Note: Descriptions are shown in the official language in which they were submitted.
79268
i:
IMMUNOLOGICAL MATERIALS
. The present invention encompasses a cytotoxic
agent comprising an irnmunoglobulin specific for antigens
on the surface of cells to be killed having 1-10 polymer
carrier molecules covalently bound thereto, said polymer
carrier having about 5-500 molecules of a cytotoxic drug
covalently bound thereto and said polymer carrier having
a molecular weight of 5000-500,000 and free carboxyl,
amino or cycloimidocarbonate groups for convalently bond-
ing, said cytotoxic crug also having amino or carboxylgroups available for covalent bonding.
Cytotoxic agents of the present invention are
represented by the formula
r~ Ig ~ . :
~ m ~
wherein Ig represents an immunoglobulin specific to antigens
15 on the surface of cells to be killed; the polymeric carrier :.
has a molecular weight of 5000-500,000 and is selected from
the group of polymers comprising:
a) dextran activated with cyanogen brornide to
form cycloimidocarbonate and functionalized with -NH(CH2)XCO2H,
0 or -NH-(CH2)X-NH2, wherein x is 3-8,
b) aminoethylated dextran,
c) polyglutamic acid,
d) polyaspartlc acid,
e) polyarginine,
-2-
` 1~79Z68 ~
f) serum albumen,g) fibrinogen, .
h) y-globulin,
i) polylysine (solubilized by reaction with :
citraconic anhydride)~
~) copolymers of lyslne-phenylalanine, lysine-
tyrosine, and glutamic acid-tyrosine; .
CD represents a covalently bound cytotoxic drug selected
from the group comprising:
0 ~ p-[N,N-bis(2-chloroethyl)]phenylenediamine, `
P-[bis(2-chloroethyl)amino]phenylalanine,
4-p-~bis(2-chloroethyl)amino]phenyl butyric acid,
2-amino-N-[p-bis(2-chloroethyl~amino~phenyl-3-
hydroxypropionamide,
2-amino-N-[p-bis(2-chloroethyl)amine~phenyl-3-
hydroxy-2-hydroxymethyl propionamide,
N-{p~[(2,4-diamino-6-pteridinyl)methyl]methylamino~-
benzyl~-L-glutamic acid,
cytosine arabinoside, cytosine arabinoside . .
20 monophosphate, :
o-[N,N-bis(2-chloropropyl)]carbamoylglycollic acid,
daunomycin, doxorubicin, and diphtheria toxin;
Bl represents a covalent bond between Ig and polymer carrier
of the formula
-N-C, -C-N, -N=CH~CH~3CH=N-;
B2 represents a covalent bond between the polymer carr~er
and CD of the formula . .
" l~)'~9Z6~
H H
- N C - , - C~rN - , - N - ;
Il 11 1 . .
O O H
m - 5-500; `
n = 1-10.
Polymer carriers of the present invention have a
molecular weight range of between 5000 and 500,000 and are
functionalized so as to react with both cytotoxic drugs and`
amino or carboxylic acid groups of immunoglobulins. Suitable
polymer carrier and functional binding groups are polypeptides
having free amino or carboxyl group for bonding such as: poly-
glutamic acid (carboxyl), polylysine (amino), polyethylene-
imine(amino), polyaspartic acid (carboxyl), polyarginine (amino);
copolymers such as:
glutamic acid-tyrosine (carboxyl)
lysine-phenylalanine(amino)
lysine-tyrosine(amino);
polypeptidyl proteins and multichain polyamino acids (carboxyl
and amino) as described by Sela et al., Biochem J. 85 223 (1962);
proteins such as:
human serum albumin)
human fibrinogen ) carboxyl and amino
human y-globulin
Also suitable are functionalized dextrans such as aminoethylated
dextran as described by Harding, Am. N.Y. Acid. Sci. 186, 270
(1971) (amino).
Dextran activated with cyanogen (amino or carboxyl)
bromide and functionalized with
H2N ~ (CH2)x- NH2 or H2N - (CH2)x - CO2H to
--4--
~ 1~'7~Z68
form dextrans represented by: -
H H
-N - (CH2)x-NH2 or -N - (CH2)X - C02H
wherein x is 3-8, preferably 6. The cyanogen bromide forms
a reactive cycloimidocarbonate
N H
C\ ' :
f
- CH - CH -
when reacted with dextran.
It is generally preferred to have only 1-10 polymer
carrier molecules covalently linked to the Ig so as not to
disturb the antibody activity of the Ig. In this manner
the immunoglobulin specifically directs or homes large .
volumes of cytotoxic drug to the surface of the cell to be
killed.
Cytotoxic drugs useful for practicing the present
inVentiQn are as follows: -
~CH2--CH2--Cl . ~
H2~ ~ \ CH -CH - Cl
p-[N,N - bis(2-chloroethyl)]phenylenediamine
CD(a)
1~)79Z68
IH2 ~ ~ CH2- CH2- Cl
H02C -CH -CH2~\ ~ ~ N~
~ CH2- CH2 - Cl
p - [bis(2-chloroethyl)amino]phenylalanine
CD(b)
- ~ ~ CH2- CH2- Cl
. H02C--(CH2)3~ CH2--CH2--Cl
4-[p-[bis(2~chloroethyl)amino]phenyl]butyric acid
CD(c)
OI H
CH2 H
I I ~ ~ CH2 -CH2 -Cl
H2N- ~ N ~ ~ CH2 -CH2 -Cl
2-amino-N-[p-bist2-chloroethyl)amino~phenyl-3-
- hydroxypr~pionamide
CD(d)
~ 79 2 6 8
IOH
CH2 H ~_N~
1H
:
2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-
hydroxy-2-hydroxymethylpropionamide
CD~e) ~ -
C02H
NH2 ",
2 ~ N
IH2 1~ ~ fH3 ~ H2
CN- NN- C - ~ N -CH2 ~ ~ /
N-{p{[(2,4-diamino-6-pteridinyl~methyl]methylamino}-
benzyl}-L-glutamic acid.
CD(~)
(methotrexate)
N 3
O N
~ CH2- OH
1~ .
