CA2117775A1 - Process for preparing macrocyclic chelating agents and formation of chelates and conjugates thereof - Google Patents
Process for preparing macrocyclic chelating agents and formation of chelates and conjugates thereofInfo
- Publication number
- CA2117775A1 CA2117775A1 CA 2117775 CA2117775A CA2117775A1 CA 2117775 A1 CA2117775 A1 CA 2117775A1 CA 2117775 CA2117775 CA 2117775 CA 2117775 A CA2117775 A CA 2117775A CA 2117775 A1 CA2117775 A1 CA 2117775A1
- Authority
- CA
- Canada
- Prior art keywords
- chelating agent
- polyaza
- chelate
- activated
- hydrogen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6524—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having four or more nitrogen atoms as the only ring hetero atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
- A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
- C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6515—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having three nitrogen atoms as the only ring hetero atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2123/00—Preparations for testing in vivo
Abstract
The present invention is directed to an improved process for preparing macrocyclic chelating agents and for conjugating the macrocyclic chelating agents to biological molecules.
Description
3 ^~ ~ 7 r~ 7 -WO 93/20~52 PCl`/US93/03483 PROCESS FOR PREPARING MACROCYCLIC CHELATING AGENTS AND FORMATION OF CHELATES
AND CONIUGATES THEREOF
The present invention relates to a process ~or preparing isothiocyanato 5 functionalized rnacrocyclic chelating agents and to a pro~ess for conjugating the macrocycl ic cheiating agentsto biological molecules.
Metal ions may ~e attached to biological molecules by means of bifunctional chelating agents. Such chelating agents are compoundswhich contain a metal-binding moiety which forms a chelate with metal ions and a second functional group, which is chernical Iy 10 reactive in na~ure and is capable of forming a covalent bond with biological molecules. The reactive functionality is usually one of the various known useful chemically reactive groups such as bromoacetyl group, a diazonium ion, an isothiocyanate or a carboxylic acid derivative, the reactive functionalities being capable of binding to an amino acid of a protein (e.g., the Iysine moiety of an antibody~. The biological molecules usually recognize distinctive e~ernal or internal cell markers and, thus, act as target directing groups for the metal ion.
When the bifunctional chelatins~ agent is covalently attached to an antibody having specificity for cancer or tumor cell epitopes or antigens, radionucl ide chelates o~ such anffbodylchelating agent conjugates are useful in diagnostic and/ot therapeutic applications as - a means of conveying the radionuclide to a cancer or tumor cell. See, for example, 2~ Meares et al ., Anal. ~iochem., ~, 68-78 (19~4~, and Krejcarek et al., Biochem. and Biophys.
Res Comrn., 77. 581-585 (1977).
IsothiocyanatG functionalized derivatives of ethylenediaminetetr~acetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) are reported in the literatJre and are being used to conlugate radioactive isotopes to anti bodies. For example see Gansow et al ., 25 Inorg. Chem., ~, 2n2-2781 (1986); Meares et al., Anal. Biochem., ~, 6~78 ~1984); and U.S.
Pa~ent 4,454, 106.
VVhen using short-lived radionuclides, i~ is desirable to chelate the radionuclide to thetarget directing group as close as possibte to the time of injection into the patient to provide maximum specific radioactivity and minimum degradation of the 30 radioimmunoglobulin. When using chelating agents such as EDTA and DTPA, the rapid sequestration of metal ions by these chelating agents aliows the preparation of the radionuclidelantibody/chelating agent conjugates (referred to herein as conjugates) to be prepared by activating the chelating agent, reacting the chelating agent with an antibody and then chelating the radionuclide to form a complex followed by purification of the complex.
However, although chelating agents such as EDTA and DTPA rapidly chelate the radionuclide, they suffer from the disadvantage that such binding is kinetically labile. In addition, the use of labile radionuclides for antibody labeling in this manner allows substitutionally labile trace metals (which may not be radioactive) to be incorporated into the , WO 93/20852 PCI'/US93/03~183 chelate. Competition for such non-active trace metals diminishes the biological efficacy of the antibody/chelate complex since a lower quantity of radionuclide is delivered to the target site.
A disadvantage associated with chelating agents such as EDTA and DTPA is the premature release of the chelated radioactive isotope. To lower the rate of metal release 5 in vivo, bifunctional chelating agents, based on macrocyclic ligands, such as DOTA
(1 ,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid), have been used. See, for example, Mirzadeh et al., Biocon~ugate Chem., 1 5~65 (1g90); and Deshpande et al., J. Nucl.
Med., ~, 473-479 (t990).
A disadvantage in the use of macrocyclic chelating agents is the relatively slow10 chelation rates which take place at room temperature, see for example, Mirzadeh et al., Bioconjugate Chem., ~, 5~65 ~1990). To reduce the radiolysis which occurs with prolonged chelation tirnes, the medium in which the chelation takes place is often heated to increase the rate of chelation. As the isothiocyanate functionality is heat sensitive, U.S. Patent 5,006,643 discloses the preparation of isocyanato functionalized macrocycles containing primary or ,sçcondary amines by first reacting the chelati ng agent with rhodium and then activating the chelate. U.S. Patent 4,885,363 discloses chelating gadolinium to a tetraazamacrocycle contai ning tertiary amines prior to activating with an isothiocyanate functional group. Using t his procedure for preparation of metai chelate-protein conjugates to be used in radioimmunotherapy or radiodiagnostics requires the formation of the chelate, activation of 20 the chetate~and~then conlugation of the activated chelate to an antibody in a short period of time to avoid a substantial loss in activity.
Another disadvantage in the use of macrocyclic chelati ng agents is that duri ngtheir synthesis and purification, the reagents and materials used must be free of extraneous metal lons. Metal on contami nants present during these steps may become tightly bound to 25~ the~macrocycles and not easily displaced during chelation of the desired metal ion. Metal ion contamination dùring~synthesis and purifkation will reduce the purity oF the product obtained durlngchelationofthedesiredmetal~ionaswellasreducethepurityofproductobtainedin subsequent steps in producing the conjugate.
~ .
Consequently, it would be advantageous to have a process to prepare purifi ed macrocyclic chelating agents which are substantially pure of contaminating metal ions.
It would also be advantageous to have a process to prepare the isothiocyanate activated chelating agent in an aqueous environrnent with an increased yleld and higher punty thai~ can oe prepared vvhen the reaction is performed in an organic solvent/water environment.
It would be desirable to provide a chelating agent which will react ra~idly with a radionuclide at rc,om temperature formlng a chelate whlch does not readily dissociate, allowing the chelating agent to be activated to an issthiocyanate prlor to forming the chelate Activating the chelating agent prior ~o chelation of the metal ion allows the bifunctlona :
:;
WO 93/21~852 2 1 ' 7 7 7 ,` PCI'/IJS93/03483 chelating agent to be prepared in an activated form in advance of when it is needed to produce a conjugate.
It would be further advantageous to have a process which allows rapid conjugation of the chelate with an antibody to reduce radiolysis and antibody aggregate 5 formation. Rapid formation s:~f the conjugate with a reduction in undesired reactions would result in a more complete conjugation reaction and result in easier product purification. Also, having a process wherein the yield and purity from each step is increased is particularly advantageous in preparing cc~mpounds and compositionswhich will be used in pharmaceutical applications where the pharmaceutical composition must meet the purity criteria set-forth by 10 the U. S. Food and Drug Administration.
The present invention provides a chromatographic process for prevention of extraneous metal ion incorporation in a rnacrc cyclic chelating agent during purification. The process is advantageous over existing procedures in tha~ multigram quantities of macrocyclic chelating agent free of divalent cations can be produced and purified in a single unit 15~ operation~
The invention also provides a pro~ess for preparing isothiocyanate compounds which comprises reacting thiophosgene with a polyaza ch~lating agent in an aqueous environment in the absence of an organic solvent to ~orm an isothiocyanato derivatized polyaza chelating agent, wherein the polyaza chelating agent is of the ~ormula Q
~5 ~C~(cH2)--C--(CH2)r Q
(I) :
wherein:
each Q is independentiy hydrogen, (CHR5)pC02R or (CHR5~pPO3H2;
Ql is hydrogen or (cHR5)wco2R;
each R independently is hydrogen, benzyl or Cl-4 alkyl;
with the proviso that at least two~of the sum of Q and Q1 rnust be other than hydrogen;
each R5 independently is hydrogen, C1-4 alkyl or -(C1-C2 alkyl)phenyl;
X and Y are each independently hydrogen or may be taken with an adjacent X
35 ~nd Y to form an additional carbon-c~arbon bond;
nisOorl;
m is an integer from O to t O inclusive;
AND CONIUGATES THEREOF
The present invention relates to a process ~or preparing isothiocyanato 5 functionalized rnacrocyclic chelating agents and to a pro~ess for conjugating the macrocycl ic cheiating agentsto biological molecules.
Metal ions may ~e attached to biological molecules by means of bifunctional chelating agents. Such chelating agents are compoundswhich contain a metal-binding moiety which forms a chelate with metal ions and a second functional group, which is chernical Iy 10 reactive in na~ure and is capable of forming a covalent bond with biological molecules. The reactive functionality is usually one of the various known useful chemically reactive groups such as bromoacetyl group, a diazonium ion, an isothiocyanate or a carboxylic acid derivative, the reactive functionalities being capable of binding to an amino acid of a protein (e.g., the Iysine moiety of an antibody~. The biological molecules usually recognize distinctive e~ernal or internal cell markers and, thus, act as target directing groups for the metal ion.
When the bifunctional chelatins~ agent is covalently attached to an antibody having specificity for cancer or tumor cell epitopes or antigens, radionucl ide chelates o~ such anffbodylchelating agent conjugates are useful in diagnostic and/ot therapeutic applications as - a means of conveying the radionuclide to a cancer or tumor cell. See, for example, 2~ Meares et al ., Anal. ~iochem., ~, 68-78 (19~4~, and Krejcarek et al., Biochem. and Biophys.
Res Comrn., 77. 581-585 (1977).
IsothiocyanatG functionalized derivatives of ethylenediaminetetr~acetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) are reported in the literatJre and are being used to conlugate radioactive isotopes to anti bodies. For example see Gansow et al ., 25 Inorg. Chem., ~, 2n2-2781 (1986); Meares et al., Anal. Biochem., ~, 6~78 ~1984); and U.S.
Pa~ent 4,454, 106.
VVhen using short-lived radionuclides, i~ is desirable to chelate the radionuclide to thetarget directing group as close as possibte to the time of injection into the patient to provide maximum specific radioactivity and minimum degradation of the 30 radioimmunoglobulin. When using chelating agents such as EDTA and DTPA, the rapid sequestration of metal ions by these chelating agents aliows the preparation of the radionuclidelantibody/chelating agent conjugates (referred to herein as conjugates) to be prepared by activating the chelating agent, reacting the chelating agent with an antibody and then chelating the radionuclide to form a complex followed by purification of the complex.
