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The interaction of chlorambucil with human γ-globulin
THE INTERACTION
OF CHLORAMBUCIL
WITH HfJMAN y-GLOBULIN
Gef p~r~~~~i~~~~~~~~~~~r~~h~ of solutions containing c~~~rarnbu~~~ and human pglobulin indicates that there is littte, if any physical binding of the drug but a progressive chemical j~tera~tjon. The rate of reaction of ~h~~rambnci1 is not signi~~ant~yabated by the presence of the globulin. The implications of these endings for current attempts at imm~~o~hemotherapy are discussed.
Recent claims for the en~n~ment of the anti~neopIastic activity of chgorambucil when admixed with immunoglobu~~ns (GHO~Eet uLXJ and FLECHNER~)has stimulated interest in the nature of the ant~b~y-drug complex. Pre~~rn~narywork here had indicated that the binding of Gh~or~buci~ to globulins was less marked than that implied in the afore-mentioned publications, For example, the passing of a solution contajning ~hlorarnb~~jl and human ~-globulin in the proportion l-10 by weight through a coiumn of Sepbadex G 25 resulted in the almost complete separation uf the components. This was not consistent with the 90% binding of the drug expected by the workers cited. There appears to be a misapprehension since fsRAr3.sAND L~NDFURI~~ du not appear to studied the binding of chlorambucil to y-glob&ins but only to mixed serum proteins. Their work using axr ultrafiltration technique at 5”-to minimise the reaction of the alkylating agent-relates to the amount of binding at a steady ehlorambucili concentration. It was conside ered that, if the components could be separated by gel filtration, which is electively a dialysis technique in which the co~~entra~o~ of drug is pro~essive~y reduced, then following injection of ~hlorambu~~~-g~abu~i~mixtures and s~b~quent dilution in tissue fluids a similar dissolution would occur. There were afso suggestions that the enhanced biological activity was due to rll
262
W.
C. J. ROSS
reduction in the rate of reaction of chlorambucil in the presence of globulin. This was not, in fact, supported by the quoted work of HOPWOODAND STOCKY. Accordingly, it was decided to examine the interaction of chlorambucil with globulin. A gel permeation chromatographic resolution of antibody-drug mixtures and a spectrophotometric method of assaying the components were developed. MATERIALS
AND METHODS
The sodium salt of chlorambucil was prepared by adding an exact equivalent of sodium methoxide to a methanolic solution of chlorambucil and evaporating to dryness under vacuum. The human lyophilized y-globulin was supplied by AB Kabi (Stockholm, Sweden). Sephadex G 25 (fine) and K9/60 columns (Pharmacia) were used for chromatographic analysis and the absorbance of eluates at 280 nm was monitored with an LKB Uvicord II UV absorptiometer. The absorbance data for pooled eluates was obtained using 1 cm silica cells in an SP 500 UV spectrophotometer. The pH 8 saline buffer was prepared by dissolving sodium chloride (IO g) in Fison’s pH7 M/20 phosphate buffer (1 1) and adjusting the pH to 8 by the addition of aqueous sodium hydroxide (18 ml N). Spectrophototnetric
data
A solution of chlorambucil
in pH 8 saline buffer (0.02589 mg/ml) immediately after mixing exhibited E:
The sodium salt of chlorambucil (34.7 mg equivalent to 32.36 mg acid) was dissolved in pH 8 saline buffer (5 ml) at 20”-the sodium salt was used as it dissolved much more rapidly than the free acid. Within 5 min of mixing 1 ml of the solution was applied to the Sephadex column which had been equilibriated with pH 8 saline buffer. Elution was carried out using the same buffer. The flow rate was 0.5 ml/min and each fraction contained 2 ml. The elution profile is shown in Fig. 1. The chromatographic resolution was repeated after keeping the mixture for 2 and 24 h at 20” (Figs. 2 and 3). To a solution of human y-globulin (250 mg) in pH 8 saline buffer (5 ml) at 20”
INT&%l.ACTlGN OF CHLGRAMRWCILWITH y-GLOBWLKN
I
263
SW.2
Figs. l-3. Chromatographic profile of chlorambucil(32.36 5 min. 2 h and 24 h after mixing.