H
cytosine arabinosi.de or monophosphate
CD(g)
-7-
.
` 1~7~268
The above are preferred agents against neoplastic
disease. 5-Fluorodeoxyuridine, o-(N,N-bis-2-chloropropyl)-
carbamoylglycollic acid, daunomycin, doxorubicin, and dipth-
eria toxin are likewise useful. It is, however, to be recog-
nized that other cytotoxic drugs with suitable functional
groups can be bound to polymer carriers which, in turn, are
bound to an immunoglobulin. The latter serving as a means of
directing the drug to the cell to be killed.
The bond between the cytotoxic drug and the polymer
carrier should be such that the cytotoxic drug is cleaved
enzymatically on the target cell surface. The peptide bond
is especially preferred-since proteases are on cell surfaces
and therefore the cytotoxic drug which is peptide bound to a
polymer carrier which in turn is bound to Ig can be select-
ively delivered to the surface of the cell to be killed and
then released to provide a high concentration of cytotoxic
drug at the target cell.
Structures of immunoglobulins are remarkably similar,
Science 189, 1075 (1975), in that gross structural features,
and amino acid content varies only slightly from one immuno-
globulin to another.
Immunoglobulins specific to antigens on the surface
of cells to be killed and techniques for their production are
known. Some representative immunoglobulins are: -
(a) goat anti-CEA Ig from human colonic adenocarcinoma,
Hsu-Fu Chao et al., Res. Comm. in Chem., Path, and Pharmacol
9, 749 (1974).
(b) Ig from rabbit anti-acute lymphoblas~ic leukemia
serum, Greaves et al., Clin. Immunol. and Immunopath. 4, 67
(1975).
'',,
-8- -
':
, , .- . . . . . . . .: .
9Z6~
(c) Ig from various primate anti-leukemia antisera,
anti-acute lymphoblastic leukemia, acute myleoblastic leukemia,
chronic lymphoblastic leukemia, and chronic granulocytic
leukemia, all as described by Mohanakumar, et al., J. Nat.
Cancer Inst. 52, 1435 (1974).
(d) human anti-human meningioma Ig., Winters and Rich,
Int. J. Cancer 15, 815 (1975).
(e) rabbit anti-human prostate tumour Ig, Moncure ~-
et al., Cancer Chemotherapy Reports 59, 105 (1975).
(f) rabbit anti-human chorionic gonadotropin serum
(g) goat anti-human lymphoid cell Ig.
(h) rabbit anti-mouse (EL4) lymphoid cell Ig.
(i) Ig against lymphocyte surface antigens for
example Ia antigens. Staines et al., Tissue Antigens 393
(1975).
(j) Ig against surface antigens on antibody pro-
ducing cells.
(k) Ig against fungal cell surface antigens.
Fukazawa et al., J. Bact. 95 754 (1968).
(l) Ig against Bacterial cell surface~antigens.
(m) Ig against antigen on the surface of cells
involved in inflammatory response.
Polymer carriers are covalently bound to the above
set out immunoglobulins by way of free carboxyl or amino
groups on the immunoglobulin. Water soluble carbodiimides
such as l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide con-
veniently forms a peptide bond between a carboxyl group on the
immunoglobulin and an amino group on a polymer carrier or
between an amino group on the immunoglobulin and a carboxyl
containing polymer carrier.
,
~7926~3
Alternatively immunoglobulins (Ig) are covalently linked
to polymer carriers having free amino groups by condensation
with glutaraldehyde, forming a diimide linkage as shown in the
following scheme.
Ig-NH2 + H2N polymer carrier
~ H - C - (CH2)3-C- H
Ig -N= CH- (CH2)3- CH-N - polymer carrier
Some preferred embodiments of the present invention
are set out in the formula B:
_ Ig -I
N _ .
ICH `
'f 2)3
N
( f 2)6 ::
~ ~ m ::
_ n :
B ¦
-10-
~7g268 :
wherein m is 5-100, n is 1-10 and dextran molecular weight,
500-500,000, prererably 5000-50,000 and most preferably
.
15,000-20,000, activated by cyanogen bromide and function
alized with 1,6-diaminohexane. 17,000 Is a very desirable
molecular weight and H2N -(CH2)6 - NH2 can be replaced with
_ (CH2)X, x = 3-8.
In a similar manner cytotoxic drugs, CD(b), CD(d), .
and CD(e) are bound to dextran as polymer carrier and m, n,
x and molecular weight variables are the same as in B.
. C represents another preferred embodiment:
- ~ - H
_1. _
0-1
CH2 Pol ~ tamis~id
O CH2 H O H .
_ -Cl~ - CH-ICl- N - -fH- C- ~ -CH - N - l
- I L CH2 ~ IH2 \ ~ l
---C~ =~ U
. CIH2 . IH2 /
~ \ IH2 CH ~ ¦
wherein m is 5-100, n is 1-10, the polymer carrier is
pol.yglutamic acid havin~ a molecular weight of 5,000-500,000,
preferably 5,000-100,000, and most preferabl~ about 35,000.
~ .
-11-
- 1~79Z68
or C' using CD(b) is preferred:
Ig ~
: 7 - H - _
.~ O-CI
., CIH2 , . .
¦ ~ C -C -N - CH- Q ~ CH c ~
H - CH2 H CH2 H
¦ L~ . CO2~ 1 o \ ~ ¦
clç`F/
n : ~
. .
C ' ~:. ,. .. ::
wherein m, n and molecular weigh~ of tne polyglutamic acid ::
is as described above in C.
In a similar manner cytotoxic dru~s (d) and (e)
(C" and C' " ) are bound to pol~glutamic acid wherein the
molecular weight parameters of the polymer carrier, m, and
n, are as earlier defined in C. The peptide linkages are
-12-
. ~ 1~)~926~
preferably achieved by use of l-ethyl-3-(3-dimethylamino-
propyl)carbodiimide. Generally 10-100 GD are bound to
: each Ig by way of the polymer carrier.
.
¦-- Ig
.~
. Nl- H
O=C
' CH2
. _ I .
CH2 H . O O
HN - H- C - N -CH- C -N - CH- C- N I
¦ O CH2 H fH2 H I
¦ CH2 fH,2.