However, although chelating agents such as EDTA and DTPA rapidly chelate the radionuclide, they suffer from the disadvantage that such binding is kinetically labile. In addition, the use of labile radionuclides for antibody labeling in this manner allows substitutionally labile trace metals (which may not be radioactive) to be incorporated into the , WO 93/20852 PCI'/US93/03~183 chelate. Competition for such non-active trace metals diminishes the biological efficacy of the antibody/chelate complex since a lower quantity of radionuclide is delivered to the target site.
A disadvantage associated with chelating agents such as EDTA and DTPA is the premature release of the chelated radioactive isotope. To lower the rate of metal release 5 in vivo, bifunctional chelating agents, based on macrocyclic ligands, such as DOTA
(1 ,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid), have been used. See, for example, Mirzadeh et al., Biocon~ugate Chem., 1 5~65 (1g90); and Deshpande et al., J. Nucl.
Med., ~, 473-479 (t990).
A disadvantage in the use of macrocyclic chelating agents is the relatively slow10 chelation rates which take place at room temperature, see for example, Mirzadeh et al., Bioconjugate Chem., ~, 5~65 ~1990). To reduce the radiolysis which occurs with prolonged chelation tirnes, the medium in which the chelation takes place is often heated to increase the rate of chelation. As the isothiocyanate functionality is heat sensitive, U.S. Patent 5,006,643 discloses the preparation of isocyanato functionalized macrocycles containing primary or ,sçcondary amines by first reacting the chelati ng agent with rhodium and then activating the chelate. U.S. Patent 4,885,363 discloses chelating gadolinium to a tetraazamacrocycle contai ning tertiary amines prior to activating with an isothiocyanate functional group. Using t his procedure for preparation of metai chelate-protein conjugates to be used in radioimmunotherapy or radiodiagnostics requires the formation of the chelate, activation of 20 the chetate~and~then conlugation of the activated chelate to an antibody in a short period of time to avoid a substantial loss in activity.
Another disadvantage in the use of macrocyclic chelati ng agents is that duri ngtheir synthesis and purification, the reagents and materials used must be free of extraneous metal lons. Metal on contami nants present during these steps may become tightly bound to 25~ the~macrocycles and not easily displaced during chelation of the desired metal ion. Metal ion contamination dùring~synthesis and purifkation will reduce the purity oF the product obtained durlngchelationofthedesiredmetal~ionaswellasreducethepurityofproductobtainedin subsequent steps in producing the conjugate.
~ .
Consequently, it would be advantageous to have a process to prepare purifi ed macrocyclic chelating agents which are substantially pure of contaminating metal ions.
It would also be advantageous to have a process to prepare the isothiocyanate activated chelating agent in an aqueous environrnent with an increased yleld and higher punty thai~ can oe prepared vvhen the reaction is performed in an organic solvent/water environment.
It would be desirable to provide a chelating agent which will react ra~idly with a radionuclide at rc,om temperature formlng a chelate whlch does not readily dissociate, allowing the chelating agent to be activated to an issthiocyanate prlor to forming the chelate Activating the chelating agent prior ~o chelation of the metal ion allows the bifunctlona :
:;
WO 93/21~852 2 1 ' 7 7 7 ,` PCI'/IJS93/03483 chelating agent to be prepared in an activated form in advance of when it is needed to produce a conjugate.
It would be further advantageous to have a process which allows rapid conjugation of the chelate with an antibody to reduce radiolysis and antibody aggregate 5 formation. Rapid formation s:~f the conjugate with a reduction in undesired reactions would result in a more complete conjugation reaction and result in easier product purification. Also, having a process wherein the yield and purity from each step is increased is particularly advantageous in preparing cc~mpounds and compositionswhich will be used in pharmaceutical applications where the pharmaceutical composition must meet the purity criteria set-forth by 10 the U. S. Food and Drug Administration.
The present invention provides a chromatographic process for prevention of extraneous metal ion incorporation in a rnacrc cyclic chelating agent during purification. The process is advantageous over existing procedures in tha~ multigram quantities of macrocyclic chelating agent free of divalent cations can be produced and purified in a single unit 15~ operation~
The invention also provides a pro~ess for preparing isothiocyanate compounds which comprises reacting thiophosgene with a polyaza ch~lating agent in an aqueous environment in the absence of an organic solvent to ~orm an isothiocyanato derivatized polyaza chelating agent, wherein the polyaza chelating agent is of the ~ormula Q
~5 ~C~(cH2)--C--(CH2)r Q
(I) :
wherein:
each Q is independentiy hydrogen, (CHR5)pC02R or (CHR5~pPO3H2;
Ql is hydrogen or (cHR5)wco2R;
each R independently is hydrogen, benzyl or Cl-4 alkyl;
with the proviso that at least two~of the sum of Q and Q1 rnust be other than hydrogen;
each R5 independently is hydrogen, C1-4 alkyl or -(C1-C2 alkyl)phenyl;
X and Y are each independently hydrogen or may be taken with an adjacent X
35 ~nd Y to form an additional carbon-c~arbon bond;
nisOorl;
m is an integer from O to t O inclusive;
wo 93/20852 ~ -- 3- 7 ~ PCI'/US93/03483 p= lor2;
r = Oor1;
w = Oor 1;
with the proviso that h is only I when X andlor Y form an additional carbon-carbon bond and 5 thesumof randwisOor 1;
R2 and R4 are independently hydrogen, or amino;
R3 Ct-C4 alkoxy, -OCH2CO2H, hydroxy and hydrogen;
with the proviso that R2 and R4 ~annot both be hydrogen but one of R2 and R4 must be hydrogen.
The present invention also describes a process for preparing a conjugate which comprises reacting an isothiocyanato activat~d chelate with an antibody at between 25C and 40C; wherein the chelate is a chelating agent cc)mplexed with a metal ion, the chelating agent isasdescribed in Formuia I andthe metal ionis 1535m, l66Ho, 175Yb, 177Lu, 159Gd, 140La, 142Pr, t ~spm, goy or l l 1 In~
The current invention also provides an improved process for formation of a chelate-antibody conjugate wherein the improvement comprises contacting the chelate with an antibody at 25C to about 40C.
The present Invention provides an improved process to prepare polyaza metal conjugates.~ In particular, the present invention is a process for preparing conjugates having at ~: 20 least the steps Of (a) purifying a polyaza chelating agent, the polyaza chelating agent being as defined in Formula l;
b) contacting the purified polyaza chelating agant with thiophosgene to form an~is~thiocyanato activated polyaza chelating agent and recovery of ttle isothiocyanato : 2~: activatedpolyaza:chelatingagent;
(c) adding a metal ion to the isothiocyanto activated polyaza chelati ng agent to form~an isothiocyanato activated polyaza chelate; and (d) contacting the isothiocyanato adivated polyaza chelate with a biological molecule to form a conjugate;
30~ the improvementwhich cornprises:
in step (a): purifying the polyaza chelating agent by chromatography on a silicaget column j wherein the silica gel has been acid washed pri or to loading the polyaza chelati ng agent onto the column;
in step ~b): contacting the pùrified polyaza chelating agent with thiophosgene in 35 an aqueous environme~nt in the absence of an organic solvent at a pH from 1 to 5; and ;~ i n step (d) contacting the isothiocyanato activated polyaza metal chelate with a biological molecule at between 25C to 40C.
r = Oor1;
w = Oor 1;
with the proviso that h is only I when X andlor Y form an additional carbon-carbon bond and 5 thesumof randwisOor 1;
R2 and R4 are independently hydrogen, or amino;
R3 Ct-C4 alkoxy, -OCH2CO2H, hydroxy and hydrogen;
with the proviso that R2 and R4 ~annot both be hydrogen but one of R2 and R4 must be hydrogen.
The present invention also describes a process for preparing a conjugate which comprises reacting an isothiocyanato activat~d chelate with an antibody at between 25C and 40C; wherein the chelate is a chelating agent cc)mplexed with a metal ion, the chelating agent isasdescribed in Formuia I andthe metal ionis 1535m, l66Ho, 175Yb, 177Lu, 159Gd, 140La, 142Pr, t ~spm, goy or l l 1 In~
The current invention also provides an improved process for formation of a chelate-antibody conjugate wherein the improvement comprises contacting the chelate with an antibody at 25C to about 40C.
The present Invention provides an improved process to prepare polyaza metal conjugates.~ In particular, the present invention is a process for preparing conjugates having at ~: 20 least the steps Of (a) purifying a polyaza chelating agent, the polyaza chelating agent being as defined in Formula l;
b) contacting the purified polyaza chelating agant with thiophosgene to form an~is~thiocyanato activated polyaza chelating agent and recovery of ttle isothiocyanato : 2~: activatedpolyaza:chelatingagent;
(c) adding a metal ion to the isothiocyanto activated polyaza chelati ng agent to form~an isothiocyanato activated polyaza chelate; and (d) contacting the isothiocyanato adivated polyaza chelate with a biological molecule to form a conjugate;
30~ the improvementwhich cornprises:
in step (a): purifying the polyaza chelating agent by chromatography on a silicaget column j wherein the silica gel has been acid washed pri or to loading the polyaza chelati ng agent onto the column;
in step ~b): contacting the pùrified polyaza chelating agent with thiophosgene in 35 an aqueous environme~nt in the absence of an organic solvent at a pH from 1 to 5; and ;~ i n step (d) contacting the isothiocyanato activated polyaza metal chelate with a biological molecule at between 25C to 40C.
- 4~
,, .
wO 93/20852 2 ~ ~ ~ r~ 7 ~ PCI`/US93/03483 As used herein, theterms "chelating agent" or nligand" mean a compound capable of chelatin~ or sequestering a metal ion. The term Nchelate" means a chelating agent which has sequestered a metal ion.
The term "bifunctional chelating agent" refers to compounds that have a moiety 5 capable of chelating a metal ion and a linker/spacer moiety covalently bonded to the chelati ng moiety that is capable of being activated or function~ ecl to serve as a means to covalently attach to a biological molecule.
The term "biological molecule" refers to any protei n, antibody, antibody fragment, hormone, antigen or hapten which functionsto recognize a specific biological 10 target site. Such a biological molecule, when attached to a functionalized chelate, serves to carry the attached metal ion to specific targeted tissues. Preferably, the bislogical material is an antibody or antibody fragment.
As used herein, "antibody" refers to any polyclonal, monoclonal, chimeric antibody, heteroantibody, or recorr~binant or derivative thereof, preferably a monoclonal 15 an~ibody. As used herein the term "antibody fragment" includes Fab fragments and F(ab'~2 fragrnents and any portion of an antibody, including recombinants and derivatives thereof, having specificity toward a desired epitope or epitopes. The antibody fragments may be produced by onventional enzymatic methods or by genetic or protein 0ngineering techniques, such asthe production of single chain antibodies.
The terms ~activateJ~ or "activating!' in relatiorl to a chelating agent means the c helating agent has been modified with a functional group which is capable of forming a covalent bond with a biological molscule.
The term "conj ugate~ as used herein refers to a complex of a ~iological material attached to a bifunc~ional chelating agent or bifunctional chelate. The term "antibody/chelate 25 conjugate" refers to an antibody which is covalently attached to a bifunctional chelate, (i.e., the bifunctional chelating agent having a chelated rnetal ion).