mg) dissolved in pH 8 saline buffer (5 ml)
was added the sodium salt of chlorambucil(34.1 mg = 31.8 mg acid) and 1 ml of the mixture was sub~t~d to ~hromatographic resohrtion after 5 mm, 2 and 24 h (Figs. 4, 5 and 6). The El cmvalues of the various pooled eiuates, suitably diluted, were measured at 258 nm and 280 nm and the amounts of chlorambucil or derived product therein were calculated as indicated below. Example of calculation
For the chromatogram represented by Fig. 5:
fractions 7-I 3 in 100 ml fractions M-27 in 50 ml fractions 28-42 in 100 ml
I em E 258 nm
f em E 280 nm
0.886
0.696
0.976
0,138
I A54
0.16
w. c.
264
FRACTION
J. ROSS
NUMBERS
Figs. 4-6. Chromatographic profile of chlorambucil (31.8 mg) dissolved in pH 8 saline buffer (5 ml) human y-globulin (250 mg) 5 min. 2 h and 24 h after mixing.
containing
The amount of chlorambucil in fractions 28-42 is 0.16 - 100 * 0.216 or 3.45 mg (53.3 ,I/,), Fractions 14-27 contain products derived from chlorambucil equivalent to 0.138 * 50 *0.216 or 1.49 mg (23.0%). In fractions 7-13 there is 50 mg of globulin so that the absorbance due to the protein (0.5 mg/ml) is 0.625 at 280 nm. The absorbance attributable to the chlorambucil is 06960.625 or 0.071 and the amount of chlorambucil bound is 0.071 - 100 mO.216or 1.53 mg (23.6%). The total recovery of chlorambucil is 6.47 mg (101.7 %). The number of molecules of chlorambucil of the globulin (mol. wt. I50 000) is: 1.53 * 150 000 - or 15.1 50 * 304
(mol. wt. 304) bound to one molecule
INTBRACTION OF CHLORAMBUCIL
WITH y-GLOBULIN
265
RESULTS
Figs. 1,2 and 3 show the efuticm profiles of solutions of Ghlo~buciI in #I g saline buffer when applied 5 min, 2 h and 24 h after mixing, respectively. The data in Table I give the percentage of chlorambucil or derived product associated with various fractions. These values do not represent the com~sition of the mixtures as applied to the column since reaction will be proceeding during the elution which is at the rate of one fraction (2 ml) per 4 min. Nevertheless the runs with and without globulin are comparable since they were performed under identical conditions. Fig. I shows two components, chIorambuci1 (I, 84.5%) in fractions 28-42 and a faster moving Gompone~t in fractions M-27 (I 5.5 %). Fig. 2 shows a chIorambuci1 peak (58.6%) and the second area of absorption is split into 2 peaks (total 41.4%). After 24 h (Fig. 3) only 6.3% of the original Gh1orambucil is present and the fastest moving peak of the second region predo~nates (total 93.6%). It is probable that the peak (fractions 22-28) represel~ts half hydrolysed GhlorambuGi1(II) and the peak (fractions 14-22) contains fully hydrolysed agent (III) since the former peak decreases at the expense of the latter with time. ,CH&H,OH HOOCKH,)J
N (CH&H&l
Ia
N \
HOOCKH2&
0
0
W&-H&I
(ItI
(1)
iIOCX(CH&
N KH$H20H
12
Figs. 4,s and 6 show the elution profiles of solutions of chlorambucil(31.8 mg) in S “/::human ~-globulin (5 ml in pIi 8 saline buffer). The ch1orambucil~globu1inratio by weight is l/7.85. Fig. 4 shows that the protein emerges after th:: void volume in TABLE
I
THE PERCENTAGE NUMBER
(HyG)
OF
(Figs. 4, 5 and 6) ___-_..~-~-l
Time
____~.-w-.-5 min (Fig. 1) 2 h 24 h
OF APPLIED
MOLECULES
(Fig. 2) (Fig. 3)
5 min (Fig. 4) 2 h (Fig. 5) 24 h (Fig. 6) .______.. ~_-.---
OF
CNLO~AMBUCIL CHLORAMBUCIL
-
PRESENT
(CB)
IN THE VARlOUS
BOUND
TO
ONE
-___
_._-
FRACTIONS
MOLECULE
OF
-. .- -..- l_____-l
Percentage of applied chlorambucii ..___-I-~-Fraction No. Fracfion No. Fraction No. 7-13 M-27 2842 protein h~~u~~~ed u~~rea~ted chlorambwil chlorambucil __-..
