CA1079268A - Immunological materials - Google Patents
Immunological materialsInfo
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- CA1079268A CA1079268A CA314,487A CA314487A CA1079268A CA 1079268 A CA1079268 A CA 1079268A CA 314487 A CA314487 A CA 314487A CA 1079268 A CA1079268 A CA 1079268A
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- Prior art keywords
- amino
- bis
- chloroethyl
- polymer carrier
- phenyl
- Prior art date
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Abstract
ABSTRACT OF THE DISCLOSURE
The present invention relates to a polymer carrier, suitable for binding to immunoglobulin, having 5 to 500 molecules of a cytotoxic drug covalently bound thereto, said carrier having a molecular weight of 5000 to 500,000 and free carboxyl, amino or cycloimidocarbonate groups for covalent bonding, the cytotoxic drug also having amino or carboxyl groups available for covalent bonding.
The present invention relates to a polymer carrier, suitable for binding to immunoglobulin, having 5 to 500 molecules of a cytotoxic drug covalently bound thereto, said carrier having a molecular weight of 5000 to 500,000 and free carboxyl, amino or cycloimidocarbonate groups for covalent bonding, the cytotoxic drug also having amino or carboxyl groups available for covalent bonding.
Description
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,
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 ~ 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
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~
:
2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxy-2-hydroxymethylpropionamide CD~e) ~ -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) O N
~ CH2- OH
1~ .
H
cytosine arabinosi.de or monophosphate CD(g) .
` 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).
'',, ':
, , .- . . . . . . . .: .
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 ¦
~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. _ 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.
~ .
- 1~79Z68 or C' using CD(b) is preferred:
Ig ~
: 7 - H - _ .~ O-CI
., CIH2 , . .
¦ ~ C -C -N - CH- Q ~ CH c ~
¦ 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 . ~ 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 . . . . .
', '' ' .
'~, .
1~79Z68 -- N--H
O=C
.
--HN--CH--C--N--CH--C--N--CH--C--N ' 11 1 1 1 1 1 ' ..
CH2 ICH2 1 ~ . ~ :
.~ _ CO7H C--O
~UOCH2 G CH2--OH
. ~N
' ' CH2 7H2 .- ~
fH2 ~H2 ' . ' . Cl 1 n ~ :
_ ' - ' ,_ ' ' .
C''' ~;.,.
'' ... . ..
` ` 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.
~" ' ' _ 1 B2 Polymer Carrier CD
Polyglutamic acid a,b 7 d,e :
_N-- C-- --C--N--.,.:
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 ~ 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. ~.
.
)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.
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 ,, , . . - .
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 ' 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.
:
,.,, ., . , , ~ .
, .
- ~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 .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 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.
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.
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 ~ 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
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~
:
2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxy-2-hydroxymethylpropionamide CD~e) ~ -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) O N
~ CH2- OH
1~ .
H
cytosine arabinosi.de or monophosphate CD(g) .
` 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).
'',, ':
, , .- . . . . . . . .: .
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 ¦
~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. _ 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.
~ .
- 1~79Z68 or C' using CD(b) is preferred:
Ig ~
: 7 - H - _ .~ O-CI
., CIH2 , . .
¦ ~ C -C -N - CH- Q ~ CH c ~
¦ 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 . ~ 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 . . . . .
', '' ' .
'~, .
1~79Z68 -- N--H
O=C
.
--HN--CH--C--N--CH--C--N--CH--C--N ' 11 1 1 1 1 1 ' ..
CH2 ICH2 1 ~ . ~ :
.~ _ CO7H C--O
~UOCH2 G CH2--OH
. ~N
' ' CH2 7H2 .- ~
fH2 ~H2 ' . ' . Cl 1 n ~ :
_ ' - ' ,_ ' ' .
C''' ~;.,.
'' ... . ..
` ` 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.
~" ' ' _ 1 B2 Polymer Carrier CD
Polyglutamic acid a,b 7 d,e :
_N-- C-- --C--N--.,.:
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 ~ 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. ~.
.
)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.
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 ,, , . . - .
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 ' 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.
:
,.,, ., . , , ~ .
, .
- ~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 .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 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.
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.
was added.
6) The white gelatinous precipitate formed was centrifuged cold and washed by three repeated suspensions ~;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.
, .
-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:
- ~` 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) : ' , : ', ' ' - 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 For any given concentration, the PDM-PGA-RIg complex demonstrates greated cytostatic properties than the other materials.