A general overall procedure giving the steps for preparation of conjugates used in the present invention is given in the following 5cheme 1. Steps (b) and (d of the process scheme may be reversed. When sequestering metal ions which are radionuclides with short half lives, it is preferred that the anivation occurs before formation of a chelate to avoid radiolysis which would occur during the subsequent activating step and any subsequent purification.
W093/~0852 h ~ ~ 7 7 ~ PC~/US93/O~B3 Scheme 1 Synthesi~ of a Chelating Agent Purification of the Chelating Agent on Silica Gel Step (a~
I
1t Activation of the Chelating Agent Step ~b~
1' . I
Chelation of a Metal Ion by the Activated Chelating Agent to Form a Chelate Step tc) I
2t Conjugation o~ the Chelate to a Biological Material to Form a Conjugate Step ~d) 2' ~ .
Puri~ication of the Conjugate :
3t :
. .
-ç,-WO 93/20852 2 1 ~ 7 r~ 7 ~ PCI`/US93/03483 The present invention provides a process for reducing extraneous metal ions, specifically calcium ions (Ca2 +), during the purification of macrocyclic chelating agents by flash chromatography using silica gel. It has been found that commercially aYailable silica gel used to purify the macrocyclic chelating agents unexpectedly contai ns a source of undesired metal 5 ions, particularly divalent cations such as calcium, which become tightly bound to the chelating agentduringpurification. Thewashingofthesilicagelwithastrongmineralacid,suchas hydrochloric, nitric, sulfuric, perchloric or hydrobromic, prior to use substantially reduces the amount of undesired metal ions bound ts the chelating agent during the purification process.
Preferably, the mineral acid used to wash the silica gel is hydrochloric acid. Procedures for 10 washing silica gel with a strong acid to remove impurities are known in the art, see, for example, Ralph K. Iler, The ChemistryofS~lica, John Wiley & Sons (1979).
The elimination of undesired rnetal ions from the macrocyclic chelating agents during the initial purification results in several advantages in the subsequent steps for forming a conjugate. The elimination of undesired bound metal ions from the macrocyclic chelating 15 agents allows rapid chelation of the desired metal ions at room temperature as opposed to heating or long reaction times required to displace the calcium. The ability ~o perform the cnelation step at room temperature, rather than at temperatures which would destroy the functionalizing group, allows the activation of the macrocyclic bifunctional chelating agent prior to chelation of the metal ion. The ability to functionalize the macrocyclic chelating agent ;~o prior to chelation of the me~al ion is particularly advantageous as the activated chelating agent can be synthesized, stored and then s~questration of the metal ion performed shortly before conjugation to the biological material. This is particularly advantageous when the metal ion is a radionuclide with a half li~e of 10 days or less, as a significant amount of radiolysis can occur during ~he time n~cessary for functionalizing the chelate, conjugation, and purification of the 25 products from each step. Preclusion of undesired metal ions is particularly important in insuring rapid ~Jptake of radioactive metals of the 13nthanide (111) series.
The abilityto rapidly chelate the radionuclide after activation of the chelatingagent also allows for simpler 3nd more efficient purification procedures to be used in preparation of the conj ugate. ~
The present process for purifying bifunctional chelating agents can be used for many classes of ligands including any tri to hexa -(carboxylated), -(phosphonomethylated), or -(phosphinomethylated) polyazamacrocycle where the polyazamacrocycle has 3 to 6 nitrogens in the ring and the total number of atoms in the ring is 9 to 24 atoms. Of particular interest are aminocarboxylic acid chelaeing agents, amlnophosphonit chelating agents and polyaza 35 chelating agents containing secondary and tertiary amines. Preferred polyazamacrocyclic chelating agents are of the formula WC)93/20852 ~1 7775 PCI/~JS93/03~183 2~ (CH2)--C--(CH2)r ~ ,~
(I) wherein:
each Q is independently hydrogen, (CHR5)pC02R or (CHR5)pPO~H2;
Q1 is hydrogen or (CHR5)wCO2R;
each R independently is hydrogen, benzyl or C1-C4 alkyl;
with the proviso that that at least two of the sum of Q and Ql must be other than hydrogen;
each R5 independently is hydrogen, C1-C4 alkyl or -(C1-C2 alkyl)phenyl;
X and Y are each independently hydrogen or may be taken with an adjacent X
and Y to form an additionaJ carbor~carbon bond;
nisOorl;
~0 m is an integer from O to 10 inclusive;
p= lor2;
r= oGr1;
w = Oor1;
with the prsviso that n is only 1 when X and/or Y form an additional carbon-carbon bond, and 25 the sum of r and w is O or 1;
R2 and R4are independen~ly hydrogen, nitro, amirlo or carboxyl;
~: R3isC1-C4alkoxy,-OCH2CO2H,hydroxyorhydrc\gen; :~
with the proviso tha~ R2 and R4 cannot both be hydrogen but one of R2 ~nd R4 must be hydrogerl.
30~ Preferred amines of the polyaza chelating agents of Formula I are tertiary amines, preferably where r is O and each Q is (CHRS)pCO~R.
Procedures fw synthesizing polyaza macrocycles are well known in the art.
Examples of preferred chelating agents are given in Table I and are named as foilows:
I A is 1 -(4-aminophenyl)- 1,4,7,1 O-tetra-azacyclododecane-1,4,7, 1 0-tetraacetic acid;
I B is 1-~2-~4-aminophenyl)ethyl] -1,4,7,10-tetraazacyclododecane--4,7,10-triacetic acid;
f'~ 7 ~ I v WO 93/2~852 PCr/US93/~34$3 ."
I C is 1-[2-(~aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane--1,4,7,10-tetraacetic acid;
I D is 1-(5-amino-2-rnethoxybenzyl)-1,4,7,10-tetraazacyclododecane--1,4,7,10-tetraaeetic acid;
I E is 1-(5-amino-2-hydroxybenzyl)-1,4,7,10-tetraazacyclododecane--4,7,10-triaceticacid; and I F is 1-[2-(4-aminophenyl)ethyl] -1,4,7,10-tetraazacyclododecane--1-(R,S,-acetic-4,7,10-tris-(S-methylacetic) acid, the preparation of which is given in European Patent Publication No. 0420942, published April 10, 1991.
: 10 The preparation of IA-IE is given in European Patent Publication No. 03S3450, published February 7, 1990, and European Patent Publication No. 0420942, published April 10, 1991.
''''' ' ;
: .:
: ~25 .
, `: : : :
::~ 35 -W(:~93/2~852 f,~ ~ 3 777~ PCI/US93/03483 TABLE I
r COOH
CO2~
N COOH
H2N ~COOH
rcoo~l .
lSr /@ ~ ~\> I B
H2N N~>
--COOH
.
COOH r COOH
)~0 ~ COOH I C
2~ ` COO~
:
: :
.'.
: , WO 93/21~852 2 i 1 7 7 7 :3 PCr/US93/03483 TABLE I CONT ' D
r COOH
COOH
~N ~<~ ~,~
OCH3 \~COOH
0 r COOH
H!!N ~ COO~ I E
N ....
OH ~ COOH
:
COOH /'--COOH I F
~,J~ ~ N ~
I O I N ~r--COOH
H N ~ ~ H3 fOOH
25 ~ CH3 ` : ~ : .
: :
:: : , Methods for the carbc~xylation of an arnine of a ligand to give amine derivatives containing a carboxyalkyl group are well known, as are the methods which give alkyl 3q ~ phosphonic and hydroxyalkyl~substituents on the amine nitrogens. ~See, for example, U.S.
Patents 3,726,g12 and 3,398,198.l Aminophosphonic acid derivatives of ligands can be prepared by a number of known s~nthetic techniques. Of particular importance isthe reaction of a compound containing at least one reactive amine hydrogen with a carbonyl compound (aldehyde or 35 ketone) and phosphorous a~id or derivative thereof. [See the procedure of Moeoritzer and Irani,J Org. Chem.,~, 1603(1966~.]
- 1 1 - .
.WO93/20852 ~ 777~ PCI/US93/03483 The polyaza chelating agents of the present invention purified by flash chrornatography on acid washed silica gel can be activated with any of the known functional groups capable of forming a covalent bond with a biological molecule. Examples of such functional groups are isothiocyanate, bromoacetamide, maleimide, imidoester, 5 thiophthalamide, diazonium and carboxyl. The use of polyaza chelating agents which are essentially free of undesired metal ions are particularly important when using functionalizing groups which are pH andlor thermally unstable, such as isothiocyanate. The activating functional grc ups are located at positions R2 or R4 of Formula I on the macrocyclic chelating agent.
It has been unexpectedly found that when reacting the polyaza chelating agent with thiophosgene to activate the chelating agent with an isothiocyanate group, conducting the reaction in an aqueous environment, preferably in a di lute acid, with vigorous mixing i n the absence of an organic solvent results in an activated chelating agent of greater purity than that obtained in the presence of an organic solvent. This is in contrast to the known procedures of 15 using a pH of 8. 5 and/or adding the thiophosgene in the presence of an organic solvent. See, for example, Brechbiel et al., Inorg. Chem., ~, 2772-2781 (1986); Keana and Mann, J. Org.
Chem., 55, 2868-2871 (1990); Westerberg et al.jJ. Med. Chem., ~ 23~243 (1989); and Kline et al., Bioconjugate Chem., 2, 2~31 (1991). The reaction of the thiophosgene with the - ~ chelatlng agent is~at a pH from 1 to 7. Preferably, the pH is frorn 1 to 5. More preferably, the 20~ pH is from 1 to 3 The reaction between the~ chelating agent and thiophosgene is preferably ~arriedout in an aqueous environment having an acid concentration of from about 0.005 to about 0.S~normal (N), preferably 0.01 to 0 2 N. ~ The acid can be any mineral acid, preferably hydrochloric acid. The reaction betweèn the cheiating agent and thiophosgene in a dilute acid 25 ;~with~ vigorous mixing and excess~thiophosgene is very fast and usually complete i n less than 5 minutes at room temperature~(1 5C to 25C). Higher or lower temperatures can be used (e~g., 0Cto S0C) but room temperature is preferred.
The amount of excess thiophosgene added to the mixture depends on the concentration of the polyaza cheiating agent. The lower the concentration of chelating agent, 30: the larger the excess of thiophosgene to insure the rapid and complete conversion of amine to isothiocyanate. For exarnple, if the concentration of chelating agent is 10-3 M, the ratio of thiophosgene to chelating agent is S-20: 1. If the concentration of chelating agent is 10-8 M, the ratio of thiophosgene to chelating agent is several thousand times larger (i.e., 105:1). The excess thiophosgene is removed by conventionai techniques such as evaporati on, 35 ~hromatography or extraction.
Rapid mixing of thiophosgene with the aqueous solution may be accomplished using conventional equipment known to those in the art which is capable of producing sufficient shear to produce dispersion of the thiophosgene within the aqueous solution.
, .
WO 93/20X52 '~ 1 ~ 7 7 ~ 5 PCI/US93/03483 Illustrative of such mixing means is the use of a Waring blender for large scale preparations and with a Mixxor~ type mixer available from Alltech tnc., for smaller scale preparations.