MOI. CB ..-.__Mol. HyG
_-_-
-
15.5 41.4 93.6
84.5 58.6 6.3
-
3.3 23.6 59.4
15.6 23.0 37.2 ~_I
81.3 53.3 3.3
2.0 15.1 37.0 _..I_ -
~
_...~
(Figs. l-6) HUMAN
AND THE:
‘1,-GLOBULIN
266
W. C, 1. RO!B
fractions 7-13 and calculations indicate that 3.3 % of the applied chlorambucil is attached to this protein. Then follows hydrolysis products (15.6%) and unchanged chlorambucil(8 1.3 %). Figs. 5 and 6 give the comparable values for mixtures run 2 hr and 24 h after preparation. DISCUSSION
It has been shown that the reaction of chlorambucil in buffered saline can be followed by gel permeation chromatography. As already indicated the figures do not represent the composition of the mixtures as applied to the columns but if a comparable experiment is carried out with added globulin then various conclusions can be drawn. Firstly, there is no appreciable change in the rate of reaction of chlorambucil in the presence of human ~-glubuIin. This result agrees with that of HOPWOODAND STOCKY using bovine ~-globulin. Secondly, there is no evidence of strong binding of unreacted chlorambucil with the globulin. Only 3.3 “/, of the chlorambucil is bound in the run carried out within 5 min of mixing and even this amount can be accounted for by any alkylation reaction which must occur before the protein and the agent are separated on the column. Despite the higher ratio of chlorambucil/globulin (l-7.85) used compared with that of previous workers (I-10 or less) the percentage of agent bound is certainly less than 3 7:. It is appreciated that the extent of binding in eluates does not necessarily represent the situation in the mixture as applied to the column where the drug/protein ratio is l-7.85 but it probably does indicate that relatively little of the drug will be associated with the globulin soon after injection. If the claimed enhancement of anti-tumour activity is substantiated the present work indicates that the time of injection after preparation of the so-called conjugate is important. Should an appreciable time interval occur some of the chlorambucil will be combined with the globulin by an alkylation reaction* and certainly some of the bound agent will still have a reactive chloroethyl group which could react further after antibody-antigen combination. ACKNOWLEDGEMENTS
This work was supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research:RoyaI Cancer Hospital) from the Medical Research Council and the Cancer Research Campaign.
* There is ample evidence that alkylating proteins~.
agents of the mustard gas type combine covalently with
REFERENCES 1 T. GHOSE;,S. T. NORVELL, A. GWCLV,D. CAMERON,A. BODURTMA AND A. S. ~AC~ON~~~ Immunochemotherapy of cancer with chlorambucii-carrying antibody, Btit. hJed X, 3 (1972) 49s-499. 2 T. G~osa AND S. P. N~GA&%, Antibody as carrier of chlorambucil, Cancer, 29 (1972) 1398-I 3 I. FK.§c~ER, The cure and ~oncomit~t imm~~t~oo of mice bearing Ehrlich ascites tomom by treatment with an antibody-alkylating agent complex, Ewopean J. Cancer, 9 (1973) 743-745. 4 L. G. ISRAE~SAND J. H. LMFORD,Some observations on the reactions of chlorambucil, azomustard (CB 1414) and cyc~ophosph~ide, in R. W. BEGG(Ed.), Proceedings of the 5th Cordial Cancer C~nfereence, Academic Press, New York, 1963, pp. 399-415. 5 W. J. Ho~w~~ ANDJ. A. STOCK,The effect of mac~moI~u1~ upon the rates of hydrol~~s of aromatic nitrogen mustard derivatives, Chess.. Bioi. Interactions, 4 (1971172) 31-39, 6 W. C. J. Ross, 3jofogica~ ~~kyiuting Age&+ Buti~rwo~h, Landon, 1962, pp. 33-39.