,' ' , -- 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.
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 . . . .. .
' ` 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~
- ~079Z68 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.
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.
Using equivalent quantities of polyethyleneimine .
.
~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
; 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.
, .
-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:
- ~` 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) : ' , : ', ' ' - 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 For any given concentration, the PDM-PGA-RIg complex demonstrates greated cytostatic properties than the other materials.
,' ' , -- 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.
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 . . . .. .
' ` 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~
- ~079Z68 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.
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.
Using equivalent quantities of polyethyleneimine .
.
~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.
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.
. . . ~
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.
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 . ' .~
.
.:
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 \
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 , 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
OH
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
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 .
. : , - . . ~ : ... - -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.
~ .
~ ~ ' ",, ''.: . ' .~ . . .
'' ~:, .' ~"'~' : ' .
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.
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.
. . . ~
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.
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 . ' .~
.
.:
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 \
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 , 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
OH
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
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 .
. : , - . . ~ : ... - -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.
~ .
~ ~ ' ",, ''.: . ' .~ . . .
'' ~:, .' ~"'~' : ' .
Claims (18)
1. A process for the preparation of a polymer carrier, suitable 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 represented by the formula:
wherein the polymer carrier is selected from the group of polymers comprising:
a) dextran activated with cyanogen bromide to form cycloimidocarbonate and functionalized with -NH(CH2)xCO2H, -NH-(CH2)x-NH2, wherein x is 3 - 8, b) aminoethylated dextran, c) polyglutamic acid, d) polyaspartic acid, e) polyarginine, f) serum albumen, g) fibrinogen h) .gamma.-globulin, i) polylysine (solubilized by reaction with citraconic anhydride), j) copolymers of lysine-phenylalanine, lysine-tyrosine and glutamic acid-tyrosine, k) polyethyleneimine;
CD represents a covalently bound cytotoxic drug selected from the group comprising:
N-{p-{[(2,4-diamino-6-pteridinyl)methyl]methylaminno}-benyl}
-L-glutamic acid, 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- hydroxy-propionamide, 2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxy-2-hydroxymethylpropionamide, cytosine arabinoside, cytosine arabinoside monophosphate, o-[N,N-bis(2-chloropropyl)]carbamoylglycollic acid, daunomycin, doxorubicin, and diphtheria toxin;
B2 represents a peptide linkage between a free carboxyl group of the polymer carrier and the free amino group of CD, or between a free amino group of the polymer carrier and the free carboxyl group of CD, or the covalent linkage between a cyclo-imidocarbonate group of the polymer carrier, when the polymer is activated dextran, and the free amino group of the appropriate CD; and m = 5 - 500;
which comprises contacting the polymer carrier with CD in the presence of a suitable carbodiimide to form the resultant peptide linkage B2.
wherein the polymer carrier is selected from the group of polymers comprising:
a) dextran activated with cyanogen bromide to form cycloimidocarbonate and functionalized with -NH(CH2)xCO2H, -NH-(CH2)x-NH2, wherein x is 3 - 8, b) aminoethylated dextran, c) polyglutamic acid, d) polyaspartic acid, e) polyarginine, f) serum albumen, g) fibrinogen h) .gamma.-globulin, i) polylysine (solubilized by reaction with citraconic anhydride), j) copolymers of lysine-phenylalanine, lysine-tyrosine and glutamic acid-tyrosine, k) polyethyleneimine;
CD represents a covalently bound cytotoxic drug selected from the group comprising:
N-{p-{[(2,4-diamino-6-pteridinyl)methyl]methylaminno}-benyl}
-L-glutamic acid, 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- hydroxy-propionamide, 2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxy-2-hydroxymethylpropionamide, cytosine arabinoside, cytosine arabinoside monophosphate, o-[N,N-bis(2-chloropropyl)]carbamoylglycollic acid, daunomycin, doxorubicin, and diphtheria toxin;
B2 represents a peptide linkage between a free carboxyl group of the polymer carrier and the free amino group of CD, or between a free amino group of the polymer carrier and the free carboxyl group of CD, or the covalent linkage between a cyclo-imidocarbonate group of the polymer carrier, when the polymer is activated dextran, and the free amino group of the appropriate CD; and m = 5 - 500;
which comprises contacting the polymer carrier with CD in the presence of a suitable carbodiimide to form the resultant peptide linkage B2.