Il: L C02H Cl = O l
/ H - N ~ : :
H
. CIH2 ICH2 n
: IH2 IH2 . . . . .
', ''
'
.
'~,
.
13
1~79Z68
-- N--H
O=C
CH2
.
--HN--CH--C--N--CH--C--N--CH--C--N '
11 1 1 1 1 1 ' ..
O 7H2 H ICH2 H I
CH2 ICH2 1 ~ . ~ :
.~ _ CO7H C--O
~UOCH2 G CH2--OH
. ~N
' ' CH2 7H2 .- ~
fH2 ~H2 ' . '
. Cl 1 n ~ :
_ ' - ' ,_ ' ' .
C'''
~;.,.
--14--
''
... . ..
` ` 1~7926~3 ~
;~ D represents another preferred embodiment
~ .
Ig _
' T- CH
1 b -
h"' ' ' ,;. ~H. H 0 / C02H
., .~. ~ N - - CH = C
~ CH2 lH2 CH3
;~ CH2 - lH2 l
H IH2 I fH? I .
_ - C N- CH -C -N - CH - C -N - CH ~ r
.' . l .
~/ CU2--CH2--CHz ~
' CH2--CH2-- ¦
. . .
D
.. . .
wherein m is 5-100, n is 1-10~ and the molecular weight of ,
the polylysine polymer carrier is 5,000-500,000, preferably .
5000-100,000. The cytotoxic drug (b) or (f) may be used
in place of 4-{p-[ bis(2-chloroethyl)amino]phenyl}butyric
acid. In addition a preferred embodiment is obtained fixing
the parameters in formula D as shown and replacing polyl~ysine
with polyethylenimine of the above set out molecular weight
ranr;e with molecular weight 20,000 being especially preferred.
~ree amino groups on the polymer carrier can be reacted
. .
1~79268
further with citraconic acid anhydride to function the
amine and aid in solubility of the cytotoxic agent
Table I illustrates preferred embodiment of the :
present invention.
TABLE 1
~" ' '
_ 1 B2 Polymer Carrier CD
Polyglutamic acid a,b 7 d,e :
_N-- C-- --C--N--
0
.,.:
H H
-C-N - - N- C - Polylysine b,c,f -
M.W. 5000-100, 000
O O solabilized by reaction
or with citraconic anhydride ::
-N=CH--(CH2) 3 CH=N
.. . ..
H
-C-l~-- '- ::
Il . ' .
O .,',:
or H
'~N=CH - (CH2) 3 CH=N - - C-C - Polyethyleneimine b,c,f
¦¦ M.W. 5,000-100,000
~H . H
C-N - -N- C - aminoethylaled dextran b,c,f -
O O
or
-N--CH- (CH2) 3 CH~N-
.
H : :
~ dextran activated with
-C- ~N - CNBr and functionalized b,c,f
ll ¦ with
O or H
H --N--(CH ) --NH
-N= CH--(CH2) 3 CH-N-- 2 2 6 2
~~~- dextran activated wlth
-N ~C- ~ N - CNBr and functionalized a,b,d,e
2 ( 2 ) 6 2
--16--
~ 1~79268
~-
The present invention also encompasses polymer carrierssuitable for binding to immunoglobulin having a molecular
weight between 5,000 and 500,000 and having 5-500 molecules of
cytotoxic drug covalently bound thereto. These embodiments of
the present invention are represented by the following formula:
L POLYMER CARRIER ¦ :~
,1, . :
( 72
\ CD~ m
where the terms polymer carrier, B2, CD, and m are as previously
defined.
A preferred embodiment of polymer carrier is polyglutamic
acid having a molecular weight of 5,000 - 100,000, most
preferably 35,000; B2 is - c- N - ;
O
CD represents p-lN,M-bis(2-chloroethyl)]phenylenediamine,
p-lbisl2-chloroethyl)amino]phenylalanine~ 2-amino-N-[p-bis~2-
chloroethyl)amino]phenyl-3-hydroxypropionamide, or 2-amino- :~
. N-[p-bis(2 chloroethyl)amino]phenyl-3-hydroxy-2-hydroxymethyl
propionamide;
m = 5-100.
.~,. . .
~7~268 :
Another preferred embodiment is dextran activated with : .
. .
cyanogen bromide and functionalized with 1,6-diaminohexane hav- ~
ing a molecular weight of 5,000 - 100,000, most preferably 17,000, ~.
B2 is ~N -;
CD represents p-[N,N-bis(2-chloroethyl)]phenylenediamine, p-[bis-
t2-chloroethyl)amino]phenylalanine, 2-amino-N-[p-bis(2-chloro- ~ :
ethyl)amino]phenyl-3-hydroxypropionamide, or: 2-amino-N-[p-bis(2--
chloroethyl)amino]phenyl-3-hydroxy-2-hydroxymethyl propiona~ide; ~;
and
m = 5-100.
Another preferred embodiment is aminoethylated dextran
having a molecular weight of 5,000-500,000 in which CD
represents p-~bis(2-chloroethyl)amino]phenylalanine or
4-~p-[bis(2-chloroethyl)amino]phenyl~butyric~acid,
.~ .
,~
H
. ,1 .
`IL .
Bl = ~ ; and
O .~ .:
20 m = 5-100
Another preferred embodiment is polylysine, molecular
weight of 5,000 - 100,000, reacted with citraconic acid anhydride
- in which
.
CD represents p-[bis(2-chloroethyl)amino]phenylalanine,
4-~p-[bis(2-chloroethyl)amino]phenyl~butyric acid, or
N-~p~[(2,4-diamino-6-pteridinyl)methyl]methylamimo~-benzyl~-L-
glutamic acid;
H
Bl = -N -C - ; and
O
m = 5-100. ~.
-18-
.
)7926i8
.;;. ^.~ .
,
,. .
Also preferred is polyethyleneimine having a
:
molecular weight Or 5,000-100,000, most prererably 20,000
t~ in which
, . . . .