By conducting the reaction of the chelating agent with thiophosgene as described above, the obtained purity of the isothiocyanato activated chelating agent is about 5 90 to about 95 percent as measured by high performance liquid chromatcgraphy (HPLC).
A~ter the chelating agent has been activated, the activated chelating agent is placed in contact with a metal ion to form the chelating agenVmetal ion chelate. Although any metal ion, whether a radioactive metal ion or not, can be used which is sequestered by the chelating agent, the chelates formed should have reasonable stability such that the metal 10 complex is not readily disassociated. P~adionuclides are preferred because of the use of the resulting products in a radiopharmaceuticai drug for therapy and/or diagnosis. Especially preferred radioactive isotopes are those of samarium (Sm-153), holmium (H~166), ytterbium (Y~175), lutetium (Lu-177), gadol;nium (Gd-159), lanthanum (La-140), praseodymium (Pr-142), promethium (Pm-149). yttrium (Y-90) and indium (In-111).
Radionuclides can be produced in s~veral ways. In a nuclear reactor a nuclide isbombarded with neutrons to obtain a radionuclide, e.g., Sm-t52 ~ neutron ~ Sm-153 ~ gamma Another methocl of obtaining radionuclides is to bombard nuciides with particlesproduced by a linear accelerator or a cyclotron. 'Yet another way is to isolate the radionuclide 20 from a mixture of fission products. The method of obtaining the nuclides employed in the present invention is not critical thereto.
The radionuclides can be complexed with the bifunctional chelating agent by adding the bifurctional chelating agent to a solution of the radionuclide. Chelates form readily upon mixing in an aqueous solution at a pH of 1 to 10. Preferably, the reaction is carried 25 out in a rnedium having a pH of 1 to 7 and more preferably 5to 7. Ambienttemperatures of about t 0C to 4ûC can be readily employed for metal ion chelation. The amount of metal ion employed may be from trace amounts to an amount in excess of equimolar with the chelate.
Preferablyj the formation of the chelate occurs at room temperature, between 1 5C to 25C.
Chelation proceeds rapidly and yields of about 90 percent and greater as 30 measured by high performance liquid chromatography are obtained when using a chelating agent which has been initially purified on subs~antially calciurn free silica gel and activated with thiophosgene prior to chelation of the desired metal ion.
The ability to rapidly form chelates over a wide pH range is advantageous to obtain a high chelation yield of the desired metal ion. When it is necessary to displace divalent 35 cations such as calcium prior to chelation of the desired metal ion, yields are variable due to the pH sensitive nature of the displacement reanion. Displacement of the calcium ion requires mnninS} the reaction at a pH sufficiently low to displace ~he calcium, yet the pH cannot be too low as ~o adversely affect the chelation of the desired metal ion. Util izi ng the procedure of the r~ 7 ~
WO 93/~0852 PCI'/US93/03483 present invention allows the chelating reaction to proceed with high yields over a broad pH
range.
The ability to activate the chelating agent prior to forming the chelate and therapid formation of the chelate with a high yield also has tl~e advantage in that it reduces the 5 purification necessary prior to the conjugation step. When the metal ion is chelated prior to activation using thiophosgene, it is necessary to extract the unreacted thiophosgene with an organic solvent and then purify the chelate prior to conjugation. The low concentration of the chelate in the elution volume obtained after purification using an ion exchange column necessitates concentrating the chelate prior to conjugation. During the process of extracting 10 the thiophosgene and concentrating the eluent by reduced pressure and/or heat, some degradation of the product occurs along with radiolysis creating additional impurities.
Following the chelation step of the present i nvention, the activated chelate can be rapidly purified by column chromatography and the eluent used directly, without concentration, in the conjugation step.
t5 ~ The bifunctional chelate is preferably coniugated to a biological molecule which carries out a specific target function. In a preferred embodiment, the biomolecule is a monoclonal antibody or fragmentthereof which is specific against a selected cell-surface target site. Such antibodies may be commercially available or may be made by standard somatic cell hybridi~ation techniques. An example of a suitable monoclonal antibody is CC49, one of a 20~ series of monoc!onai antibodies specific for TA6-72 (tumor associated glycoprotei n3 descri bed in~published PCT Application No. WO 8~!00692, on January 26, 1989, and published PCT
app!ication WO 90104410, on May 3, 1990. Generally, the chelate and protein are mixed in a ., molar ratio of greater than 1:10 and less than about 100:1 depending on the protein and protein concentration. Ratios of 0 5:1 to 4:1 are preferred.
~ Methods for conjugating thiocyanate derivatized chelates to antibodies are well known in the art. The procedure generally involves reacting the functionalized chelating agent orchelatewiththeantibodyfrom2tol8hoursinanaqueousbufferedsolutionatpH6-10at roc m temperature.
Theincreaseinpurityofthechelateobtainedbytheprocessofthepresent invention allows the chelate to be conjugated to a biological material, preferably a protein, at temperatures from 25C to 40C, preferably 30C to 40C, to obtain a protein/chelate containing an activity from 0.5 millicuries to ~0 miliicuries per mg protein without the expected increase in degradation of the chelate activated group (i.e., hydrolysis of the isothiocyanate) or protei n.
As the temperature of the chelation reaction is i ncreased~ an increase i n the rate of formati on of protein aggregates and hyd rolysis of the chelate activated grou ps would be expected i n addition to-an increased conju~ation rate. U nexpectedly, elevated temperature results i n a much faster conjugation rate increase relative tothe rate of increase of undesired by-product formation (e.g., chelate hydrolysis and protein aggregates). Performing the conjugation at ~ ~ -:
: ~
~. .
WO 93/21D852 2 ~ ~ 7 r~ 7 5 PCI/US93/03483 about pH 9.5 and 37C, the reaction is approximately 92 percent complete in one hour with less than 4 percent of the radioactivity being associated with protein aggregates.
The formation of fewer by-prodùcts duringthe conjugation coupled with a more complete reaction leads to easier product purification, production of a product with higher 5 purity, and gives a greater overall recovery of the initial radioactivity. Utilizing a conjugation temperature of 37C also allows a radiochemical yield of greater than 90 percent during purification by columrl chromatography as compared to a radiochemical yield of approximately 80 percent when the conjugation is done at 20C.
The invention will be further clarified by a consi~eration of the following 10 examples, which are intended to be purely exenlplary of the present invention.
In the following examples, the following terms are used.
BFC = bifunctional chelating agent.
PA-DOTA = 1-12-(4-aminophenyl)ethyl]-1,4,7,10--tetraazacyclododecan~ 1,4,7, t O-tetraacetic acid.
HEPES = N-2-hydroxyethylpiperazine-N'-2~thane sulfonic acid.
General Ex~erimental Mass spectra (fast atom bombardment with xenon) were obtained on a Vacuum Gen~r~tors ZAB HS mass spectrometer. Sarrlples for mass spectral analysis were prepared by dissolution in a 3:1 mixlure of dithiothreitol:dithioerythritol (magic bullet) unless othervvise 20 Stated.
Analytical high performance liquid chromatograph (HPLC) of non-radioactive sampleswas performed on a Hewlett Packard 1090 liquid chromatograph with a 4.6 x tOO mm Alltech Econosphere C18 31I column with a flow rate of 1 rnL per minute and detection at 254 nm. Samples were eluted with a gradient of 0.05 M, pH 6.0, sodium acetate/ acetonitrile as given for the samples.
HPLC of radioa~tive samples (i.e., those in which l77Lu was incorporated) was performed using a DuPont Zorbax GF-250 ~9.4 x 250 mrnkolumn with a flow rate of 1.5 mL per minute and radioactivity detection. The mobile phase was O.25 M, pH 6.0 sodi um citrate/tO percentacetonitrile.
3(~ ExamE~ Purification of PA-DOTA by Flash Chromatography A 1.5 x 14 inch ~1ash chromatography column was packed with 17.0 g of Merck 60 A silica gel, 230-400 mesh ~AIdrich Chemical Co.) which had been acid-washed by Ailtech Inc. Crude PA-H4DOTA-3HCI (3.2 9, 75 percent pure by HPLC analysis) was applied to the column and eluted with a mobile phase of 2:2:1 chloroform:methanol:concentrated 35 ammonium hydroxide. Fractions containing only PA-DOTA(NH4)2 as assayed by thin layer chromatography were collected and pooled to give PA-DOTASNH4)2 which was greater than 98 percent area purity as assayed by HPLC analysis. The absence of caicium in the recovered :
. ' WO93/2085~ 2 ~ ~ ~ I I rj PCr/US93/034~3 PA-DOTA(NH4~2 was confi rmed by mass spectrometry and HPLC using an elution buffer gradientof95t5sodiumacetate~.05M,pH=6)/acetoni~rileto30nOin15minutes.
Chelation of yttri um to the PA-DOTA obtai ned above was performed by dissolving 3 mg of PA-DOTA in 3 mL of O.S of sodium acetate buffer ~pH = 6) and 0.2 mL of 40 millimolar Y~OAC)3.4H20, this represents approximately a 5 fold excess of yttrium. The extent of chelation was monitored by HPLC as described above.
Chelation with PA-DOTA which was purified using acid washed silica gel was complete in less than S mi nutes at room temperature ( 1 ~25C).
tO Chelation with PA-I:)OTA purified using sitica gel which had not been acid washed was 4 percent complete in l S minutes at room temperature and was 86 percent complete i n 10 minutesat90C.
These results show acid washing the silica gel to remove extraneous metal ions prior to use for purifying the chelating agent ~reatly enhances the rate at which the desired 15~ metal is sequestered at room tempe!ature.
ExamPle 2 Preparation of SCN-PA-H4DOTA.2HCI.2NH4CI
To 100 mL of 0.01 M hydrochloric acid was added 250 mg of calcium free PA-H2(NH4)2DOTA (0.448 millimoies, 2 of the carboxylated groups are protonated and 2 of the carboxylated groups are ammonium salts) as prepared in Example l~ and the solution placed in 2~ ~ a 40-oz. Waring blender. Thiophosgene (170 microliters, 2.23 millimoles) was added and the biender qukkly started. After 2 minutes of mixing, the mixture was added to a separatory funnel and excess thiophosgene was extracted with three 50 rnL portions of chloroform. The aqueous layer was added to~ 1 ûO mL of acetonitrile and the solution reduced to dryness on a ;~ rotary evaporator (fitted with~a vacuum~pump) at room temperature. The solid obtained was 25 ~further dried far 2 hours at room temperature on a vacuum line. The yield was 321 mg (9O pe~cent yield) of an off-white~solid.
Characterization of th~ product by mass spectrometry and by HPLC showed the -product was 96 percent byweight an isothiocyanate derivatized PA-DOTA
SCN-PA-H4DOTA-2HCI.2NH4Cl).~ The HPLC elutlon gradient being sodium acetate/acetonitrile 30 ~ 9S/S to 70/30 in 15 minutes and~then to 4ûl60 in 20:rninutes.
ComPar e Example B
Samples pre~ared in an identical manner except that the reaction was carried outby the~addition of 20 to SO per~ent by vol ume chloroform prior to mixing were 84 to 8~ percent pure as measured~by HPLC.
