f~om Norit A treatment, is not isolated but is allowed to react
Lmmediately with succimc anhydride to mask the amino gTOUpS which tend
to be oxi~ to colored pr~duct. Such a "one-pot" synthesis increased the
10 yield o~ 3 in Figure 9, because all crude 2 is used for the second step instead
of the sO-60æ of isolated product 2 that was converted in the method
described above. In addition, the use of methanol as cIystallization solvent
` for 3~ was excluded to avoid ~he fonnation of methyl es~er of 3.: l~ç 5
~; ~y.nthesis of P~S-4~00.
PAS-4200 (Pigure 9, 4b) was pr~pared using the integlated approach
, ::` above.
~: : 20 ~a~
; ~ Ceion to_Activated Polvmenzation A~ents: Polvam e
Synthesis of:polyamide bis(maleimidopr~pionate) (desig~a~d PAM-
4080) was completed as a "~ne-pot" tw~st~p synthesis w~ich is summarized
in~Figule 10. Crude~polyamide bisamine (~;igure 1~,-2B) was obtained by
heatiDg BMDAB and DGBE in refluxing methanol for 24 hours f~llowed by
decolorizing ~ith N~it A and was immediately treated with N-
hydroxysucGinimid~3 maleimidopr~pionate (S~P, Flgure lû, 5) to give 6b.-~
The firs~ ~*empt to ca~y ~ut the latter st~p by mixing 2b and 5 in a molar
o ra~ of 1:2 in chlorofonn in the presence of triethylamine gave a higher
~ molecular weight product. Since SMP is a bifunctional cross-l}nking
.~ eagent, it could polymelize 2b under these conditions. Increasing the
: SMP/2b molar ratio to 4.5 and- the slow addition of 2b in chloroform
c ontaining t~iethylamine to a solution of SMP in chlo~ofo~n eliminated the
35 polymelization of 2b by SMP. Ihus, cJude product 6b, obtained by this
- procedure, was mixed witl1 cation-exchange resin (AGSOW-X8) to remo~e
! :
:`
Wo ~4112220 PcT/uss3/li47~?
æ~3S~
- 20 - ~
unreact~ polyamide amine and the Ry~i~d polyamide polymezization ageslt
6b (designated PAM-4080) was obtained by crystallization of crude product
from methanol/acetone. In contract to PAS derivatives~ PAM de~ivatives
are stable in water.
~ymç~z~ion of DCLHb with Polvarnide Polyme~ization Rea~ents.
A t~pical polymerization of DCLHb with PAS denvatives of the type
:~ ~ descr~bed irl ~amples 4 and 5~above or PAM deriva~ives of the type
lo described in l~xample 6 above was completed as follows. A solution of
~: : DC~Ib (10 g/dL ior PAS and 20 g/dL for PAM~ was deoxygenated by
successive:~racuum/::~itrogen cycles for 1.5 hours at room temperature.
ramide:~eagent in deoxygenated~water was added immedia~ely to the
D(~ :solution.~ :: The~;reaction ~nixture was stiIT~d at room temperatu~e:
15:~ u~der~ni~g~and:~the caurse~of the Icacti~ was followed by S~C.:
Pol~enza on was ~completed~ wi~n~2 to 3 hours for PAS: d~riva~ives and
ovemight: f~r PA~derivatives. The reac~ion mi~bire was ~oled to 5C;
thè~olutio~ ;was~:adjus1:e~d~ to: 8.0~wi~h 1 mo1ar HE~ pH 9.0~a~d a
solution~of I~M ~N~ L-cy:st~e, pH;9.0 (molar ratio N~C/I:)CI~ of:
20 ~ 5) was~ded.:~The~solution was sti~Ted at SC under iitrogen overDight~a~d
n ~d ~against~ ated Ri~er's solution to :gi~e the final pr~duct.
