Note: Descriptions are shown in the official language in which they were submitted.
., 2.~ 3~9
POLYMERIC CARRIERS FOR RELEASE OF COVALENTLY LINKED AGENTS
~iAC~ROUND OF T}IE INVENTION
1`. Field of the Invention
S The invention relates to chemically defined polymeric
carriers that provide advantageous properties for in vivo
imaging and therapy. The polymeric carriers consist of
~-amino acids that contain side chains covalently joined
to (i) diagnostic and therapeutic molecules and (ii)
chelating agents capable of binding diagnostic or
therapeutic radionuclides.
2. Related art
Monoclonal antibodies have been developed that localize in
cancerous tissue, due to their high specificity and
affinity for antigens on tumor cell surfaces. This
development has increased the prospect of clinical
applications, if such antibodies can be linked to
diagnostic and therapeutic agents. The high specificity
of the antibodies makes them desirable candidates as
targeting molecules for delivering a diagnostic or
therapeutic agent to a cancer site.
Unfortunately, the direct linkage of such agents to an
antibody weakens its immunoreactivity. Any deriv- -
atization of the antibody~weakens its immunoreactivity.
Therefore, an antibody with multiple linkages to
diagnostic or therapeutic agents is an antibody with low
specificity. At the present time, chelating agents, which
bind to diagnostic and therapeutic radionuclides, are
'
- i - .
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., , ,~ .
,~
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,
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directly linked to antibodies. This precludes the
controlli_d relcasc of the radionuclide from the
antibodies. Due to the large size of the antibody
molec~le, it is difficult, using conventional detection
techniques, to determine the exact nature of the chelating
agent linkages to the antibody, and to determine the
number of chelating agents linked to the antibody. A lack
of such precise information presents problems for getting
regulatory approval of chelating agents. ~egulatory
agencies require that all substances subject to their
approval must include information that clearly identifies
the structure o~ the substance, which is to be introduced
into the body.
What is needed is an approach that derivatizes a targeting
molecule, such as an antibody, at a minimum number of
sites to carry larger amounts of diagnostic and
therapeutic agents. Also needed, is an approach that can
determine the nature of the chelating agent linkage to the
2Q antibody and the number of chelating agents linked to the
antibody.
., . : . ,
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~ . .
SUMI~RY OF T~IE INVENTION
The present invention provides a chemically defined
polymeric carrier. It comprises a series of ~-amino
acids, in any combination, containing side chains to which
diagnosticltherapeutic and chelating agents can be
covalently joined through cleavable linkers. The agents
can be covalently joined to the side chains through
cleavable linkers either directly or covalently joined to
the side chains through cleavable linkers after chemical
modificat.ion of the side chains. Hydrazone, disulfide, and
ester linkages, in any combination, can be present in the
polymeric carrier between the side chains of the ~-amino
acids and the agents~ The selection of a particular
covalent linkage between the side chain and the agent in
the polymeric carrier is determined by the functional
group in the ~-amino acid side chain and the reactive
functional group in the agent. The ~-amino acids with side
chains to which agents do not covalently join can function
as spacers, to minimize interaction between the bulky
molecules attached to the polymeric carrier. In addition,
those ~-amino acids with charged or hydrophilic side
chains to which agents do not covalently join can pro~ide
incraased solubility to the polymeric carrier.
N-terminal protecting groups which are optional for the
polymeric carrier include all the standard amine
protecting groups. C-terminal conjugation groups ~hich
are optional for attachment of the polymeric carrier to
the targeting molecule include all conjugation groups
known in the art. In order to provide efficient
attachment of the polymeric targeting molecula, a spacer
' " ~
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.
- 4 ~ 7~
group is present in the polymeric carrier between the
~-amino acids and the conjugation group. The spacer group
presents any steric hindrance to the attachment by any
agent appended from the C-terminal end of the carrier.