OF CHLORAMBUCIL
WITH HfJMAN y-GLOBULIN
Gef p~r~~~~i~~~~~~~~~~~r~~h~ of solutions containing c~~~rarnbu~~~ and human pglobulin indicates that there is littte, if any physical binding of the drug but a progressive chemical j~tera~tjon. The rate of reaction of ~h~~rambnci1 is not signi~~ant~yabated by the presence of the globulin. The implications of these endings for current attempts at imm~~o~hemotherapy are discussed.
Recent claims for the en~n~ment of the anti~neopIastic activity of chgorambucil when admixed with immunoglobu~~ns (GHO~Eet uLXJ and FLECHNER~)has stimulated interest in the nature of the ant~b~y-drug complex. Pre~~rn~narywork here had indicated that the binding of Gh~or~buci~ to globulins was less marked than that implied in the afore-mentioned publications, For example, the passing of a solution contajning ~hlorarnb~~jl and human ~-globulin in the proportion l-10 by weight through a coiumn of Sepbadex G 25 resulted in the almost complete separation uf the components. This was not consistent with the 90% binding of the drug expected by the workers cited. There appears to be a misapprehension since fsRAr3.sAND L~NDFURI~~ du not appear to studied the binding of chlorambucil to y-glob&ins but only to mixed serum proteins. Their work using axr ultrafiltration technique at 5”-to minimise the reaction of the alkylating agent-relates to the amount of binding at a steady ehlorambucili concentration. It was conside ered that, if the components could be separated by gel filtration, which is electively a dialysis technique in which the co~~entra~o~ of drug is pro~essive~y reduced, then following injection of ~hlorambu~~~-g~abu~i~mixtures and s~b~quent dilution in tissue fluids a similar dissolution would occur. There were afso suggestions that the enhanced biological activity was due to rll
262
W.
C. J. ROSS
reduction in the rate of reaction of chlorambucil in the presence of globulin. This was not, in fact, supported by the quoted work of HOPWOODAND STOCKY. Accordingly, it was decided to examine the interaction of chlorambucil with globulin. A gel permeation chromatographic resolution of antibody-drug mixtures and a spectrophotometric method of assaying the components were developed. MATERIALS
AND METHODS
The sodium salt of chlorambucil was prepared by adding an exact equivalent of sodium methoxide to a methanolic solution of chlorambucil and evaporating to dryness under vacuum. The human lyophilized y-globulin was supplied by AB Kabi (Stockholm, Sweden). Sephadex G 25 (fine) and K9/60 columns (Pharmacia) were used for chromatographic analysis and the absorbance of eluates at 280 nm was monitored with an LKB Uvicord II UV absorptiometer. The absorbance data for pooled eluates was obtained using 1 cm silica cells in an SP 500 UV spectrophotometer. The pH 8 saline buffer was prepared by dissolving sodium chloride (IO g) in Fison’s pH7 M/20 phosphate buffer (1 1) and adjusting the pH to 8 by the addition of aqueous sodium hydroxide (18 ml N). Spectrophototnetric
data
A solution of chlorambucil
in pH 8 saline buffer (0.02589 mg/ml) immediately after mixing exhibited E:
The sodium salt of chlorambucil (34.7 mg equivalent to 32.36 mg acid) was dissolved in pH 8 saline buffer (5 ml) at 20”-the sodium salt was used as it dissolved much more rapidly than the free acid. Within 5 min of mixing 1 ml of the solution was applied to the Sephadex column which had been equilibriated with pH 8 saline buffer. Elution was carried out using the same buffer. The flow rate was 0.5 ml/min and each fraction contained 2 ml. The elution profile is shown in Fig. 1. The chromatographic resolution was repeated after keeping the mixture for 2 and 24 h at 20” (Figs. 2 and 3). To a solution of human y-globulin (250 mg) in pH 8 saline buffer (5 ml) at 20”
INT&%l.ACTlGN OF CHLGRAMRWCILWITH y-GLOBWLKN
I
263
SW.2
Figs. l-3. Chromatographic profile of chlorambucil(32.36 5 min. 2 h and 24 h after mixing.