2. A process according to claim 1 for the preparation of a polymer carrier, suitable for binding to immunoglobulin, wherein the polymer carrier is dextran activated with cyanogen bromide and functionalized with 1,6-diaminohexane and having a molecular weight of 5,000 - 100,000;
B2 represents a covalent linkage between the activated portion of the dextran and the free amino group of CD;
CD represents p-[N,N-bis(2-chloroethyl)]phenylene-diamine,p-[bis(2-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-hydroxy-methylpropionamide; and m - 5 - 100;
which comprises contacting the above dextran with CD in the presence of a suitable carbodiimide, thereby forming a covalent linkage between the amino group on CD and a cyclo-imidocarbonate group of the activated dextran represented in B2.
B2 represents a covalent linkage between the activated portion of the dextran and the free amino group of CD;
CD represents p-[N,N-bis(2-chloroethyl)]phenylene-diamine,p-[bis(2-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-hydroxy-methylpropionamide; and m - 5 - 100;
which comprises contacting the above dextran with CD in the presence of a suitable carbodiimide, thereby forming a covalent linkage between the amino group on CD and a cyclo-imidocarbonate group of the activated dextran represented in B2.
3. A process according to claim 2 for the preparation of a polymer carrier wherein the dextran has a molecular weight of 17,000.
4. A process according to claim 1 for the preparation of a polymer carrier, suitable for binding to immunoglobulin, wherein the polymer carrier is aminoethylated dextran having a molecular weight of 5,000 - 500,000;
CD represents p-[bis(2-chloroethyl)amine]phenyl-alanine or 4-{p-[bis(2-chloroethyl)amino]phenyl}butyric acid;
B2 represents a peptide linkage between a free amino group of the aminoethylated dextran and the free carboxyl group of CD; and m = 5 - 100;
which comprises contacting the above aminoethylated dextran with CD in the presence of a suitable carbodiimide, thereby forming a peptide linkage between an amino group on the aminoethylated dextran and the free carboxyl group of CD, represented by B2.
CD represents p-[bis(2-chloroethyl)amine]phenyl-alanine or 4-{p-[bis(2-chloroethyl)amino]phenyl}butyric acid;
B2 represents a peptide linkage between a free amino group of the aminoethylated dextran and the free carboxyl group of CD; and m = 5 - 100;
which comprises contacting the above aminoethylated dextran with CD in the presence of a suitable carbodiimide, thereby forming a peptide linkage between an amino group on the aminoethylated dextran and the free carboxyl group of CD, represented by B2.
5. A process according to claim 1 for the preparation of a polymer carrier which, when dissolved in citraconic anhydride, is suitable for binding to immunoglobulin, wherein the polymer carrier is polylysine having a molecular weight of 5,000 - 100,000;
CD represents p-[bis(2-chloroethyl)amino]phenylala-nine, 4-{p-[bis(2-chloroethyl)amino]phenyl}butyric acid,or N-{p-{[(2,4-diamino-6-pteridinyl)methyl]methylamino}-benzyl}-L-glutamic acid;
B2 represents a peptide linkage between a free amino group of the polylysine and the free carboxyl group of CD; and m = 5 - 100;
which comprises contacting the above polylysine with CD in the presence of a suitable carbodiimide, thereby forming a peptide linkage between an amino group on the polylysine and the free carboxyl group of CD represented by B2.
CD represents p-[bis(2-chloroethyl)amino]phenylala-nine, 4-{p-[bis(2-chloroethyl)amino]phenyl}butyric acid,or N-{p-{[(2,4-diamino-6-pteridinyl)methyl]methylamino}-benzyl}-L-glutamic acid;
B2 represents a peptide linkage between a free amino group of the polylysine and the free carboxyl group of CD; and m = 5 - 100;
which comprises contacting the above polylysine with CD in the presence of a suitable carbodiimide, thereby forming a peptide linkage between an amino group on the polylysine and the free carboxyl group of CD represented by B2.
6. A process according to claim 1 for the preparation of a polymer carrier, suitable for binding to immunoglobulin, wherein the polymer carrier is polyethyleneimine having a mole-cular weight of 5,000 - 100,000;
CD represents p-[bis(2-chloroethyl)amino]phenyl-alanine, 4-{p-[bis(2-chloroethyl)amino]phenyl}butyric acid, or N-{p{[(2,4-diamino-6-pteridinyl)methyl]methylamino}-benzyl}-L-glutamic acid;
B2 represents a peptide linkage between a free amino group of the polyethyleneimine and the free carboxyl group of CD; and m = 5 - 500 which comprises contacting the above polyethylene-imine with CD in the presence of a suitable carbodiimide, thereby forming the peptide linkage B2.