ExpGrimentalresu~1ts~esummaTized~Figuresllto18 ~ :
~ o1yme~i~atiorl o~ DCI~b w~th the ac~rated ester PAS denv~ es
may~ summar follows. ~a) The de~ee of polymenzation and the~
` y~:of :oligomo~s ~incre;lsed with ~e m~ atio ~of PAS used.~ (b) ~ :
ConcuITent wi~h~increases in ~e molar Iatio of PAS used, ~e e11ltion time of
DCl~ monomer~deo~ased, suggesting that d~a~on of DCI~b,by P~S,
~.. ~, ,; . ~ ,
is occumng. (c~ Polyme~on was fast; it was comp1ete within 2 to 3
hours.~ (d~ The~ SEC~profiles: of polymeric p~oduct obtained by~employing
:30 ~f1vé :equiivalents of PAS-3070 and t~ree equivalents of P~S-4200 ar~ very
si~nil~ ~This also~ demonstrates that longer reagents facilitate polymerization
of DCL~lb. (e) Pour:equivalents of PAS-3û70 at d 2.5 eqjuivalents of PAS-
4200 gave the bes~ product mixnlres under these expenmental condi~ions.
PAS derivat1ives do not affect the P50 values of DCLHb: the Pso values
~-;. WO 94/12220 213 5 8 8 0 PCT/US93/11470
- 21 -
of polymenzed product arë'in the ~nge of 29 to 36 mm Hg. (g) RP-~IPLC
analyses (lFigure 13) indicate that bath B and cYa chains are modified by
PAS, but acY chains to a lesser extent than B chains.
DCLHb polymeIization by PAM derivatives may also be
s summanzed. (a) As was ~ue of the PAS derivatives, the yield of oligomer
increased with the number of molar equivalents of PAM used. (b) l~lutivn
times of the mollomer decreased with the number of molar equivalents of
PAM used; thus, decoration of I)CLHb by PAM is lLkely. (c) Two
equivalents of PAM give the best product mixtures. (d) ~-HPLC profiles
o (Pigure 15) suggest tbat reagent reacted specifically. A specific B' peak,
which could be a modifled B peak, was detected at all ratios of PAM test~d.
Specific reaction with t~e~,B subunits was also supported by t~e decrease in
fit~le thiol residues. Reagent P~M is expected to b~d ~pecifically to
cysteine-B93 resi~ues, ~d about 65% and 90% of thiol groups are modified
when I and 2 equivalents of P~M are used, re~pectiv~ly. (e) T~e binding of
PAM ~o the cysteine residue results in a decrease in P50 values of the
; polyme~ed products to 18 to 20 mm Hg. (f) acY-Chains are also modified,
but much less extensi~fely than the ,B chains.
20;~ ~BIOLOGIC~ ING
e~ples 8 through 12 we quenched the polyamide P~-:4200 by
~ction ~with ~-acetyl-L cys~eine and tested a sterile7 non-pyrogenic solution
of the:~p~lyamide :(PM~080) in Ri~ger's lactate solutio~. 'rhe polyam~de
: con~ ation was 5g/dL of solution. The p~I of tlhe polyamide solution was
adjusted to physiologic v~lues. The osmolality of the solution was withi~ ~he
physiologic ~a~ge. The concentration of the polyamide was selected t~
exceed ipro~ected use levels~ by~ at least-an order ~fimagnitude,~
Example 8
0In Iq~posure of isolated mammalian cells. CCL 1 NCTC 929
clone of strain L cells, mouse connective tissue) were cultured aseptically in
sterile media until confluency. The L-929 cell concentration was adjusted ~o
about 1.3 x 105 cellslmL, and aliquots were transferred to wells of a tissue
` ~ culture plate. The plates were covered and incubated for approximately
3~twenty four hours. Ihen the culture medium was aspirated from each well
WO 94/12220 ' o PCT/US93/1147~
35~
- 22
~ r~`
and aliquots of the test article s.oiiution and dilutions havLng PAM-4080
concentrations of 2.5 and 1 g/dI~spectively, were added to duplicate wells
of the prepared pla~es. After incubation of the plates for approximately forty
eight hours, the wells were stained with 2% crystal violet stain. The toxicity
5 was rated on a scale from 0 to 4+, where a rating of 0 corresponded to the
~ ~presence of disc ete intrac~toplasmic gIanules and the absence of cell lysis
;~ Iand a ~ating of 4+ corresponded to nearly complete destruction of the cell
layers. At the highest concentration, a moderate toxicity rating of 2+
applied. At the two lower concentrations, a toxicity rating of 0 applied, i.e.,
10 the polyamide caused no adverse biological response.