mg) dissolved in pH 8 saline buffer (5 ml)
was added the sodium salt of chlorambucil(34.1 mg = 31.8 mg acid) and 1 ml of the mixture was sub~t~d to ~hromatographic resohrtion after 5 mm, 2 and 24 h (Figs. 4, 5 and 6). The El cmvalues of the various pooled eiuates, suitably diluted, were measured at 258 nm and 280 nm and the amounts of chlorambucil or derived product therein were calculated as indicated below. Example of calculation
For the chromatogram represented by Fig. 5:
fractions 7-I 3 in 100 ml fractions M-27 in 50 ml fractions 28-42 in 100 ml
I em E 258 nm
f em E 280 nm
0.886
0.696
0.976
0,138
I A54
0.16
w. c.
264
FRACTION
J. ROSS
NUMBERS
Figs. 4-6. Chromatographic profile of chlorambucil (31.8 mg) dissolved in pH 8 saline buffer (5 ml) human y-globulin (250 mg) 5 min. 2 h and 24 h after mixing.
containing
The amount of chlorambucil in fractions 28-42 is 0.16 - 100 * 0.216 or 3.45 mg (53.3 ,I/,), Fractions 14-27 contain products derived from chlorambucil equivalent to 0.138 * 50 *0.216 or 1.49 mg (23.0%). In fractions 7-13 there is 50 mg of globulin so that the absorbance due to the protein (0.5 mg/ml) is 0.625 at 280 nm. The absorbance attributable to the chlorambucil is 06960.625 or 0.071 and the amount of chlorambucil bound is 0.071 - 100 mO.216or 1.53 mg (23.6%). The total recovery of chlorambucil is 6.47 mg (101.7 %). The number of molecules of chlorambucil of the globulin (mol. wt. I50 000) is: 1.53 * 150 000 - or 15.1 50 * 304
(mol. wt. 304) bound to one molecule
INTBRACTION OF CHLORAMBUCIL
WITH y-GLOBULIN
265
RESULTS
Figs. 1,2 and 3 show the efuticm profiles of solutions of Ghlo~buciI in #I g saline buffer when applied 5 min, 2 h and 24 h after mixing, respectively. The data in Table I give the percentage of chlorambucil or derived product associated with various fractions. These values do not represent the com~sition of the mixtures as applied to the column since reaction will be proceeding during the elution which is at the rate of one fraction (2 ml) per 4 min. Nevertheless the runs with and without globulin are comparable since they were performed under identical conditions. Fig. I shows two components, chIorambuci1 (I, 84.5%) in fractions 28-42 and a faster moving Gompone~t in fractions M-27 (I 5.5 %). Fig. 2 shows a chIorambuci1 peak (58.6%) and the second area of absorption is split into 2 peaks (total 41.4%). After 24 h (Fig. 3) only 6.3% of the original Gh1orambucil is present and the fastest moving peak of the second region predo~nates (total 93.6%). It is probable that the peak (fractions 22-28) represel~ts half hydrolysed GhlorambuGi1(II) and the peak (fractions 14-22) contains fully hydrolysed agent (III) since the former peak decreases at the expense of the latter with time. ,CH&H,OH HOOCKH,)J
N (CH&H&l
Ia
N \
HOOCKH2&
0
0
W&-H&I
(ItI
(1)
iIOCX(CH&
N KH$H20H
12
Figs. 4,s and 6 show the elution profiles of solutions of chlorambucil(31.8 mg) in S “/::human ~-globulin (5 ml in pIi 8 saline buffer). The ch1orambucil~globu1inratio by weight is l/7.85. Fig. 4 shows that the protein emerges after th:: void volume in TABLE
I
THE PERCENTAGE NUMBER
(HyG)
OF
(Figs. 4, 5 and 6) ___-_..~-~-l
Time
____~.-w-.-5 min (Fig. 1) 2 h 24 h
OF APPLIED
MOLECULES
(Fig. 2) (Fig. 3)
5 min (Fig. 4) 2 h (Fig. 5) 24 h (Fig. 6) .______.. ~_-.---
OF
CNLO~AMBUCIL CHLORAMBUCIL
-
PRESENT
(CB)
IN THE VARlOUS
BOUND
TO
ONE
-___
_._-
FRACTIONS
MOLECULE
OF
-. .- -..- l_____-l
Percentage of applied chlorambucii ..___-I-~-Fraction No. Fracfion No. Fraction No. 7-13 M-27 2842 protein h~~u~~~ed u~~rea~ted chlorambwil chlorambucil __-..