CD represents p-[bis(2-chloroethyl)amino~phenylalanine,
4-{p-~bis(2-chloroethyl)amlno]phenyl}butyric acid, or
N-{p{[(2,4-diamino-6-pteridinyl)methyl]methylamino}-
,
; benzyl}-L-glutamic acid;
." ' ` .
r, Bl = -N- 1- ; and
O
m = 5-100.
,~, - . .
~10 Compounds of the present invention are preferably -
administered intravenously and they are especially advan-
tageous by reason o~ their low toxicity. For example,
'
'il p-[N,N-bis(2-chloroethyl)]phenylenediamine unbound has an
;, LDso f 5 compared to an LDso of 200 for that compound
'j~15 bound to polyglutamic acid. Dosages of compounds of the
present invention can be based on dosages of known cytotoxic
~ agents. For example (Melphalan) p-[~,N-bis(2-chloroethyl)-
; amino]-phenylalanine is recommended at a dose Or 6 mg daily
for 2-3 weeks whereas 1-20 times of this drug bound to a
~20 polymer carrier according to the present invention for a
like period of time would constitute an effective dose.
In a similar manner (chloroambacil) 4~{p-[bis(2-chloroethyl)-
amino]phenyl}butyric acid ls recommended at 0.1-0.2 mg/kg/day
for 3-6 weeks. That dosage can be increased up to 20 times
tqhen bound according to methods of the present invention.
Compounds of the present invention preferably administered
in a suitable manner by intraperitaneal in~ection Or a dose
,
.: --19--
~ 1~)79Z~i8
; having 0.1-2.0 m~/kg/day of cy~otoxic agent bound to the
polymer carrier and immunoglobulin to a warm blooded
animal. The utility of the present compound is further
disclosed in the hereinafter set forth examples.
The following examples are presented to further
illustrate the present invention. They should not be
construed as limiting it either in spirit or in scope.
In these examples quantities are indicated in parts by
weight unless parts by volume are specified, and tempera-
tures are indicated in degress Centigrade (C). The
relationship between parts by weight and parts by volume
is the same as that existing between grams and milliliters.
EXAMPLE 1
500 Mg of dextran (Molecular Weight 17,700) was
dissolved in 500 ml of distilled water and the pH was
ad~usted to 11.0 with sodium-hydroxide. 400 Mg of cyanogen
bromide in 1.6 ml of acetonitrile was added to the dextran
;~ solution dropwise with rapid stirring at 23C. This mix- -~
ture was stirred for 10 minutes with the pH maintained at
?O 11. 0 by addition of sodium hydroxide and then 100 mg of
diaminohexane dissolved in 2.5 ml of water was added with
stirring; the pH was lowered to 9.0 with hydrochloric acid
and stirring was continued for 5 minutes. This procedure
provided dextran having three hydroxyl groups functionalized
with
H
- N- (CH2)6 - NH2
To this functionalized dextran solution was added
dropwise a solution of 250 mg of p-[N,N-bis(2-chloroethyl)]-
phenylenediamine hydrochloride in 5 ml of ethanol containing
-20-
,, , . . - .
11:)7~2~i8
2% w/v hydrochloric acid and was then diluted with 20 ml of
60% aqueous propylene glycol containing 1.2% w/v potassium
hydrogen phosphate. During the addition the pH was allowed
to fall to 6.5 and was maintained at that level by the add-
ition of sodium hydroxide. The mixture was stirred for 15minutes at 23C, cooled to 4C, and transferred to membrane
ultra filtration apparatus for diafiltration with water.
Diafiltration was continued until the effluent was free of
uncombined p-[N,N-(bis(2-chloroethyl)]phenylenediamine.
The solution was concentrated by ultrafiltration to 1/5 the
reaction volume and then was freeze dried to provide dextran
having 3 hydroxyls functionalized with
O H
C N (CH2)6 NH2
and about 15 molecules of p-[N,N-(di-2-chloroethyl)]phenyl-
enediamine mustard bound as
H ~ /(CH2)2 Cl
-N ~ (CH2)2 Cl
This functionalized dextran polymer having N,N-[bis(2-
chloroethyl)]phenylenediamine covalently linked thereto was
then bound to immunoglobulin (Ig) specific to the cell to
be killed; i.e., rabbit antiserum against mouse lymphoma cells
(EL4) made specific by repeated absorption with normal mouse
spleen cells, Br. J. Cancer 30 297-304 (1974).
6 Ml of phosphate buffered saline pH 7.2 containing
180 mg of functionalized dextran polymer carrier was
-21-
'
1~79~68
mixed with 6 ml of the same buffer containing 18 mg of rabbit
anti-mouse EL4 ly~phoma Ig. To this solution ~Jas added 1.2
ml of 0.1% w/v glutaraldehyde in 12 x 0.1 ml increments over
a 5 minute time period. The mixture was rotated at 4C for
30 minutes, then centrifuged at 35,000 x g for 10 minutes
and the supernate discarded. The precipitate was resuspended
in 25 ml of 40~ ammonium sulfate and recentrifuged a& above.
The precipitate was redissolved in 4 ml of phosphate buffer-
ed saline and dialysed exhaustively to remove ammonium sulfate.
The resulting cytotoxic agent - polymer carrier -
Ig, was characterized as having 31 mg/ml Or protein Folin and
Cioculteau, J. Biol. Chem. 73 627 (1927), and the alkylating -
activity was determined by the method of Epstein et al. J.
Analyt. Chem. 27 1435-1439 (1955), to be 2.25 mg/ml for the
dextran carrier-cytotoxic agent moiety. Thus the complex
contained 36 p-[N,N-[bist2-chloroethyl)]phenylenediamines
per Ig, indicating about 2 dextran carriers per Ig. ;
To show the effectiveness of the complex as an
antitumor agent in vivo several groups of five C56BL/6 mice
were inoculated with 5 x 104EL4 cells intraperitoneally.
This is approximately 10,000 times the LD50 challenge dose.
After 24 hours the mice received the first of four daily
in;ections of the complex or controls. The results are
shown in the following table.
-22-
:
,.,, ., . , , ~ .
, .