CD represents p-[bis(2-chloroethyl)amino]phenyl-alanine, 4-{p-[bis(2-chloroethyl)amino]phenyl}butyric acid, or N-{p{[(2,4-diamino-6-pteridinyl)methyl]methylamino}-benzyl}-L-glutamic acid;
B2 represents a peptide linkage between a free amino group of the polyethyleneimine and the free carboxyl group of CD; and m = 5 - 500 which comprises contacting the above polyethylene-imine with CD in the presence of a suitable carbodiimide, thereby forming the peptide linkage B2.
7. A process according to claim 6 for the preparation of a cytotoxic agent wherein the polyethyleneimine has a mole-cular weight of about 20,000.
8. A process according to claim 1 for the preparation of a polymer carrier, suitable for binding to immunoglobulin, wherein the polymer carrier is polyglutamic acid having a molecular weight of 5,000 - 500,000;
CD represents p-[N,N-bis(2-chloroethyl)]phenylene-diamine, p-[bis(2-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-hydroxy-methylpropionamide;
B2 represents a peptide linkage between a free carboxyl group of the polyglutamic acid and the free amino group of CD; and m = 5 - 100;
which comprises contacting the above polyglutamic acid with CD in the presence of a suitable carbodiimide, there-by forming a peptide linkage between a free carboxyl group of the polyglutamic acid and the free amino group of CD represented by B2.
CD represents p-[N,N-bis(2-chloroethyl)]phenylene-diamine, p-[bis(2-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-hydroxy-methylpropionamide;
B2 represents a peptide linkage between a free carboxyl group of the polyglutamic acid and the free amino group of CD; and m = 5 - 100;
which comprises contacting the above polyglutamic acid with CD in the presence of a suitable carbodiimide, there-by forming a peptide linkage between a free carboxyl group of the polyglutamic acid and the free amino group of CD represented by B2.
9. A process according to claim 8 for the preparation of a cytotoxic agent wherein the polyglutamic acid has a molecular weight of about 35,000.
10. A polymer carrier, suitable for binding to immuno-globulin, having a molecular weight between 5,000 and 50,000 and having 5-500 molecules of cytotoxic drug covalently bound thereto represented by the formula wherein the polymer carrier is selected from the group of polymers comprising:
a) dextran activated with cyanogen bromide to form cycloimidocarbonate and functionalized with - NH(CH2)xCO2H, - NH- (CH2)x -NH2, wherein x is 3 - 8, b) aminoethylated dextran, c) polyglutamic acid, d) polyaspartic acid, e) polyarginine, f) serum albumen, g) fibrinogen, h) .gamma.-globulin, i) polylysine (soluhilized by reaction with citraconic anhydride), j) copolymers of lysine-phenylalanine, lysine-tyrosine, and glutamic acid-tyrosine, k) polyethyleneimine;
CD represents a covalently bound cytotoxic drug selected from the group comprising:
N-{p-{[(2,4-diamino-6-pteridinyl)methyl]methylamino}
-benzyl}-L-glutamic acid, 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-hydroxy-propionamide, 2-amino-N-[p-bis(2-chloroethyl)amine]phenyl-3-hydroxy-2-hydroxymethyl propionamide, cytosine arabinoside, cytosine arabinoside monophosphate, o-[N,N-bis(2-chloropropyl)]carbamoylglycollic acid, daunomycin, doxorubicin, and diphtheria toxin;
B2 represents a peptide linkage between a free carboxyl group of the polymer carrier and the free amino group of CD, or between a free amino group of the polymer carrier, and the free carboxyl group of CD, or the covalent linkage between a cycloimi-docarbonate group of the polymer carrier, when the polymer is activated dextran, and the free amino group of the appropriate CD; and m = 5 - 500;
whenever prepared according to the process of claim 1.