35 ~ These results show that~a high purlty is:othiocyanato activated chelating agent can be obtained by mixing the thiophosgene and chelating agent in an aqueous systerr in the absenceofanorganicsolvent. ~
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:
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~ ~ i 7 1 7 ~
WC) 93/20852 PCI'/US93/03483 Examele 3 Preparation of [l77Lu(SCN-A-DOTA)]
To 20 ~L of SCN-PA-DOTA (4.88 x 10-3 moles) prepared as described in Example 2 was added 100 llL of Lu- 177 ~50 microcurries) to give a molar ratio of chelati ng agent to Lu- 177 of about 1: t . The solution was mixed on a vortex mixer for about S seconds, 100 ,uL of H EPES
5 buffer (0.5 M, ph 7) were added and the solution mixed for 5 minutes. The pH was measured and adjusted to between pH ~7 with HEPES buffer if necessary. The reaction was allowed t proceed for another 5 minutes at room temperature and the yield of [l77Lu(SCN-A-DoTA)] was 91.4 percent as determined by HPLC.
The [177Lu(SCN-PA-DOTA)] complex was purifie~ by placing the sample on a PRP-10 1 mini-clean~ column (80 }~L) which had been pretreated with ~00 }lL acetonitrile, 400 IIL of water and ~00 ~L 10 percent by volume acetonitrile in 20 mM carbonate buffer, pH 9.5. After !oading the complex onto the column, the reaction vial was rinsed with a 200 ~lL and then a 600 }lL volume of 10 percent acetonitrile in carbonate buffer, the washes also being placecl on the column.
Thecomplexwasthenelutedfromthecolumnusinga 1:2ratioofcarbonate buffer ~20 rnM, pH 9.5~: acetonitril~. Approximately 80 percent of the radioactivity was recovered in the second 50 ~lL elution volume.
Example 4 t:onjugation of 1177Lu(SCN-PA-DC)TA)l With IgG CC49 at P~oom Temperature (21-22~C) To 257 IlL of an an~ibody solution (IgG CC49, 14.5 rng/mL in 50 mM carbonate bwffer,pll9.5)wasadded25}lLof[l77Lu(SCN~PA-DOTA)I preparedasdescribedabove,giving a BFC:antibody molar ratio ~f about 1.36. The activated chelate and antibody solution was mixed on a vortex mixer for about 10 seconds and allowed to stand at room temperature for approximately 2 hours with mixing about every 10 to 15 minutes. The disappearance of the [177Lu(SCN-PA-DOTA)] W35 measured by HPLC. After 120 minutes, about 62 percent c,f the 177Lu activity was associated with the antibody, about 7 percent was non-antibody associated impurities, and unreacted t177LU(SCN-PA-DOTA)I was about 30 percent.
ExamD!e 5 Conjugation of ~177Lu(SCN-PA-DOTA)] With IGg C~9 at 37C
To an antibody solution (15.7 mg CC49 IgG (30 nanomoles) in 2~6 }IL of carbonatebuffer, 20 mM, pH 9.S) was added 21 nanomoles of [177~u(SCN-PA-~OTA)] complex (9.7 millicuries of 2ctivity) in 1 1 ~lL of ~he eluent from Example 3. The mixture was mixed on a vortex mixer and allowed to stand in ~ 37C oven fs~r one hour with mixing about every 10 to 15 minutes. The progress of the reaction was monitored by HPLC analysis for the disappearance of the complex as described in Example 4. After 1 hour the 177Lu activity 35 associated with the antibody as measured by HPLC was 91 percent, less than 4 percent of the 177Lu activity was associated with impurities, and S percent was unreacted [l77LU(SCN-PA-DOTA)]'.
WO 93/2~85~ ~ 1 1 7 7 ~ ~3 PC~/US93/03483 The conjugation reaction was terminated by isolation and purification of the conjugate on a PD-10column (Sephadex G-25, medium, 9 mL) which had been equilibrated with phosphate buffered saline.
Homogeneity of the labeled antibody was examined by HPLC and by SDS PAGE
5 electrophoresis (sodium dodecyl sulfate-polyacrylamide electrophoresis~ coupled with autoradiography. Immunoreactivity was determined by IRMA and affinity column binding and was found to be comparable to that conjugated at 25C. In vivo biodistribution ~in rats) of the conjugate prepared at 37C was not significantly different from that prepared at 25C.
The results show that the chelate can be rapiclly conjugated to an antibody at 10 37C without affecting the immunoreactivity or biodistribution of the conjugate.
Other embodiments of the invention will be apparent to those skilled in the art from a consideratiorl of this specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.
~15 ' ,:
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wO 93/20852 2 ~ ~ ~ r~ 7 ~ PCI`/US93/03483 As used herein, theterms "chelating agent" or nligand" mean a compound capable of chelatin~ or sequestering a metal ion. The term Nchelate" means a chelating agent which has sequestered a metal ion.
The term "bifunctional chelating agent" refers to compounds that have a moiety 5 capable of chelating a metal ion and a linker/spacer moiety covalently bonded to the chelati ng moiety that is capable of being activated or function~ ecl to serve as a means to covalently attach to a biological molecule.
The term "biological molecule" refers to any protei n, antibody, antibody fragment, hormone, antigen or hapten which functionsto recognize a specific biological 10 target site. Such a biological molecule, when attached to a functionalized chelate, serves to carry the attached metal ion to specific targeted tissues. Preferably, the bislogical material is an antibody or antibody fragment.
As used herein, "antibody" refers to any polyclonal, monoclonal, chimeric antibody, heteroantibody, or recorr~binant or derivative thereof, preferably a monoclonal 15 an~ibody. As used herein the term "antibody fragment" includes Fab fragments and F(ab'~2 fragrnents and any portion of an antibody, including recombinants and derivatives thereof, having specificity toward a desired epitope or epitopes. The antibody fragments may be produced by onventional enzymatic methods or by genetic or protein 0ngineering techniques, such asthe production of single chain antibodies.
The terms ~activateJ~ or "activating!' in relatiorl to a chelating agent means the c helating agent has been modified with a functional group which is capable of forming a covalent bond with a biological molscule.
The term "conj ugate~ as used herein refers to a complex of a ~iological material attached to a bifunc~ional chelating agent or bifunctional chelate. The term "antibody/chelate 25 conjugate" refers to an antibody which is covalently attached to a bifunctional chelate, (i.e., the bifunctional chelating agent having a chelated rnetal ion).
A general overall procedure giving the steps for preparation of conjugates used in the present invention is given in the following 5cheme 1. Steps (b) and (d of the process scheme may be reversed. When sequestering metal ions which are radionuclides with short half lives, it is preferred that the anivation occurs before formation of a chelate to avoid radiolysis which would occur during the subsequent activating step and any subsequent purification.
W093/~0852 h ~ ~ 7 7 ~ PC~/US93/O~B3 Scheme 1 Synthesi~ of a Chelating Agent Purification of the Chelating Agent on Silica Gel Step (a~
I
1t Activation of the Chelating Agent Step ~b~
1' . I
Chelation of a Metal Ion by the Activated Chelating Agent to Form a Chelate Step tc) I
2t Conjugation o~ the Chelate to a Biological Material to Form a Conjugate Step ~d) 2' ~ .
Puri~ication of the Conjugate :
3t :
. .
-ç,-WO 93/20852 2 1 ~ 7 r~ 7 ~ PCI`/US93/03483 The present invention provides a process for reducing extraneous metal ions, specifically calcium ions (Ca2 +), during the purification of macrocyclic chelating agents by flash chromatography using silica gel. It has been found that commercially aYailable silica gel used to purify the macrocyclic chelating agents unexpectedly contai ns a source of undesired metal 5 ions, particularly divalent cations such as calcium, which become tightly bound to the chelating agentduringpurification. Thewashingofthesilicagelwithastrongmineralacid,suchas hydrochloric, nitric, sulfuric, perchloric or hydrobromic, prior to use substantially reduces the amount of undesired metal ions bound ts the chelating agent during the purification process.
Preferably, the mineral acid used to wash the silica gel is hydrochloric acid. Procedures for 10 washing silica gel with a strong acid to remove impurities are known in the art, see, for example, Ralph K. Iler, The ChemistryofS~lica, John Wiley & Sons (1979).
The elimination of undesired rnetal ions from the macrocyclic chelating agents during the initial purification results in several advantages in the subsequent steps for forming a conjugate. The elimination of undesired bound metal ions from the macrocyclic chelating 15 agents allows rapid chelation of the desired metal ions at room temperature as opposed to heating or long reaction times required to displace the calcium. The ability ~o perform the cnelation step at room temperature, rather than at temperatures which would destroy the functionalizing group, allows the activation of the macrocyclic bifunctional chelating agent prior to chelation of the metal ion. The ability to functionalize the macrocyclic chelating agent ;~o prior to chelation of the me~al ion is particularly advantageous as the activated chelating agent can be synthesized, stored and then s~questration of the metal ion performed shortly before conjugation to the biological material. This is particularly advantageous when the metal ion is a radionuclide with a half li~e of 10 days or less, as a significant amount of radiolysis can occur during ~he time n~cessary for functionalizing the chelate, conjugation, and purification of the 25 products from each step. Preclusion of undesired metal ions is particularly important in insuring rapid ~Jptake of radioactive metals of the 13nthanide (111) series.
The abilityto rapidly chelate the radionuclide after activation of the chelatingagent also allows for simpler 3nd more efficient purification procedures to be used in preparation of the conj ugate. ~
The present process for purifying bifunctional chelating agents can be used for many classes of ligands including any tri to hexa -(carboxylated), -(phosphonomethylated), or -(phosphinomethylated) polyazamacrocycle where the polyazamacrocycle has 3 to 6 nitrogens in the ring and the total number of atoms in the ring is 9 to 24 atoms. Of particular interest are aminocarboxylic acid chelaeing agents, amlnophosphonit chelating agents and polyaza 35 chelating agents containing secondary and tertiary amines. Preferred polyazamacrocyclic chelating agents are of the formula WC)93/20852 ~1 7775 PCI/~JS93/03~183 2~ (CH2)--C--(CH2)r ~ ,~
(I) wherein:
each Q is independently hydrogen, (CHR5)pC02R or (CHR5)pPO~H2;
Q1 is hydrogen or (CHR5)wCO2R;
each R independently is hydrogen, benzyl or C1-C4 alkyl;
with the proviso that that at least two of the sum of Q and Ql must be other than hydrogen;
each R5 independently is hydrogen, C1-C4 alkyl or -(C1-C2 alkyl)phenyl;
X and Y are each independently hydrogen or may be taken with an adjacent X
and Y to form an additionaJ carbor~carbon bond;
nisOorl;
~0 m is an integer from O to 10 inclusive;
p= lor2;
r= oGr1;
w = Oor1;
with the prsviso that n is only 1 when X and/or Y form an additional carbon-carbon bond, and 25 the sum of r and w is O or 1;
R2 and R4are independen~ly hydrogen, nitro, amirlo or carboxyl;
~: R3isC1-C4alkoxy,-OCH2CO2H,hydroxyorhydrc\gen; :~
with the proviso tha~ R2 and R4 cannot both be hydrogen but one of R2 ~nd R4 must be hydrogerl.