;~ ~No toxicity was observed at the lower doses and moderate toxicit~!
was observed at the }lighe~ dose. Acco~ingly, the polyamides of the
present invention ~are expected to be nontoxic when administered as
con~ugates o~ thc~apeutically~-usefui suhstrates.
Acute toxicit~ testing in rodents. Doses of S00 or 1500 mg of
quonched PAM~080/kg~body weight wer~ infused at a rate of 1 mL/kgl~.
to t}~e~taiI vein~of male, Splague-Dawley rats. Each test g~up csnsisted of
20;~ six~ s;~six undosed animalsrserved as contr~ls. ~ ~imals ~vere
moni~ored fo~ se~y two~ hours ~for signs of overt ~xlcity; none~ were
ol~ed. The~animals were saclificed. No evidence of toxicity was seen at
tho~ e~of necropsy. ~ Tissues ~rom the liveF, hdney, lung weIe subjected to
bi ~ical a lalysis. No adverse histopa~ology $mdings were noted.
J ~ Exa~mp!e 10 : ;'`f
Compatibil3~ ~lth human e~hro~es. To determine the
biocompatibility ~ of PAM 4080 ~ith human erythrocytes, the~ stock~
polyamide solution was~diluted five-fold with lactated Ringer's solution.~ A
30~ volume of this`pr~paraDon was n~ixed with an e~ual volume of ~heparil~ized
human~ blood, vortexed gently, and placed in an incubator (37C) ove~ght.
Afier arl incubation pe}iod of 16 hours, a 100-~L aliquot of the supenlatant
was removed from the top of the test sample; care was taken Dot to disturb
he sedimented red cells below. The aliquot was mixed with 5000 ~L of
.j. WO 94/12220 213 5 ~ 81~ PCT/U~93/11470
SEC mobile phase, filtered through a 0.2 ~m pore-s~e fllter and injecte~ on
a SuperoseTM 12 column for S:E~C analysis for native hemoglobin. The
expemnental data indicated that less than 0.1% hemolysis had occu~ed.
This amount of hemolysis was considered negligible.
s
~xample 1 1
Com~atibil;tv with huma~i monocvtes. The potential of PAM-4080
for causing white blood cell activation was evaluated. The stock polyamide
solution was dilute~ five-fold with lactated Ringer's solution. A volume of
lo this preparation was mixed with an equal volume of penpheral blood
mononucle~r celI prepa~ation and vortexed gently. An aliquot of this test
prepaIation was remoYed and diluted with trypan blue. Toxicity was
det~ined by microscopic detection of cells that could no lo~ger exclude
: ~ ; ` the dye. Percent viability was measured by a Iatio of live/dead cells.
5: PAM~080 caused no decrease in cell viability. The remain~g test
p~eparat~on: was plac~ an ~cubator (37C) overnight. After an
incubation :peliod of 16 hours, cytokines were analyzed by pipet~ing an
aliquot of ~he sample into microtiter wells and quantitation by EL~SA. The
concentrations ~of Tumor Necrosis Factor (IN~a), Interleulcitl-lB and
20 ~: ~terleukin-6 detem~ined were no dif~ereDt from those ~ound ~y exposure OI
human monocytes to lactated Ri~ger's solution. Thus, PAM-408û is
c ompatible with human monocytes.
amp~o 12
Compatib_t,~f PA-DCL~Ib with human monoGvtes. Ihe potential
of polyamide decorated and polymerized DCLHb ~PA-DCLEIb) to i~duce
~::: ' cytokine producdon by human monocytes was ev~luated. liactat~ Ringer's
solutio~l was used as the control a~ticle. The test articles were seven
different preparations of PA-DCLHb in lactated Ringer's solution. Test and
30 ~ cQntrol solutions were made by mix~g a volume of each test and control
: article with an ~ual volume of peripheral blood mononuclear cell
preparation. A~ter incubation of each resulting test and control solution at
37C for about 16 hours, an aliquot of each sample was transfesred into
separate wells of microtiter plates and the concentra~ions of Tumor Necrosi~
. ~ ~
~ -
~: :
WO 94/~æ0 ~a~3Ss~ PCT/US93/1147~
.