- ~79'~8
Dose per _ .
injection
Drug Alky- Median
.~ lating Survival % Mice free .
activity Ig Time of tumor at
Treatment ~g/in~ection. (mg) (days)day 60
::
Saline _ _ 13 0
RIg alone _ 6.2 25 20
PDM DEX 450 _ 16 0
PDM DEX 450 ~ -
RIg unlinked 6.2 28 0
PDM DEX RIg 450 6.2 ~ 60 lO0
PDM= p-[N,N-bis(2-chloroethyl)]phenylenediamine (CDa)
DEX= dextran polymer carrier activated with CNBr and
. 15functionalized with H2N (CH2)6 NH2
RIg= rabbit antiserum against mouse EL4 lymphoma
EXAMPLE 2 -
.Following the procedure set out in Example l and
replacing p-[N,N-bis(2-chloroethyl)]phenylenediamine with
20 an equivalent quantity of p-~bis(2-chloroethyl)amino]-
phenylalanine provided a polymer carrier having a cytotoxic
;drug bound thereto as
7 ICO2H ~ /CH2 -CH2 Cl :-
- N - CH- ~ \
CH2 - CH2 - Cl
~.
In a similar manner and using an equivalent quantity
of 2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxy-
propionamide provided dextran having a cytotoxic drugbound thereto as
~79268
H fH2}l ~ ~ CH2 - CH2 Cl
CH ~ H ~ N \
CH2 - CH2 - Cl
~sin~ an equivalent quantity Or 2-amino-N-tp-bis(2-
chloroethyl)amino]phenyl-3-hydroxy-2-hydroxymethyl
propionamide provided a dextran polymer carrier having
cy~totoxic drug bound as:
H 7H2H ~ / CH2 - CH~ - Cl
- N - C-~ - NH ~ N \ ;
H2 C}~2--Cl
CH2H
Also rollowing the procedures in Example`s 1 -
and 2 for binding rabbit anti-sera against mouse EL4,
the rollowing Ig were bound in a like manner using
equivalent quantities.
(a) Goat anti-CEA Ig from human colonic
adenocarcinoma, Hsu. Fu Chao et al. Res. Co~m. in Chem.
Path. and Pharmacol. 9, 749 (1974).
(b) Ig from rabbit anti-acute lymphoblastic
leukemia serum, Greaves et al, Clin. Immunol. and
Immunopath. 4 67 (1975).
(c) Ig from various primate anti-leukemia
an,isera, anti-acute lymphoblastic leukemia, acute
myleoblastic leukemia, chron c lymphoblastic leukemia,
and chronic granulocytic leukemia cell as described by
Mohanakumar et al., J. Nat. Cancer Inst. 52 1435 (1974).-
~d) Human anti-human meningioma Ig. l~'inters
and Rich, Int. J. Cancer 15, 815 (1975).
' '
. -2~
~, ' : '
... ,.,~ ., .
. ' .
- ` 1079268
. .
(e) Rabbit anti-human prostate tumour Ig, Moncure
et al., Cancer Chemotherapy Reports 59 105 (1975).
(f) Rabbit anti-human chorionic gonadotropin
serum.
EXAMPLE 3
Preparation of Drug-Carrier p-[N,N-(bis-2-chloroethyl)]-
(PDM - PGA) phenylenediamine -
polyglutamic acid
1) 250 mg polyglutamic acid PGA (mol. wt. 35,000)
was dissolved in water by addition of lN NaOH dropwise until
all of the PGA was in solution. The pH was adjusted to 7.0
by the addition of lN NaOH dropwise until all of the PGA was
in solution. The pH was adjusted to 7.0 by the addition of
lN hydrochloric acid. (The PGA used was poly-L-glutamic
acid).
2) 1 g of 1-Ethyl-3-(3-dimethylaminopropyl)-
carbodiimide (EDC) was added to the PGA solution at room
temperature.
3) 140 mg of PDMp-[N,N-(bis-2-chloroethyl)]-
phenylenediamine-HCl was dissolved in 3.5 ml ethanol con-
taining 2% w/v hydrochloric acid and was diluted with 10.5
ml 60% aqueous propylene glycol containing 1.2% w/v K2HPO4.
4) The above PDM solution was added dropwise to
the stirred PGA and EDC mixture, the pH being maintained
; 25 at from 6 to 7 by the addition of sodium hydroxide.
5) The above mixture was cooled to 4C and an
equal volume of cold 0.2 M Na-acetic acid buffer ~ph 4.0]
was added.
6) The white gelatinous precipitate formed was
centrifuged cold and washed by three repeated suspensions
-25-
~;079Z68
and centrifugations in cold acetate buffer.
7) The washed precipitate was re-dissolved in
lN ~aOH to a final volume of 5 ml, the pH being ad~usted to
- 7 with lN hydrochloric acid.
; 5 The drug~carrier combination had the following
characteristics: -
1) The preparation was soluble in water above
pH 4.5--
2) When scanned in the U.V. range the prepara-
t~on had an absorption maximum at 277 nm due to the presence
~, of covalently bound PDM (ymax of free PDM is 258 nm).
From the extinction of PDM the degree of substitution of
PDM molecules per PGA molecule was calculated at approximate-
ly 50:1.
` 15 3) The 'mustard' moiety was shown to be present
.~, .. .
by determination of alkylating activity of the preparation.
This was done by reacting the preparation with nitrobenzyl-
pyridine in acetone at 80C and measuring the intensity
Or blue dye formed, by spectroscopy (Epstein J, Rosenthal,
R. W. and Ess, R. J., Anal. Chem. 27, 1435 (1955).
4) When chromatographed on ion-exchange resins
the preparation showed polyanionic behavior indicating the
~ presence of free carboxyl residues remaining on the carrier.
; For example, elution on DEAE Sephadex A50 or QAE Sephadex A50
with a gradient from 0.1 M Tris-HCl pH 6.5 to lM sodium
chloride showed a peak elution profile at a concentration
of 0.43 M sodium chloride.
,
.
-26-
-
1079Z68
Method of Linking Drug-Carrier to Immunoglobulin
1) The PDM-PGA solution was concentrated by mem-
brane ultrafiltration to 200 mg/ml.
2) 0.6 ml was allowed to react with 20 mg EDC
at room temperature for 2 minutes, then diluted to 20 ml
with phosphate-buffered saline pH 7.2.