30~ Preferred amines of the polyaza chelating agents of Formula I are tertiary amines, preferably where r is O and each Q is (CHRS)pCO~R.
Procedures fw synthesizing polyaza macrocycles are well known in the art.
Examples of preferred chelating agents are given in Table I and are named as foilows:
I A is 1 -(4-aminophenyl)- 1,4,7,1 O-tetra-azacyclododecane-1,4,7, 1 0-tetraacetic acid;
I B is 1-~2-~4-aminophenyl)ethyl] -1,4,7,10-tetraazacyclododecane--4,7,10-triacetic acid;
f'~ 7 ~ I v WO 93/2~852 PCr/US93/~34$3 ."
I C is 1-[2-(~aminophenyl)ethyl]-1,4,7,10-tetraazacyclododecane--1,4,7,10-tetraacetic acid;
I D is 1-(5-amino-2-rnethoxybenzyl)-1,4,7,10-tetraazacyclododecane--1,4,7,10-tetraaeetic acid;
I E is 1-(5-amino-2-hydroxybenzyl)-1,4,7,10-tetraazacyclododecane--4,7,10-triaceticacid; and I F is 1-[2-(4-aminophenyl)ethyl] -1,4,7,10-tetraazacyclododecane--1-(R,S,-acetic-4,7,10-tris-(S-methylacetic) acid, the preparation of which is given in European Patent Publication No. 0420942, published April 10, 1991.
: 10 The preparation of IA-IE is given in European Patent Publication No. 03S3450, published February 7, 1990, and European Patent Publication No. 0420942, published April 10, 1991.
''''' ' ;
: .:
: ~25 .
, `: : : :
::~ 35 -W(:~93/2~852 f,~ ~ 3 777~ PCI/US93/03483 TABLE I
r COOH
CO2~
N COOH
H2N ~COOH
rcoo~l .
lSr /@ ~ ~\> I B
H2N N~>
--COOH
.
COOH r COOH
)~0 ~ COOH I C
2~ ` COO~
:
: :
.'.
: , WO 93/21~852 2 i 1 7 7 7 :3 PCr/US93/03483 TABLE I CONT ' D
r COOH
COOH
~N ~<~ ~,~
OCH3 \~COOH
0 r COOH
H!!N ~ COO~ I E
N ....
OH ~ COOH
:
COOH /'--COOH I F
~,J~ ~ N ~
I O I N ~r--COOH
H N ~ ~ H3 fOOH
25 ~ CH3 ` : ~ : .
: :
:: : , Methods for the carbc~xylation of an arnine of a ligand to give amine derivatives containing a carboxyalkyl group are well known, as are the methods which give alkyl 3q ~ phosphonic and hydroxyalkyl~substituents on the amine nitrogens. ~See, for example, U.S.
Patents 3,726,g12 and 3,398,198.l Aminophosphonic acid derivatives of ligands can be prepared by a number of known s~nthetic techniques. Of particular importance isthe reaction of a compound containing at least one reactive amine hydrogen with a carbonyl compound (aldehyde or 35 ketone) and phosphorous a~id or derivative thereof. [See the procedure of Moeoritzer and Irani,J Org. Chem.,~, 1603(1966~.]
- 1 1 - .
.WO93/20852 ~ 777~ PCI/US93/03483 The polyaza chelating agents of the present invention purified by flash chrornatography on acid washed silica gel can be activated with any of the known functional groups capable of forming a covalent bond with a biological molecule. Examples of such functional groups are isothiocyanate, bromoacetamide, maleimide, imidoester, 5 thiophthalamide, diazonium and carboxyl. The use of polyaza chelating agents which are essentially free of undesired metal ions are particularly important when using functionalizing groups which are pH andlor thermally unstable, such as isothiocyanate. The activating functional grc ups are located at positions R2 or R4 of Formula I on the macrocyclic chelating agent.
It has been unexpectedly found that when reacting the polyaza chelating agent with thiophosgene to activate the chelating agent with an isothiocyanate group, conducting the reaction in an aqueous environment, preferably in a di lute acid, with vigorous mixing i n the absence of an organic solvent results in an activated chelating agent of greater purity than that obtained in the presence of an organic solvent. This is in contrast to the known procedures of 15 using a pH of 8. 5 and/or adding the thiophosgene in the presence of an organic solvent. See, for example, Brechbiel et al., Inorg. Chem., ~, 2772-2781 (1986); Keana and Mann, J. Org.
Chem., 55, 2868-2871 (1990); Westerberg et al.jJ. Med. Chem., ~ 23~243 (1989); and Kline et al., Bioconjugate Chem., 2, 2~31 (1991). The reaction of the thiophosgene with the - ~ chelatlng agent is~at a pH from 1 to 7. Preferably, the pH is frorn 1 to 5. More preferably, the 20~ pH is from 1 to 3 The reaction between the~ chelating agent and thiophosgene is preferably ~arriedout in an aqueous environment having an acid concentration of from about 0.005 to about 0.S~normal (N), preferably 0.01 to 0 2 N. ~ The acid can be any mineral acid, preferably hydrochloric acid. The reaction betweèn the cheiating agent and thiophosgene in a dilute acid 25 ;~with~ vigorous mixing and excess~thiophosgene is very fast and usually complete i n less than 5 minutes at room temperature~(1 5C to 25C). Higher or lower temperatures can be used (e~g., 0Cto S0C) but room temperature is preferred.
The amount of excess thiophosgene added to the mixture depends on the concentration of the polyaza cheiating agent. The lower the concentration of chelating agent, 30: the larger the excess of thiophosgene to insure the rapid and complete conversion of amine to isothiocyanate. For exarnple, if the concentration of chelating agent is 10-3 M, the ratio of thiophosgene to chelating agent is S-20: 1. If the concentration of chelating agent is 10-8 M, the ratio of thiophosgene to chelating agent is several thousand times larger (i.e., 105:1). The excess thiophosgene is removed by conventionai techniques such as evaporati on, 35 ~hromatography or extraction.
Rapid mixing of thiophosgene with the aqueous solution may be accomplished using conventional equipment known to those in the art which is capable of producing sufficient shear to produce dispersion of the thiophosgene within the aqueous solution.
, .
WO 93/20X52 '~ 1 ~ 7 7 ~ 5 PCI/US93/03483 Illustrative of such mixing means is the use of a Waring blender for large scale preparations and with a Mixxor~ type mixer available from Alltech tnc., for smaller scale preparations.
By conducting the reaction of the chelating agent with thiophosgene as described above, the obtained purity of the isothiocyanato activated chelating agent is about 5 90 to about 95 percent as measured by high performance liquid chromatcgraphy (HPLC).
A~ter the chelating agent has been activated, the activated chelating agent is placed in contact with a metal ion to form the chelating agenVmetal ion chelate. Although any metal ion, whether a radioactive metal ion or not, can be used which is sequestered by the chelating agent, the chelates formed should have reasonable stability such that the metal 10 complex is not readily disassociated. P~adionuclides are preferred because of the use of the resulting products in a radiopharmaceuticai drug for therapy and/or diagnosis. Especially preferred radioactive isotopes are those of samarium (Sm-153), holmium (H~166), ytterbium (Y~175), lutetium (Lu-177), gadol;nium (Gd-159), lanthanum (La-140), praseodymium (Pr-142), promethium (Pm-149). yttrium (Y-90) and indium (In-111).
Radionuclides can be produced in s~veral ways. In a nuclear reactor a nuclide isbombarded with neutrons to obtain a radionuclide, e.g., Sm-t52 ~ neutron ~ Sm-153 ~ gamma Another methocl of obtaining radionuclides is to bombard nuciides with particlesproduced by a linear accelerator or a cyclotron. 'Yet another way is to isolate the radionuclide 20 from a mixture of fission products. The method of obtaining the nuclides employed in the present invention is not critical thereto.
The radionuclides can be complexed with the bifunctional chelating agent by adding the bifurctional chelating agent to a solution of the radionuclide. Chelates form readily upon mixing in an aqueous solution at a pH of 1 to 10. Preferably, the reaction is carried 25 out in a rnedium having a pH of 1 to 7 and more preferably 5to 7. Ambienttemperatures of about t 0C to 4ûC can be readily employed for metal ion chelation. The amount of metal ion employed may be from trace amounts to an amount in excess of equimolar with the chelate.
Preferablyj the formation of the chelate occurs at room temperature, between 1 5C to 25C.
Chelation proceeds rapidly and yields of about 90 percent and greater as 30 measured by high performance liquid chromatography are obtained when using a chelating agent which has been initially purified on subs~antially calciurn free silica gel and activated with thiophosgene prior to chelation of the desired metal ion.
The ability to rapidly form chelates over a wide pH range is advantageous to obtain a high chelation yield of the desired metal ion. When it is necessary to displace divalent 35 cations such as calcium prior to chelation of the desired metal ion, yields are variable due to the pH sensitive nature of the displacement reanion. Displacement of the calcium ion requires mnninS} the reaction at a pH sufficiently low to displace ~he calcium, yet the pH cannot be too low as ~o adversely affect the chelation of the desired metal ion. Util izi ng the procedure of the r~ 7 ~
WO 93/~0852 PCI'/US93/03483 present invention allows the chelating reaction to proceed with high yields over a broad pH
range.
The ability to activate the chelating agent prior to forming the chelate and therapid formation of the chelate with a high yield also has tl~e advantage in that it reduces the 5 purification necessary prior to the conjugation step. When the metal ion is chelated prior to activation using thiophosgene, it is necessary to extract the unreacted thiophosgene with an organic solvent and then purify the chelate prior to conjugation. The low concentration of the chelate in the elution volume obtained after purification using an ion exchange column necessitates concentrating the chelate prior to conjugation. During the process of extracting 10 the thiophosgene and concentrating the eluent by reduced pressure and/or heat, some degradation of the product occurs along with radiolysis creating additional impurities.
Following the chelation step of the present i nvention, the activated chelate can be rapidly purified by column chromatography and the eluent used directly, without concentration, in the conjugation step.
t5 ~ The bifunctional chelate is preferably coniugated to a biological molecule which carries out a specific target function. In a preferred embodiment, the biomolecule is a monoclonal antibody or fragmentthereof which is specific against a selected cell-surface target site. Such antibodies may be commercially available or may be made by standard somatic cell hybridi~ation techniques. An example of a suitable monoclonal antibody is CC49, one of a 20~ series of monoc!onai antibodies specific for TA6-72 (tumor associated glycoprotei n3 descri bed in~published PCT Application No. WO 8~!00692, on January 26, 1989, and published PCT
app!ication WO 90104410, on May 3, 1990. Generally, the chelate and protein are mixed in a ., molar ratio of greater than 1:10 and less than about 100:1 depending on the protein and protein concentration. Ratios of 0 5:1 to 4:1 are preferred.