MOI. CB ..-.__Mol. HyG
_-_-
-
15.5 41.4 93.6
84.5 58.6 6.3
-
3.3 23.6 59.4
15.6 23.0 37.2 ~_I
81.3 53.3 3.3
2.0 15.1 37.0 _..I_ -
~
_...~
(Figs. l-6) HUMAN
AND THE:
‘1,-GLOBULIN
266
W. C, 1. RO!B
fractions 7-13 and calculations indicate that 3.3 % of the applied chlorambucil is attached to this protein. Then follows hydrolysis products (15.6%) and unchanged chlorambucil(8 1.3 %). Figs. 5 and 6 give the comparable values for mixtures run 2 hr and 24 h after preparation. DISCUSSION
It has been shown that the reaction of chlorambucil in buffered saline can be followed by gel permeation chromatography. As already indicated the figures do not represent the composition of the mixtures as applied to the columns but if a comparable experiment is carried out with added globulin then various conclusions can be drawn. Firstly, there is no appreciable change in the rate of reaction of chlorambucil in the presence of human ~-glubuIin. This result agrees with that of HOPWOODAND STOCKY using bovine ~-globulin. Secondly, there is no evidence of strong binding of unreacted chlorambucil with the globulin. Only 3.3 “/, of the chlorambucil is bound in the run carried out within 5 min of mixing and even this amount can be accounted for by any alkylation reaction which must occur before the protein and the agent are separated on the column. Despite the higher ratio of chlorambucil/globulin (l-7.85) used compared with that of previous workers (I-10 or less) the percentage of agent bound is certainly less than 3 7:. It is appreciated that the extent of binding in eluates does not necessarily represent the situation in the mixture as applied to the column where the drug/protein ratio is l-7.85 but it probably does indicate that relatively little of the drug will be associated with the globulin soon after injection. If the claimed enhancement of anti-tumour activity is substantiated the present work indicates that the time of injection after preparation of the so-called conjugate is important. Should an appreciable time interval occur some of the chlorambucil will be combined with the globulin by an alkylation reaction* and certainly some of the bound agent will still have a reactive chloroethyl group which could react further after antibody-antigen combination. ACKNOWLEDGEMENTS
This work was supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research:RoyaI Cancer Hospital) from the Medical Research Council and the Cancer Research Campaign.
* There is ample evidence that alkylating proteins~.
agents of the mustard gas type combine covalently with
REFERENCES 1 T. GHOSE;,S. T. NORVELL, A. GWCLV,D. CAMERON,A. BODURTMA AND A. S. ~AC~ON~~~ Immunochemotherapy of cancer with chlorambucii-carrying antibody, Btit. hJed X, 3 (1972) 49s-499. 2 T. G~osa AND S. P. N~GA&%, Antibody as carrier of chlorambucil, Cancer, 29 (1972) 1398-I 3 I. FK.§c~ER, The cure and ~oncomit~t imm~~t~oo of mice bearing Ehrlich ascites tomom by treatment with an antibody-alkylating agent complex, Ewopean J. Cancer, 9 (1973) 743-745. 4 L. G. ISRAE~SAND J. H. LMFORD,Some observations on the reactions of chlorambucil, azomustard (CB 1414) and cyc~ophosph~ide, in R. W. BEGG(Ed.), Proceedings of the 5th Cordial Cancer C~nfereence, Academic Press, New York, 1963, pp. 399-415. 5 W. J. Ho~w~~ ANDJ. A. STOCK,The effect of mac~moI~u1~ upon the rates of hydrol~~s of aromatic nitrogen mustard derivatives, Chess.. Bioi. Interactions, 4 (1971172) 31-39, 6 W. C. J. Ross, 3jofogica~ ~~kyiuting Age&+ Buti~rwo~h, Landon, 1962, pp. 33-39.