3) The diluted solution was gradually added to an
equal volume of stirred solution of rabbit immunoglobulin
(RIg) as described in Example 1 at 5 mg/ml in 0.9~ sodium
chloride. This immunoglobulin preparation was obtained from
antiserum to mouse lymphoma cells (EL4) extensively absorbed
to remove antibody to normal mouse tissues.
4) 1.2 ml of SM sodium acetate was added to quench
any excess EDC remaining.
5) The PDM-PGA-RIg preparation was dialysed over-
night against 0.9~ sodium chloride at 4C.
6) The preparation was concentrated by membrane
ultrafiltration to a final volume of 10 ml.
The drug-carrier-globulin preparation had the following
chemico-physical properties:
1) Ethanol precipitability. 0.1 ml of ethanol
was added to 0.1 ml of the PDM-PGA-RIg preparation, and also
to 0.1 ml PDM-PGA at the same concentration. After 20
minutes at room temperature the samples were centrifuged at
2500 rpm for 10 minutes on a bench centrifuge and the super-
natants diluted for U.V. spectroscopy. From the U.V. read-
ings, the percentages of PDM-PGA and RIg precipitated were
calculated:
-27-
- ~` 1079Z~8
PDM-PGA alone 0%
RIg alone 80%
PDM in PDM-PGA-RIg 61%
Hence, 61% of the drug-carrier was precipitated with 80% of
the RIg, suggesting 76.5% of the drug-carrier was linked. -
2) Behavior on ion-exchange resins. An unlinked mix- -
ture of RIg and PDM-PGA chromatographed on QAE or DEAE Sephadex
with a gradient from 0.1 M tris-HCL pH 6.5 to 1 M sodium chlor-
ide produced two elution peaks (a) RIg eluted with 0.1 M tris-
HCl, pH 6.5 and (b) PDM-PGA eluted at 0.43 M sodium chloride.
In contrast, the PDM-PGA-RIg preparation showed a much dimin- ~-
ished RIg peak and a shift of the second peak to 0.35 M.
3) Immunoelectrophoretic behavior. Electrophoresis
of 2 1 of PDM-PGA-RIg on an agar-coated slide followed by
diffusion with goat-anti-rabbit antiserum gave a precipitin
line showing rabbit immunoglobulin present in material having
marked anionic characteristics consistent with PDM-PGA.
PDM~PGA itself gave no precipitin line.
4) Alkylating activity. By testing the material with
nitrobenzyl pyridine in the manner described above, it was shown
that the full alkylating activity of the drug-carrier was re-
tained following linkage to globulin.
5) Analysls. The preparation thus contained the
following amounts of drug, carrier and globulin:
PDM 3.1 mg/ml by U.V. spectroscopy
1.9 mg/ml in terms of alkylating activity
PGA 8 mg/ml)
) calculated from starting quantities
RIg 10 mg/ml)
-28-
: ' , : ', ' '
- 1079~,68
The drug/carrier/globulin showed the following biological
properties:
1) Antibody activity. Complement-dependent cytotoxi-
city was determined by the release of radioactive chromium
from labelled mouse lymphoma cells. The titre of the PDM-PGA-
RIg was 1 in 20 compared with 1 in 30 for unlinked RIg (both
at 10 mg/ml starting concentration.
2) Cytostatic activity in vitro. The mouse
lymphoma cells were maintained in tissue culture for two
days in the presence of a range of concentrations of
PDM-PGA-RIg. Cytostasis was determined by measuring inhi-
bition of cellular tritiated thymidine incorporation
during the last four hours of culture, in comparison with
cells in the absence of drug complexes. The results of a
typical experiment shown below gave the percentage inhibi-
tion at four concentrations of the components.
~Concentrations ~ Inhibition with various
preparations
Drug (in termsGlobulin RIg PDM-PGA+ PDM-PGA~ PDM-PGA-
,~ 20of alkylating)~g/mlalone normal RIg RIg
activity-~g/ml rabbit complex
globulin
120 624 56 58 76 83
312 28 25 58 67
2530 156 14 18 38 54
78 8 9 28 43
For any given concentration, the PDM-PGA-RIg
complex demonstrates greated cytostatic properties than
the other materials.
,' ' ,
-29-
-- 10'79Z6i8
:. :
3) Antl-tumour_activity in vivo. Groups of five
C57Bl/6 strain mice were injected with 5 x 104 EL4 lymphoma
cells intraperitoneally on day 0. At 24 hour intervals on
days, 1,2,3 and 4; the mice were injected i.p. with the
drug-carrier-globulin complex and compared with mice
similarly treated with various components of the complex. ~-
The survival times of the various groups are given below~
... . ......... .. __
% increase % mi~e
Dose per in~ection Median in survlval surviving
_ Survival time com- without
Drug (alky- Globuli ¦time(MST pared with evidence
lating) mg days saline of tumor
rreatment_ activity ~g __ controls at day 45
aline _ _ 13 _ 0
~Ig alone _ 4 19 47% 0
PDM-PGA 765 _ 25 92% 20
PDnlinkeAdRI~ {765 4} 38 192~ 40
PDM-PGA-RI~765~ __ 4 >60 >360% 100
Thus doses of PDM-PGA and RIg when linked in the
complex had greater antitumor activity than either component
alone or the two components together but unlinked.
EXAMPLE 4
Goat anti-human melanoma Ig was prepared from a
melanoma cell suspension containing 2.8 x 107 cells injected
intraperitoneally into a young adult female goat. This in-
~ection was repeated four times at weekly intervals and thenthe animal was bled out one week after the final injection.
Serum was separated from the blood and this was absorbed
with 3 g wet weight pooled human spleen cells/ml for 90
minutes at 4. This absorption was repeated twice. The
-30-
. . . .. .
' ` l~g~6~
abso~rbed serum was the~ fractio~ated to yield Ig by a
standard ammonium sulphate precipitation method followed by
dialysis.