~ Methods for conjugating thiocyanate derivatized chelates to antibodies are well known in the art. The procedure generally involves reacting the functionalized chelating agent orchelatewiththeantibodyfrom2tol8hoursinanaqueousbufferedsolutionatpH6-10at roc m temperature.
Theincreaseinpurityofthechelateobtainedbytheprocessofthepresent invention allows the chelate to be conjugated to a biological material, preferably a protein, at temperatures from 25C to 40C, preferably 30C to 40C, to obtain a protein/chelate containing an activity from 0.5 millicuries to ~0 miliicuries per mg protein without the expected increase in degradation of the chelate activated group (i.e., hydrolysis of the isothiocyanate) or protei n.
As the temperature of the chelation reaction is i ncreased~ an increase i n the rate of formati on of protein aggregates and hyd rolysis of the chelate activated grou ps would be expected i n addition to-an increased conju~ation rate. U nexpectedly, elevated temperature results i n a much faster conjugation rate increase relative tothe rate of increase of undesired by-product formation (e.g., chelate hydrolysis and protein aggregates). Performing the conjugation at ~ ~ -:
: ~
~. .
WO 93/21D852 2 ~ ~ 7 r~ 7 5 PCI/US93/03483 about pH 9.5 and 37C, the reaction is approximately 92 percent complete in one hour with less than 4 percent of the radioactivity being associated with protein aggregates.
The formation of fewer by-prodùcts duringthe conjugation coupled with a more complete reaction leads to easier product purification, production of a product with higher 5 purity, and gives a greater overall recovery of the initial radioactivity. Utilizing a conjugation temperature of 37C also allows a radiochemical yield of greater than 90 percent during purification by columrl chromatography as compared to a radiochemical yield of approximately 80 percent when the conjugation is done at 20C.
The invention will be further clarified by a consi~eration of the following 10 examples, which are intended to be purely exenlplary of the present invention.
In the following examples, the following terms are used.
BFC = bifunctional chelating agent.
PA-DOTA = 1-12-(4-aminophenyl)ethyl]-1,4,7,10--tetraazacyclododecan~ 1,4,7, t O-tetraacetic acid.
HEPES = N-2-hydroxyethylpiperazine-N'-2~thane sulfonic acid.
General Ex~erimental Mass spectra (fast atom bombardment with xenon) were obtained on a Vacuum Gen~r~tors ZAB HS mass spectrometer. Sarrlples for mass spectral analysis were prepared by dissolution in a 3:1 mixlure of dithiothreitol:dithioerythritol (magic bullet) unless othervvise 20 Stated.
Analytical high performance liquid chromatograph (HPLC) of non-radioactive sampleswas performed on a Hewlett Packard 1090 liquid chromatograph with a 4.6 x tOO mm Alltech Econosphere C18 31I column with a flow rate of 1 rnL per minute and detection at 254 nm. Samples were eluted with a gradient of 0.05 M, pH 6.0, sodium acetate/ acetonitrile as given for the samples.
HPLC of radioa~tive samples (i.e., those in which l77Lu was incorporated) was performed using a DuPont Zorbax GF-250 ~9.4 x 250 mrnkolumn with a flow rate of 1.5 mL per minute and radioactivity detection. The mobile phase was O.25 M, pH 6.0 sodi um citrate/tO percentacetonitrile.
3(~ ExamE~ Purification of PA-DOTA by Flash Chromatography A 1.5 x 14 inch ~1ash chromatography column was packed with 17.0 g of Merck 60 A silica gel, 230-400 mesh ~AIdrich Chemical Co.) which had been acid-washed by Ailtech Inc. Crude PA-H4DOTA-3HCI (3.2 9, 75 percent pure by HPLC analysis) was applied to the column and eluted with a mobile phase of 2:2:1 chloroform:methanol:concentrated 35 ammonium hydroxide. Fractions containing only PA-DOTA(NH4)2 as assayed by thin layer chromatography were collected and pooled to give PA-DOTASNH4)2 which was greater than 98 percent area purity as assayed by HPLC analysis. The absence of caicium in the recovered :
. ' WO93/2085~ 2 ~ ~ ~ I I rj PCr/US93/034~3 PA-DOTA(NH4~2 was confi rmed by mass spectrometry and HPLC using an elution buffer gradientof95t5sodiumacetate~.05M,pH=6)/acetoni~rileto30nOin15minutes.
Chelation of yttri um to the PA-DOTA obtai ned above was performed by dissolving 3 mg of PA-DOTA in 3 mL of O.S of sodium acetate buffer ~pH = 6) and 0.2 mL of 40 millimolar Y~OAC)3.4H20, this represents approximately a 5 fold excess of yttrium. The extent of chelation was monitored by HPLC as described above.
Chelation with PA-DOTA which was purified using acid washed silica gel was complete in less than S mi nutes at room temperature ( 1 ~25C).
tO Chelation with PA-I:)OTA purified using sitica gel which had not been acid washed was 4 percent complete in l S minutes at room temperature and was 86 percent complete i n 10 minutesat90C.
These results show acid washing the silica gel to remove extraneous metal ions prior to use for purifying the chelating agent ~reatly enhances the rate at which the desired 15~ metal is sequestered at room tempe!ature.
ExamPle 2 Preparation of SCN-PA-H4DOTA.2HCI.2NH4CI
To 100 mL of 0.01 M hydrochloric acid was added 250 mg of calcium free PA-H2(NH4)2DOTA (0.448 millimoies, 2 of the carboxylated groups are protonated and 2 of the carboxylated groups are ammonium salts) as prepared in Example l~ and the solution placed in 2~ ~ a 40-oz. Waring blender. Thiophosgene (170 microliters, 2.23 millimoles) was added and the biender qukkly started. After 2 minutes of mixing, the mixture was added to a separatory funnel and excess thiophosgene was extracted with three 50 rnL portions of chloroform. The aqueous layer was added to~ 1 ûO mL of acetonitrile and the solution reduced to dryness on a ;~ rotary evaporator (fitted with~a vacuum~pump) at room temperature. The solid obtained was 25 ~further dried far 2 hours at room temperature on a vacuum line. The yield was 321 mg (9O pe~cent yield) of an off-white~solid.
Characterization of th~ product by mass spectrometry and by HPLC showed the -product was 96 percent byweight an isothiocyanate derivatized PA-DOTA
SCN-PA-H4DOTA-2HCI.2NH4Cl).~ The HPLC elutlon gradient being sodium acetate/acetonitrile 30 ~ 9S/S to 70/30 in 15 minutes and~then to 4ûl60 in 20:rninutes.
ComPar e Example B
Samples pre~ared in an identical manner except that the reaction was carried outby the~addition of 20 to SO per~ent by vol ume chloroform prior to mixing were 84 to 8~ percent pure as measured~by HPLC.
35 ~ These results show that~a high purlty is:othiocyanato activated chelating agent can be obtained by mixing the thiophosgene and chelating agent in an aqueous systerr in the absenceofanorganicsolvent. ~
: : :
:
:~ :
~ ~ i 7 1 7 ~
WC) 93/20852 PCI'/US93/03483 Examele 3 Preparation of [l77Lu(SCN-A-DOTA)]
To 20 ~L of SCN-PA-DOTA (4.88 x 10-3 moles) prepared as described in Example 2 was added 100 llL of Lu- 177 ~50 microcurries) to give a molar ratio of chelati ng agent to Lu- 177 of about 1: t . The solution was mixed on a vortex mixer for about S seconds, 100 ,uL of H EPES
5 buffer (0.5 M, ph 7) were added and the solution mixed for 5 minutes. The pH was measured and adjusted to between pH ~7 with HEPES buffer if necessary. The reaction was allowed t proceed for another 5 minutes at room temperature and the yield of [l77Lu(SCN-A-DoTA)] was 91.4 percent as determined by HPLC.
The [177Lu(SCN-PA-DOTA)] complex was purifie~ by placing the sample on a PRP-10 1 mini-clean~ column (80 }~L) which had been pretreated with ~00 }lL acetonitrile, 400 IIL of water and ~00 ~L 10 percent by volume acetonitrile in 20 mM carbonate buffer, pH 9.5. After !oading the complex onto the column, the reaction vial was rinsed with a 200 ~lL and then a 600 }lL volume of 10 percent acetonitrile in carbonate buffer, the washes also being placecl on the column.
Thecomplexwasthenelutedfromthecolumnusinga 1:2ratioofcarbonate buffer ~20 rnM, pH 9.5~: acetonitril~. Approximately 80 percent of the radioactivity was recovered in the second 50 ~lL elution volume.
Example 4 t:onjugation of 1177Lu(SCN-PA-DC)TA)l With IgG CC49 at P~oom Temperature (21-22~C) To 257 IlL of an an~ibody solution (IgG CC49, 14.5 rng/mL in 50 mM carbonate bwffer,pll9.5)wasadded25}lLof[l77Lu(SCN~PA-DOTA)I preparedasdescribedabove,giving a BFC:antibody molar ratio ~f about 1.36. The activated chelate and antibody solution was mixed on a vortex mixer for about 10 seconds and allowed to stand at room temperature for approximately 2 hours with mixing about every 10 to 15 minutes. The disappearance of the [177Lu(SCN-PA-DOTA)] W35 measured by HPLC. After 120 minutes, about 62 percent c,f the 177Lu activity was associated with the antibody, about 7 percent was non-antibody associated impurities, and unreacted t177LU(SCN-PA-DOTA)I was about 30 percent.
ExamD!e 5 Conjugation of ~177Lu(SCN-PA-DOTA)] With IGg C~9 at 37C
To an antibody solution (15.7 mg CC49 IgG (30 nanomoles) in 2~6 }IL of carbonatebuffer, 20 mM, pH 9.S) was added 21 nanomoles of [177~u(SCN-PA-~OTA)] complex (9.7 millicuries of 2ctivity) in 1 1 ~lL of ~he eluent from Example 3. The mixture was mixed on a vortex mixer and allowed to stand in ~ 37C oven fs~r one hour with mixing about every 10 to 15 minutes. The progress of the reaction was monitored by HPLC analysis for the disappearance of the complex as described in Example 4. After 1 hour the 177Lu activity 35 associated with the antibody as measured by HPLC was 91 percent, less than 4 percent of the 177Lu activity was associated with impurities, and S percent was unreacted [l77LU(SCN-PA-DOTA)]'.
WO 93/2~85~ ~ 1 1 7 7 ~ ~3 PC~/US93/03483 The conjugation reaction was terminated by isolation and purification of the conjugate on a PD-10column (Sephadex G-25, medium, 9 mL) which had been equilibrated with phosphate buffered saline.
Homogeneity of the labeled antibody was examined by HPLC and by SDS PAGE
5 electrophoresis (sodium dodecyl sulfate-polyacrylamide electrophoresis~ coupled with autoradiography. Immunoreactivity was determined by IRMA and affinity column binding and was found to be comparable to that conjugated at 25C. In vivo biodistribution ~in rats) of the conjugate prepared at 37C was not significantly different from that prepared at 25C.
The results show that the chelate can be rapiclly conjugated to an antibody at 10 37C without affecting the immunoreactivity or biodistribution of the conjugate.
Other embodiments of the invention will be apparent to those skilled in the art from a consideratiorl of this specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.