a) dextran activated with cyanogen bromide to form cycloimidocarbonate and functionalized with - NH(CH2)xCO2H, - NH- (CH2)x -NH2, wherein x is 3 - 8, b) aminoethylated dextran, c) polyglutamic acid, d) polyaspartic acid, e) polyarginine, f) serum albumen, g) fibrinogen, h) .gamma.-globulin, i) polylysine (soluhilized by reaction with citraconic anhydride), j) copolymers of lysine-phenylalanine, lysine-tyrosine, and glutamic acid-tyrosine, k) polyethyleneimine;
CD represents a covalently bound cytotoxic drug selected from the group comprising:
N-{p-{[(2,4-diamino-6-pteridinyl)methyl]methylamino}
-benzyl}-L-glutamic acid, 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-hydroxy-propionamide, 2-amino-N-[p-bis(2-chloroethyl)amine]phenyl-3-hydroxy-2-hydroxymethyl propionamide, cytosine arabinoside, cytosine arabinoside monophosphate, o-[N,N-bis(2-chloropropyl)]carbamoylglycollic acid, daunomycin, doxorubicin, and diphtheria toxin;
B2 represents a peptide linkage between a free carboxyl group of the polymer carrier and the free amino group of CD, or between a free amino group of the polymer carrier, and the free carboxyl group of CD, or the covalent linkage between a cycloimi-docarbonate group of the polymer carrier, when the polymer is activated dextran, and the free amino group of the appropriate CD; and m = 5 - 500;
whenever prepared according to the process of claim 1.
11. A compound according to claim 10 wherein the polymer carrier is dextran activated with cyanogen bromide and functionalized with 1,6-diaminohexane having a molecular weight of 5,000 - 100,000; B2 represents a covalent linkage between the activated portion of the dextran and the free amino group of CD; CD represents p-[N,N-bis(2-chloroethyl)]phenylenediamine, p-[bis(2-chloroethyl)amino]phenlalanine, 2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxypropionamide, or 2-amino-N-[p-bis(2-chloroethyl)amino]phenyl-3-hydroxy-2-hydrroxymethyl pro-pionamide; and m = 5 - 500;
whenever prepared by the process of claim 2.
whenever prepared by the process of claim 2.
12. A compound according to claim 10 wherein the polymer carrier is dextran activated with cyanogen bromide and functionalized with 1,6-diaminohexane having a molecular weight of 17,000; B2 represents a covalent linkage between the activa-ted portion of the dextran and the free amino group of CD; CD
represents p-[N,N-bis(2-chloroethyl)]phenylenediamine, p-[bis(2-chloroethyl)amino]phenylalanine, 2-amino-N-[p-bis(2-chloroethyl)-amino]phenyl-3-hydroxypropionamide, or 2-amino-N-[p-bis(2-chloro-ethyl)amino]phenyl-3-hydroxy-2-hydroxymethyl propionamide; and m = 5 - 500;
whenever prepared by the process of claim 3.
represents p-[N,N-bis(2-chloroethyl)]phenylenediamine, p-[bis(2-chloroethyl)amino]phenylalanine, 2-amino-N-[p-bis(2-chloroethyl)-amino]phenyl-3-hydroxypropionamide, or 2-amino-N-[p-bis(2-chloro-ethyl)amino]phenyl-3-hydroxy-2-hydroxymethyl propionamide; and m = 5 - 500;
whenever prepared by the process of claim 3.
13. A compound according to claim 10 wherein the polymer carrier is aminoethylated dextran having a molecular weight of 5,000 - 500,000 in which CD represents p-[bis(2-chloro-ethyl)amino]phenylalanine or 4-{p-[bis(2-chloroethyl)amino]-phenyl}butyric acid;
B2 represents a peptide linkage between a free amino group of the aminoethylated dextran and the free carboxyl group of CD; and m = 5 - 100;
whenever prepared by the process of claim 4.
B2 represents a peptide linkage between a free amino group of the aminoethylated dextran and the free carboxyl group of CD; and m = 5 - 100;
whenever prepared by the process of claim 4.
14. A compound according to claim 10 wherein the polymer carrier 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]methylamino}-benzyl}-L-glutamic acid;
B2 represents a peptide linkage between a free amino group of the polylysine and the free carboxyl group of CD; and m = 5 - 100;
whenever prepared by the process of claim 5.
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]methylamino}-benzyl}-L-glutamic acid;
B2 represents a peptide linkage between a free amino group of the polylysine and the free carboxyl group of CD; and m = 5 - 100;
whenever prepared by the process of claim 5.