These spacer groups are terminal aminoacids, such as
~-aminobutyric acid (Aba). In the absence of the
conjugation qroup, the spacer group, e.g., Aba through its
carboxyl group, can attach the polymeric carrier to the
targeting molecule.
The peptides that constitute the polymeric carrier are
prepared from ~-amino acids by conventional solution
methods or bv solid-phase peptide synthesis. These
peptides have been modified to carry derivatized
diagnostic/ therapeutic agents, and chelating agents that
bind to diagnostic and therapeutic radionuclides. These
agents can be released either at the target site or after
internalization by the cell.
Many advantages arise from the present invention. The
polymeric carrier can carry a maximum number of agents
while derivatizing a targeting molecule at a minimum
number of sites. Thus the biological activity of the
targeting molecule is maintained at a high level, even
though it is attached to multiple agents. For example, the
fewer the lin~ages in an antibody, the higher is its
specificity.
The rate at which agents can be released from the
polymeric carrier attached to the targeting molecule i~
controlled by manipulating the nature of the covalent
linkages in the polymeric carrier~ For example, by
adjusting the stability of the covalent linkages or by
.:
2~ 'n9
using different types of covalent linkages on a polymeric
carrier, agents -arc released at a mixed rate. This is
particularly important when the disease requires use of
long term diagnostics or therapeutics.
~ultiple agents, which may be the same or different, are
attached to the polymeric carrier. Not only can the same
agents be released at a mixed rate, but different agents
can be released at a mixed rate in the same target site.
The polymeric carrier, with its covalently linked agents,
is a relatively small molecule comparad to the targeting
molecule. Therefore, conventional detection techniques can
predetermine the exact nature of the agent linkages to the
targeting molecule before the polymeric carrier is
attached to it. In addition, radiolabeling techniques can
determine the precise number of polymeric carriers linked
to the targeting molecule.
.
- 6 ~ ~ ~,9
~RIEF DESCRIPTION OF THE FIGURES
Figure 1 is a flow chart representing the general
procedure for synthesis of a polymeric carrier with
attached agents and the conjugation of the polymeric
carrier to an antibody.
Eigure 2 is a flow chart representing a procedure for the
synthesis of a polymeric carrier with attached therapeutic
agents and the conjugation of the polymeric carrier to an
antibody.
Fisure 3 illustrates the re~oval of protecting groups from
amino acid side chains of a polymeric carrier.
Figures 4 and 6 are flow charts representing procedures
for the synthesis of polymeric carriers with attached
chelating agents and the conjugation of the polymeric
carriers to antibodies.
Figure 5 is a flow chart representing a procedure for the
synthesis of a chela'ing agent.
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- 7 ~
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a chemically defined
polymeric carrier that increases the loading of
diagnostic/therapeutic and chelating agents to targeting
molecules. The polymeric carrier comprises a series of
from 2 to about 18 ~-amino acids in any combination that
inclu~e side chains which can covalently join through
cleavable linkers to agents. The number of agents
covalently joined through cleavable linkers to the
polymeric carrier can be from 2 to about 18. This number
is determined by the number of ~-amino acid side chains in
the polymeric carrier available for covalent bonding
through cleavable linkers to the agents.
The term "polymeric carrier" as used in the invention
denotes a peptide carrier. Those -amino acids whose side
chains are not ~o-valently joined to agents can function as
spacers for the polymeric carrier. These spacers reduce
any non-bonded interactions between agents attached to
modified ~-amino acids. In addition to acting as spacers,
those ~-amino acids with charged or hydrophilic side
chains not covalently joined to agents can impart
increased solubility to the polymeric carrier.