~15 ' ,:
~. ~
Claims (10)
1. A process for preparing a chelate-antibody conjugate which comprises reacting an isothiocyante activated chelate with an antibody at between 30°C and 40°C
wherein the chelate is a chelating agent complexed with a metal ion; the chelating agent is of the formula (I) wherein:
each Q is independently hydrogen, (CHR5)pCO2R or (CHR5)pPO3H2;
Q1 is hydrogen or (CHR5)wCO2R;
each K independently is hydrogen, benzyl or C1-C4 alkyl;
with the proviso that at least two of the sum of Q and Q1 must be other than hydrogen;
each R5 independently is hydrogen, C1-C4 alkyl or -(C1-C2 alkyl)phenyl;
X and Y are each independently hydrogen or may be taken with an adjacent X
and Y to form an additional carbon-carbon bond;
n is 0 or 1;
m is an integer from 0 to 10 inclusive;
p = 1 or 2;
r = 0 or 1;
w = 0 or 1;
with the proviso that n is only 1 when X and/or Y form an additional carbon-carbon bond and the sum of r and w is 0 or 1;
R2 and R4 are independently hydrogen, or amino;
R3 C1-C4 alkoxy, -OCH2CO2H, hydroxy and hydrogen;
with the proviso that R2 and R4 cannot both be hydrogen but one of R2 and R4 must be hydrogen; and the metal ion is selected from the group consisting of 153Sm, 166Ho, 175Yb, 177Lu, 159Gd, 140La, 142Pr, 149Pm, 90Y and 111ln.
wherein the chelate is a chelating agent complexed with a metal ion; the chelating agent is of the formula (I) wherein:
each Q is independently hydrogen, (CHR5)pCO2R or (CHR5)pPO3H2;
Q1 is hydrogen or (CHR5)wCO2R;
each K independently is hydrogen, benzyl or C1-C4 alkyl;
with the proviso that at least two of the sum of Q and Q1 must be other than hydrogen;
each R5 independently is hydrogen, C1-C4 alkyl or -(C1-C2 alkyl)phenyl;
X and Y are each independently hydrogen or may be taken with an adjacent X
and Y to form an additional carbon-carbon bond;
n is 0 or 1;
m is an integer from 0 to 10 inclusive;
p = 1 or 2;
r = 0 or 1;
w = 0 or 1;
with the proviso that n is only 1 when X and/or Y form an additional carbon-carbon bond and the sum of r and w is 0 or 1;
R2 and R4 are independently hydrogen, or amino;
R3 C1-C4 alkoxy, -OCH2CO2H, hydroxy and hydrogen;
with the proviso that R2 and R4 cannot both be hydrogen but one of R2 and R4 must be hydrogen; and the metal ion is selected from the group consisting of 153Sm, 166Ho, 175Yb, 177Lu, 159Gd, 140La, 142Pr, 149Pm, 90Y and 111ln.
2. The process of Claim 1 wherein the metal ion is 177Lu.
3. The process of Claim 1 or 2 wherein the chelating agent is 1-[2-(4-aminophenyl)ethyl]-1,4,7,10--tetraazacyclododecane-1,4,7,10-tetraacetic acid.
4. In a process for preparing polyaza conjugates having at least the steps of (a) purifying a polyaza chelating agent, the polyaza chelating agent being as defined in Formula I;
(b) contacting the the purified polyaza chelating agent with thiophosgene to form an isothiocyanato activated polyaza chelating agent and recovery of the isothiocyanato activated polyaza chelating agent;
(c) adding a metal ion to the isothiocyanto activated polyaza chelating agent toform an isothiocyanato activated polyaza chelate; and (d) contacting the isothiocyanato activated polyaza chelate with a biological molecule to form a conjugate;
the improvement which comprises;
in step (a): purifying the polyaza chelating agent by chromatography on a silicagel column, wherein the silica gel has been acid washed prior to loading the polyaza chelating agent onto the column;
in step (b): contacting the purified polyaza chelating agent with thiophosgene in an aqueous environment in the absence of an organic solvent at a pH from 1 to 5; and in step (d): contacting the isothiocyanato activated polyaza chelate with a biological molecule at between 25°C to 40°C.
(b) contacting the the purified polyaza chelating agent with thiophosgene to form an isothiocyanato activated polyaza chelating agent and recovery of the isothiocyanato activated polyaza chelating agent;
(c) adding a metal ion to the isothiocyanto activated polyaza chelating agent toform an isothiocyanato activated polyaza chelate; and (d) contacting the isothiocyanato activated polyaza chelate with a biological molecule to form a conjugate;
the improvement which comprises;
in step (a): purifying the polyaza chelating agent by chromatography on a silicagel column, wherein the silica gel has been acid washed prior to loading the polyaza chelating agent onto the column;
in step (b): contacting the purified polyaza chelating agent with thiophosgene in an aqueous environment in the absence of an organic solvent at a pH from 1 to 5; and in step (d): contacting the isothiocyanato activated polyaza chelate with a biological molecule at between 25°C to 40°C.
5. The process of Claim 4 wherein the biological molecule is a protein and the metal ion is 153Sm, 166Ho, 175Yb, 177Lu, 159Gd, 140La, 142Pr, 149Pm, 90Y or 111ln.
6. The process of Claim 5 wherein the protein is an antibody.
7. The process of Claim 4, 5 or 6 wherein r of Formula I is 0 and each Q is (CHR5)pCO2R.
8. The process of Claim 7 wherein the chelating agent is .alpha.-[2-(4-amino-phenyl)ethyl]-1,4,7,10--tetraazacyclododecane-1,4,7,10-tetraacetic acid and the metal ion is 177Lu.
9. In a process for preparing polyaza conjugates having at least the steps of (a) purifying a polyaza chelating agent, the polyaza chelating agent being as defined in Formula I;
(b) adding a metal ion to the polyaza chelating agent to form a polyaza chelate;
(c) contacting the chelate with thiophosgene to form an isothiocyanato activatedchelate (d) contacting the isothiocyanato activated chelate with a biological molecule to form a conjugate;
the improvement which comprises;
in step (a): purifying the polyaza chelating agent by chromatography on a silicagel column, wherein the silica gel has been acid washed prior to loading the polyaza chelating agent onto the column;
in step (c): contacting the chelate with thiophosgene in an aqueous environment in the absence of an organic solvent at a pH from 1 to 5; and in step (d): contacting the isothiocyanato activated polyaza chelate with a biological molecule at between 25°C to 40°C.
(b) adding a metal ion to the polyaza chelating agent to form a polyaza chelate;
(c) contacting the chelate with thiophosgene to form an isothiocyanato activatedchelate (d) contacting the isothiocyanato activated chelate with a biological molecule to form a conjugate;
the improvement which comprises;
in step (a): purifying the polyaza chelating agent by chromatography on a silicagel column, wherein the silica gel has been acid washed prior to loading the polyaza chelating agent onto the column;
in step (c): contacting the chelate with thiophosgene in an aqueous environment in the absence of an organic solvent at a pH from 1 to 5; and in step (d): contacting the isothiocyanato activated polyaza chelate with a biological molecule at between 25°C to 40°C.
10. A process for purifying a tri to hexa-(carboxylated), -(phosphonomethylated), or-(phosphinomethylated) polyazamacrocyclic chelating agent comprising flash chromatography of a polyazamacrocycle on acid washed silica gel to give a polyazamacrocycle substantially free of metal ions; wherein the polyazamacrocycle has 3 to 6 nitrogens in the ring and the total number of atoms in the ring is 9 to 24 atoms.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US86807892A | 1992-04-13 | 1992-04-13 | |
| US868,078 | 1992-04-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2117775A1 true CA2117775A1 (en) | 1993-10-28 |
Family
ID=25351031
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2117775 Abandoned CA2117775A1 (en) | 1992-04-13 | 1993-04-13 | Process for preparing macrocyclic chelating agents and formation of chelates and conjugates thereof |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0662842A1 (en) |
| JP (1) | JPH07505886A (en) |
| AU (1) | AU694074B2 (en) |
| CA (1) | CA2117775A1 (en) |
| NO (1) | NO943847D0 (en) |
| SG (1) | SG52470A1 (en) |
| WO (1) | WO1993020852A2 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0783326B1 (en) * | 1994-09-07 | 2004-02-04 | The Dow Chemical Company | Process for preparing macrocyclic chelating agents and formation of chelates and conjugates thereof |
| NZ543044A (en) * | 2003-04-15 | 2010-04-30 | Algeta As | Thorium-227 for use in radiotherapy of soft tissue disease |
| US20090208421A1 (en) * | 2008-02-19 | 2009-08-20 | Dominique Meyer | Process for preparing a pharmaceutical formulation of contrast agents |
| JP5376130B2 (en) * | 2009-03-24 | 2013-12-25 | 独立行政法人日本原子力研究開発機構 | Separation and purification of carrier-free 177Lu capable of antibody labeling |
| GB201002508D0 (en) | 2010-02-12 | 2010-03-31 | Algeta As | Product |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4885363A (en) * | 1987-04-24 | 1989-12-05 | E. R. Squibb & Sons, Inc. | 1-substituted-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane and analogs |
| US4994560A (en) * | 1987-06-24 | 1991-02-19 | The Dow Chemical Company | Functionalized polyamine chelants and radioactive rhodium complexes thereof for conjugation to antibodies |
| HU219485B (en) * | 1988-06-24 | 2001-04-28 | Dow Chemical Co. | Process for producing 1,4,7,10-tetraazacyclododecane derivatives, complexes thereof, their antibody conjugates and pharmaceutical compositions containing them and diagnostic compositions containing the complexes and the conjugates |
| BR8907282A (en) * | 1988-12-23 | 1991-03-12 | Dow Chemical Co | PROCESS TO PREPARE METAL COMPLEXES OPERATED WITH ISOTIOCIANATE |
-
1993
- 1993-04-13 WO PCT/US1993/003483 patent/WO1993020852A2/en not_active Application Discontinuation
- 1993-04-13 CA CA 2117775 patent/CA2117775A1/en not_active Abandoned
- 1993-04-13 SG SG1996004996A patent/SG52470A1/en unknown
- 1993-04-13 EP EP93912231A patent/EP0662842A1/en not_active Ceased
- 1993-04-13 JP JP5518572A patent/JPH07505886A/en active Pending
-
1994
- 1994-10-12 NO NO943847A patent/NO943847D0/en unknown
-
1997
- 1997-07-09 AU AU28563/97A patent/AU694074B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| AU4285093A (en) | 1993-11-18 |
| AU2856397A (en) | 1997-10-30 |
| JPH07505886A (en) | 1995-06-29 |
| WO1993020852A2 (en) | 1993-10-28 |
| AU677006B2 (en) | 1997-04-10 |
| WO1993020852A3 (en) | 1993-11-25 |
| NO943847L (en) | 1994-10-12 |
| AU694074B2 (en) | 1998-07-09 |
| NO943847D0 (en) | 1994-10-12 |
| SG52470A1 (en) | 1998-09-28 |
| EP0662842A1 (en) | 1995-07-19 |
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