15. A polymer carrier according to claim 10 wherein the polymer carrier is polyethyleneimine having a molecular weight of 5,000 - 100,000, 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]-methylamino}-benzyl}-L-glutamic acid;
B2 represents a peptide linkage between a free amino group of the polyethyleneimine and the free carboxyl group of CD; and m = 5 - 100;
whenever prepared by the process of claim 6.
B2 represents a peptide linkage between a free amino group of the polyethyleneimine and the free carboxyl group of CD; and m = 5 - 100;
whenever prepared by the process of claim 6.
16. A polymer carrier according to claim 10 wherein the polymer carrier is polyethyleneimine having a molecular weight of about 20,000, in which CD represents p-[bis(2-chloro-ethyl)amino]phenylalanine, 4-p-[bis(2-chloroethyl)amino]phenyl)-butyric acid, or N-(p([(2,4-diamino-6-pteridinyl-methyl)methyl-amino)-benzyl)-L-glutamic acid;
B2 represents a peptide linkage between a free amino group of the polyethyleneimine and the free carboxyl group of CD; and m = 5 - 100;
whenever prepared by the process of claim 7.
B2 represents a peptide linkage between a free amino group of the polyethyleneimine and the free carboxyl group of CD; and m = 5 - 100;
whenever prepared by the process of claim 7.
17. A compound according to claim 10 wherein the polymer carrier is polyglutamic acid having a molecular weight of 5,000 - 100,000;
B2 represents a peptide linkage between a free carboxyl group of the polyglutamic acid and the free amino group of CD;
CD represents p-[N,N-bis(2-chloroethyl)]phenylene-diamine, p-[bis(2-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-hydroxy-methyl propionamide; and m = 5 - 100;
whenever prepared by the process of claim 8.
B2 represents a peptide linkage between a free carboxyl group of the polyglutamic acid and the free amino group of CD;
CD represents p-[N,N-bis(2-chloroethyl)]phenylene-diamine, p-[bis(2-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-hydroxy-methyl propionamide; and m = 5 - 100;
whenever prepared by the process of claim 8.
18. A compound according to claim 10 wherein the polymer carrier is polyglutamic acid having a molecular weight of 35,000;
B2 represents a peptide linkage between a free carboxyl group of the polyglutamic acid and the free amino group of CD;
CD represents p-[N,N-bis(2-chloroethyl)]phenylene-diamine, p-[bis(2-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-hydroxy-methyl propionamide; and m = 5 - 100;
whenever prepared by the process of claim 9.
B2 represents a peptide linkage between a free carboxyl group of the polyglutamic acid and the free amino group of CD;
CD represents p-[N,N-bis(2-chloroethyl)]phenylene-diamine, p-[bis(2-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-hydroxy-methyl propionamide; and m = 5 - 100;
whenever prepared by the process of claim 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA314,487A CA1079268A (en) | 1975-02-04 | 1978-10-27 | Immunological materials |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB4695/75A GB1496011A (en) | 1974-11-22 | 1975-02-04 | Defoaming composition |
| CA245,033A CA1062156A (en) | 1975-02-04 | 1976-02-04 | Immunological materials |
| CA314,487A CA1079268A (en) | 1975-02-04 | 1978-10-27 | Immunological materials |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1079268A true CA1079268A (en) | 1980-06-10 |
Family
ID=27164318
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA314,487A Expired CA1079268A (en) | 1975-02-04 | 1978-10-27 | Immunological materials |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1079268A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5057313A (en) * | 1986-02-25 | 1991-10-15 | The Center For Molecular Medicine And Immunology | Diagnostic and therapeutic antibody conjugates |
| US5084560A (en) * | 1986-06-30 | 1992-01-28 | Oncogen | Immunoconjugates and methods for their use in tumor therapy |
| US5162505A (en) * | 1989-09-19 | 1992-11-10 | Centocor | Proteins modified with positively charged carriers and compositions prepared therefrom |
-
1978
- 1978-10-27 CA CA314,487A patent/CA1079268A/en not_active Expired
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5057313A (en) * | 1986-02-25 | 1991-10-15 | The Center For Molecular Medicine And Immunology | Diagnostic and therapeutic antibody conjugates |
| US5084560A (en) * | 1986-06-30 | 1992-01-28 | Oncogen | Immunoconjugates and methods for their use in tumor therapy |
| US5162505A (en) * | 1989-09-19 | 1992-11-10 | Centocor | Proteins modified with positively charged carriers and compositions prepared therefrom |
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