P-[bis(2-chloroethyl)amino]phenylalaninepoly-
glutamic acid (m.w. 35,000) was prepared by methods
described in Example 3 replacing p-~N,N-(bis-2-chloroethyl)]-
phenylenediamine with p-[bis(2-chloroethyl)amino]phenyla-
lanine and using equivalen~ quantities. To this polymer
carrier cytotoxic drug was bound the above goat anti-human
melanoma Ig.
Freeze dried p-[bis-(2-chloroethyl)amino]phenyl-
alanine-polyglutamic acid (m.w. 35,000) was bound to the
goat anti-human melanoma Ig by dissolving the polymer-
carrier-cytotoxic drug complex in water to give a concen-
tration of 120 mg/ml. 200 Mg of ethyl-3-(3-dimethylamino-
propyl)carbodiimide was added to 10 ml of the polymer-
carrier-cytotoxic drug solution and when dissolved, the
solution was diluted to 400 ml. with phosphate buffered
saline, pH 7.2.
The dilute solution was added to 400 ml of a
rapidly stirring solution of goat anti-~elanoma Ig at
5 mg/ml containing 28.8 ml 5M sodium acetate. The prepara-
tion was dialysed against sterile 0.9% NaCl for 19 hours
with one change of saline after 16 hours. The volume after
dialysis was 890 ml. The preparation was membrane-filter
sterilized and analysed for drug and protein concentration:
Drug concentration by U.V. spectroscopy 697 ~g/ml
Drug concentration (alkylating activity) 317 ~g/ml
Protein concentration 2.25 mg/ml -~
Substitution ratio (active drug molecules per Ig molecule)
70~
-31-
- ~079Z68
EXAMPLE 5
Using the procedure set out in Example 4 and
using equivalent quantities, p-[N,N-bis(2-chloroethyl)]-
phenylenediamine, 2-amino-N-[p-bis(2-chloroethyl)amino~
5 phenyl-3-hydroxypropionamide, and 2-amino-N-[p-bis(2-chloro- -~
ethyl)amino]phenyl-3-hydroxy-2-hydroxymethylpropionamide
provided goat-anti-human melanoma Ig labeled with the
respective cytotoxic agent. For instance, using p-[N,N-
bis(2-chloroethyl)amino]phenyl-3-hydroxypropionamide and
polyglutamlc acid molecular weight 35,000 provided a
cytotoxic agent having 20 molecules of cytotoxic drug~Ig.
EXAMPLE 6
40 Mg of polylysine (m.w. 16,000) was dissolved
in 4 ml o~ 0.9% sodium chlorlde, and 40 mg of methotrexate
was dissolved in a similar amount of 0.9% sodium chloride
by addition o~ lN sodium hydroxide until a clear solution
was obtained. The pH of the methotrexate solution was ad-
~usted to ~.0 and the solution was added dropwise to the
polylysine solution and then 40 mg of 1-ethyl-3~3-dimethyl-
aminopropyl)carbodiimide was stirred in. The pH was loweredto 4 with hydrochloric acid and then returned to 7 with
sodium hydroxide. The methotrexate-polylysine preclpitated
and 0.1 ml of citraconic anhydride was added and the pH was
maintained above 8 with sodium hydroxide. The methotrexate-
polylysine precipitate gradually redissolved as citraconyla-
tion proceeded. The polymer carrier-cytotoxic drug complex
was bound to Ig as described in Example 3.
EXAMPLE 7
Using equivalent quantities of polyethyleneimine
-32-
.
.
~o~9~
(m.w. 20,000), 4-{p-[bis(2-chloroethyl)amino]phenyl}butyric
acid and l-ethyl-3-(3-dimethylaminopropyl)carbodllmide as
in Example 6, the cytotoxic drug was linked to the polymer
carrier. Reaction with citraconic anhydride was no~ carried
out with polyethyleneimine as it was with polylysine. To
40 mg of goat anti-human lymphoma Ig in 40 ml of buffer with
the pH ad~usted to 3 with hydrochloric acid was added, drop-
wise, 32 mg of the above polymer carrier-cytotoxic drug in
8~ml of buffer. The pH was maintained below 4. 20 Ml of
5% glutaraldehyde in saline was added and the pH was raised
to 9.5 with lN sodium hydroxide. The solution was held at .
that pH for 3-20 seconds and then returned to pH 3 by the
addition of lN hydrochloric acid. The process of addition
and pH ad~ustment was repeated three times. The pH was then
ad~usted to 6 and the preparation dialysed against saline.
EXAMPLE 8
10 Mg. of polyethyleneimine (m.w. 20,000) was dis-
solved in 0.25 ml of water and the pH was adjusted to 7 with
hydrochloric acid and 20 mg of cytosine arabinoside monophos-
20 phate was added and the pH was read~usted to 8, and then 20 ;
mg of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide was
added and the volume brought to 1 ml. This solution was
allowed to stand for 16 hours at 20C and protected from
light. The preparation was dialysed against saline buffered
to pH 7.3 with tris-acetate buffer (0.02M). This polymer
carrier having cytotoxic drug bound to it may in turn be
bound to Ig mentioned in Example 1 by the carbodiimide
method or the glutaraldehyde method.
-33-
. . . ~
107926~
E~AMPLE 9
,
Cytosine arabinoside was lin~ed through the amino
group to polymer carriers such as polyglutamic acid and dex-
tran activated with cyanogen bromide by methods set out in the -~
earlier examples.
EXAMPLE 10
2.39 Parts of N-carbobenzyloxy-L-serine and 2.69 parts
of p-[N,N-bis(2-chloroethyl)]phenylenediamine hydrochloride
were stirred together at room temperature and in the dark as
a suspension in 50 parts by volume of dry methylene chloride.
Then 1.01 parts of distilled triethylamine was added and
stirring was continued for 30 minutes. A solution of 2.16
parts of N,N-dicyclohexylcarbodiimide in 50 parts by volume of
.~
dry methylene chloride was added over 10 minutes. Stirring
1 was continued for 24 hours to provide a dark solution and a white
precipitate. The reaction mixture was filtered and the filtrate
was successively washed with aqueous sodium bicarbonate, 2 molar
hydrochloric acid, and water. The organic layer was then dried
, 20 over sodium sulfate and filtered and removal of solvent in vacuo
provided a crude solid which after recrystallization from
acetone/hexane or toluene provided 2-carbobenzyloxyamino-N-
, [p-bis(2-chloroethyl)amino]phenyl-3-hydroxypropionamide, melting
at 145-146 and having the following structural formula
':
H CH2 H CH2- CH2- Cl
,~ CH2-O-C-N-CH-C-N ~ N \
CH2- CH2- Cl
. '
.~
.