The polymeric carrier includes optionally a protecting
group at its N-terminal~end and optionally a conjugation
group at its C-terminal end. The conjugation group enables
the polymeric carrier to attach itself to a targeting
molecule. A spacer group is placed between the ~-amino
acids and the conjugation group to aid in the attachment
of the polymeric carrier to the targeting molecule. The
spacer group prevents any steric hindrance to the
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- 8 -
attachment by any agent appended from the C-terminal end
of the carricr. In addition, the spacer group, a terminal
amino acid, may attach the polymeric carrier to the
target.ing molecule without the presence of the conjugation
group. This may occur by reacting the carboxyl group of
the terminal amino acid with functional groups on the
targeting molecule to form covalent bonds, such as ester
and amide linkages.
lo In the polymeric carrier, ~-amino ac~ds having side chains
that enhance polarity and therefore, water solubility, are
desirable. The increased water solubility is believed to
further contribute to decreased hepatobiliary uptake of
radiolabeled polymeric carrier proteins. The ~-amino
acids having side chains that enhance water solubility
include those with charged side chains (lysine, arginine,
histidine, cysteine, aspartic acid, glutamic acid,
tyrosine, tyrosine-0-S03-) and those with hydrophilic side
chains (serine, threonine, asparagine, glutamine).
Standard amine protecting groups can be used for the
N-terminal protecting group of the poly~eric carrier.
Preferred em~odiments of the invention comprise acetyl,
proprionyl, phenylacylsulfonyl, substitut~d phenyl-
acylsulfonyl, and other hydrophilic protecting groups.
A conjugation group is a chemically reactive functional
group that will react with a targeting mol~cule to bind
the polymeric carrier thereto. ~hen the targeting
molecule is a protein, the conjugation group is reactive
under conditions that do not denature or otherwise
adversely affect the protein. Therefore, the conjugation
group is sufficiently reactive with a functional group on
. .
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- 9
a protein so that the reaction can be conducted in a
substantially aqueous solution and does not have to be
forced, e.g., by heating to high temperatures, which may
denature the protein. Examples of suitable conjugation
groups include but are not limited to active esters,
isothiocyanates, amines, hydrazines, maleimides or other
Michael-type acceptors, thiols, and activated halides.
Among the preferred active esters are
N-hydroxysuccinimidyl ester, sulfosuccinimidyl ester,
lO thiophenyl ester, 2,3,5,6-tetrafluorophenyl ester, and
2,3,5,6-tetrafluorothiophenyl ester. The latter three
preferred active esters may comprise a group that enhances
w~ter solubility, at the para (i.e., 4) or the ortho
position on the phenyl ring. Examples of such groups are
15 CO2H, S03-, po32-, opQ32~ OS03, N~R3 wherein each R represents
H or an alkyl group, and O(CH2CH20)~CH3 groups.
Terminal amino ~acids used as spacer groups in the invent
ion include aminocaproic acid, aminopentanoic acid,
20 ~-aminobutyric acid, ~-alanlne, glycine, and the like.
Agents containing hydra~ides, R(CO~NHNHi, react with ~-
amino acid side chains containing aldehydes, RCHO, or
ketones, R2~C0), to form polymeric carriers with hydrazone
25 linkages having the following formula:
N~ C~AGENT
H (
(PG ~
N-NH-CO-AGENT
..
. .. .
.; ; .
., `
. , ; ,
`, `:
.,
- 10 --
2g~ i9
wherein the ~-amino acids in the polymeric carrier are
from 2 to about 18 units;
PG is an N-terminal protecting group;
SG is a spacer group that by preventing steric
hindrance by agents appended from the c-
terminal end of the carrier promotes efficient
attachment of the polymeric carrier to a
targeting molec~le;
CG is a conjugation group useful for the
attachment of the polymeric carrier to a
targeting molecule;
AGENT is from 2 to about 18 units of a
diagnostic or therapeutic agent, or a chelating
aqent capable of binding diagnostic or
therapeutic radionuclides in the polymeric
carrier,
R is H, CH3, phenyl, or phenyl substituted with
electron-donating and/or electron-withdrawing
groups;
q is O or 1;
r is O or 1; and
s is O or 1.