~4
,. i:`
Pactor (TNFcY), Interleukin~ ~d` Interleukin-6 were quantitated by
BllSA. The concent~tions of e~iich cytokine deteImined are shown in the
Table below. The expelimental data indicate that induction of ~NFa~, IL~1,
and IL-6 are low and in some cases comparable to Ringers. In summar~y,
5 PA-DCLHb appears to be very compatible with human monocytes.
~ -- , . =,
. T~ST ~IICLE CYTO~E CONCENTRATION, ng!mL
TNF I l~ I IL-6
I _ _ .__, ,, _ ~ :.. , , _ _
Lactated Ringer's Solution 0.044 * 0.023 0.006 * 0.005 <
(Control) De~ection
. Limit
I _ . _ (DL)
1~1~ 0.154 ~t 0.027 0._80 :t 0.040 _DL
;~ ~b (Pr~ ation 2) 0.249 :~: 0.081 0.073 i 0.028 < DL
~5:~ 0.161 :~: 0.0 5_ 0.054 :: 0.011 < DL
~1~ 0.1~3 :t 0.043 0.058 :1: 0.012< DL
û.13g + 0.û27 0.049 :t O 9 . DL
¦ ~DC~lb ~ti~ 0_ 0.159 :: 0.012 0.042 :t 0.0~6 c DL
DC~ 0.144 :t 0.050 0.048 :~: 0.Q28< DL
lo ~ Cytoldne Induction bv PAS-DCI;Hb.
Samples of six P~-DCL~Ib p~oduct mixtures were submitted for
okine testing. ~he products sfelectfed were pre~p~ by polyme~ization of
3~ /dL; D~ O.lM E13PES buffer at ~f~I 7Ø l~ach samfple was diluted
to a DC I~b coDxntration of about 1 g/dL and passed t~rough an END X~
5~ ~end~toxin-remov~ng filter. The fil~ate was tested for cytokine induction
usi~g the method descnbed in Ex~unyle 12. ~ ~ ~
~ ~ .
:: ~
:~
~ ~ .
f
~f
~f
Wo 94/12220 ~13 5 8 8 0 PCT/US93/1~470
- 25 -
~ . , _ . ~____~_
~T SAMPLE ~ , ng/mL IL~ , ng/mL IL-6, ng/mL
Lactated Rin~er's Solution 0.044 0.006 _ _0.044
PAS-DCLHb (3-1) 0.154 0.080 0.008 ¦¦
, _ __ __
PAS-DCL~b (4~ 0.249 _ 0.073 ~
P.4S-DCL~Ib (5-1~ 0.161 0.054 0.001
~ _
PAS-DCLHb (6-1) 0173 0.058 0.017
~ , . _ _ .
PAS-DCLHb (7:1) 0.139 0.049 0.036
PAS-l:~CLHb (8:1) 0.159 _ 0.042 0.034
PAS-DCLHb ~10 1~ 0144 0.048 0.063
~ , . _ ~ , . ..
PAS-DCLHb (3:1) ;s the least decorated and polymerized product mixture,
whereas PAS-I:~CLHb (10:1) is the most exte~sively decorated and
5 polyme~ized product m~cture. The exte~t of decoration ~d polyme~zation
increases with the molar ratio of PAS employed. HoweYer, the PAS-~b
products, ~spective of the extent of decoration or polymc~zation, all yield
low ~-tx and IL-l~B re~ponses. None of the samples show an IL-6
response.
~ : 10
~ ~ ~ `As wil~ be readily appreciated, numerous Yanations and combinations
;`:;: ~ of the features set fort~ above can be utilized without departing from the
:~; presen~ invention as set fo~th iD the r,laimis. All such va~iations are initended
: . .
to be included in the scope of t~e following claims.
~ .
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