.:
-- 1079268
0.85 Parts of this material was dissolved in
85 parts by volume of distilled methanol containing 0.2
parts by volume of concentrated hydrochloric acid and the
solution hydrogenated at room temperature and atmospheric
pressure over 0.17 parts of a 5~ pallidium-on-charcoal
catalyst. The catalyst was removed by filtration, the
solvent removed in vacuo, and the product was precipitated
upon addition of dry ether. 2-Amino-N-[p-bis(2-chloro-
ethyl)amino]phenyl-3-hydroxypropionamide hydrochloride was
isolated as a hydroscopic solid having the formula
OH
CIH2 H ~ /CH2-CH2-Cl
HCl . H2N-C- IC -N ~ N \
H O 2 2
Replacement of hydrochloric acid with an equiva-
lent amount of sulfuric, phosphoric, hydrobromic, hydriodic,
sulfamic, citric, lactic, maleic, malic~ succinic, tartaric,
cinnamic, acetic, benzoic, gluconic, ascorbic acid provided
the corresponding acid addition salt. Neutralization of
the acid salt with base and extraction with ether provided -
the free base, 2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-
3-hydroxypropionamide.
fH
1 2 ¦ ~ / CH2-CE~2-Cl
H2N- C- I- N ~ N
H O -CH2-CEl2-cl ~-
. . .
'' ' ' ' ' . ' : ' ' '' ~ ' '~
107g268
EXAMPLE 11
,
5.9 Parts of DL-serine was dissolved in 1100 parts
by volume of a 0.2 molar sodium carbonate. 5.6 parts by
volume of a 1.0 molar cupric sulfate solution was then added,
followed by 34 parts by volume of 40% aqueous formaldehyde
solution. The solution was then heated at 95-100C for
20 minutes and a precipitate of copper resulted.
The reaction mixture was allowed to cool to room
temperature and then filtered to remove the precipitated copper.
After acidification with glacial acetic acid, the solution was
concentrated under reduced pressure to around 100 parts by
; volume and then poured onto a ZEOLITE 225 ion exchange column,
H form. This was washed with water until the acid band
disappeared, when the column was eluted with 2M ammonium
hydroxide, collecting and combining those fractions which gave
a positive ninhydrin reaction. These fractions were then
concentrated in vacuo, and 95~ ethanol was added to precipitate
the required product. After standing at 0C for 3 days the
crude product was filtered off, washed with ethanol and then
recrystallized from 95% ethanol to afford 2-amino-3-hydroxy-2-
hydroxymethylpropionic acid, melting at 253-254C and having ;
the following structural formula
OH
1 2
H2N--C C2H
1H2
OH
-36-
1079Z68
19.20 parts of this propionic acid is reacted with 21.56 parts
by volume of N-benzylchloroformate in 236 parts by volume of
sodium bicarbonate containing 29.8 parts of sodium carbonate.
Following the procedure set out in Example 1, 2-carbobenzyloxy-
amino-3-hydroxy-2-hydroxymethylpropionic acid, melting at 109-
112C (lit. 112-114) was isolated. This compound had the
following structural formula
OH
H CH
~ CH2--0-C-N-C-C02H
CIH2
OH
1.0 Parts of this material was placed with 1~.1 parts
of p-[N,N-bis(2-chloroethyl)]phenylenediamine hydrochloride
in 20 parts by volume of methylene chloride. 0.418 Parts of
distilled triethylamine was added with continuing stirring and
after stirring for 10 minutes 0.85 parts of N,N-dicyclohexyl-
carbodiimide in 20 parts by volume of dry methylene chloride
was added over a 10 minute period. The reaction was worked up
as in Example 1 to provide 2-carbobenzyloxyamino-N-lp-bis(2-
chloroethyl)amino]phenyl-3-hydroxy-2-hydroxymethylpropionamide,
melting at 138-141C, and having the following structural formula
, ~37~
lQ79268
: IH
H2 H ~ / CH2-CH2-Cl
CH2- O- C- N- C - C - N ~ N \
O CH2 , CH2-CH2-cl
OH
..
. .
. Using equivalent quantities and following the
procedures in Example 10, 0.5 parts of this material was
catalytically hydrogenated over 5% palladium-on-charcoal
catalyst to provlde 2-amino-N-[p-bis(2-chloroethyl)amino]- .
phenyl-3-hydroxy-2-hydroxymethylpropionamide hydrochloride
havlng the following structural formula
- , . .
OH
: CH2 H ~ / CH2-cH
HCl . H2- N- C - I - N ~ N \
7H2 CH2-CH2-Cl ..
OH
Other pharmaceutically acceptable acid addition
salts and the free base 2-amino-N-[p-bis(2-chloroethyl)-
amino]phenyl-3-hydroxy-2-hydroxymethylpropionamide are
prepared as described in Example 10.
Alternatively, 5.20 parts of 2-carbobenzyloxy-
amino-3-hydroxy-2-hydroxymethylpropionic acid and 5.95
parts of l-hydroxy benzotriazole in 130 parts by volume
of dry methylene chloride were reacted. To this reaction
mixture was added 4.03 parts of N,N-dicyclohexylcarbodi-
imide and stirring was continued for 16 hours. Then 5.7
-38-
.
. : , - . . ~ : ... - -
10'79Z6~
parts of p-lN,N-bis(2-chloroethyl)phenylenediamine hydro-
chloride and 2.99 parts of volume of triethylamine were added
and stirred for 65 hours and worked up as earlier described
to provide 2-amino-N-[p-bis(2-chloroethyl)amino]-phenyl-3-
S hydroxy-2-hydroxymethylpropionamide.
~ .
~ ~ '
",, ''.: . '
.~ . . .
''
~:,
-39-
.' ~"'~' : ' .