Hydrazone formation is an effective method of attaching
certain therapeutic agents to monoclonal antibodies (King
et al., Biochèmistry, Vol. 25:5774, 1986). Recent work in
the area of therapeutic immunoconjugates addressed the
hydrazone functionality as~a potentially cleavable linker
; between a chemotherapeutic agent and a monoclonal
antibody. Laguzza et al., (J. Med. Chem., Vol. 32:543,
1989j demonstrated that a vinca alkaloid can be conjugated
to an antibody via a hydrazone linkage and that pH
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dependency of the drug could be studied. The hydrazone
linkage approach ~as baOed on the premise that a conjugate
formed via a serum stable, yet acid-labile hydrazone
linker would serve the purpose of delivering the drug
conjugate to the tumor site and then slowly release upon
exposure to the tumor's acidic environment tTannock et
al., Cancer Research, Vol. 49:4373, 1989). This
conditional requirement necessitated the screening of
several small molecule hydrazones to evaluate their
stability in human serum and acetate buffer at pH 5.6.
The design of the polymeric carrier system with a
hydra~one linkage incorporates the observed results of a
small molecule study. Peptides of known amino acid
sequences are constructed to carry primary or secondary
hydroxyl groups (the chain may carry more than one hydroxy
amino acid (primary or secondary), which can be oxidized
to the carbonyl compound.
The results of the small molecule study indicate that hy
drazones from aromatic aldehydes may be too stable to be
useful. Hydrazones derived from aliphatic ketones have a
serum half-life of 15-20 hours (generated in the peptides
from threonine and other aminoacids containing secondary
-OH groups). Hydrazones derived from aliphatic aldehydes
(generated in the peptides from serine, homoserine and
other amino acids containing primary -OH groups) have a
serum half-life of 50-60 hours. Hydrazones derived from
aromatic ketones (generated in the peptides from
phenylserine and substituted phenylserines)) have a serum
half-life of 130 hours. By choosing an antibody or its
fragment with a half-life in human serum similar to that
of hydrazone, maximum delivery of the antlbody or its
.
.
- 12 -
Z~ i9
Eragment to the tumor is expected. After which, the
release of the therapeutic unit could occur at a rate
dependPnt on the chosen hydrazone's half-life in ~he tumor
site's acidic environment. To prevent the polymeric
carrier from possible premature degradation, the polymeric
carrier can be constructed with only D-amino acids or a
mixture of D- and L-amino acids.
Agents containing thiols, SH, react with cysteine side
chains to form polymeric carriers with disulfide linkages
having the following formula:
kR'
S AGENT
X~ ~D~SG~CG1
r q O
R~/
AGENT
, ::
", . : '. :. '
,,
., . ,~
-
- . , . -. . :, :
.
wherein the ~-amino acids in the polymeric carrier are
from ~ to about 18 units;
PG is an N-terminal protecting group;
SG is a spacer group that by preventi.ng steric
hindrance by agents appended from the C-
terminal end of the carrier promotes efficient
attachment of the polymeric carrier to a
targeting molecule;
CG is a conjugation group useful for the
attachment of the polymeric carrier to a
targeting molecule;
AGENT is from 2 to about 18 units of a
diagnostic or therapeutic agent, or a chelating
agent capable of binding diagnostic or
therapeutic radionuclides in the polymeric
carrier;
R is H or CH3;
R' is H or CH3; and
q is 1 or 2;
r is 0 or 1; and
s is 0 or 1.
The r lease rate for agents linked through disulfide bonds
to the polymeric carrier can~be decreased by replacement
of hydrogen with ~-alkyl groups ~R,R'=CH3).
:
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: . ~ :
Agents containing hydroxyl groups react with aspartic and
glutamic acid side chains to form polymeric carriers with
ester linkages h~vin~ the following formula:
O--AGENT
0:~ .
( ()q
SG-~CG~
O
O--AGENT
wherein the ~-amino acids in the polymeric carrier 2 to
about 18 units;
PG is an N-terminal protecting group;
SG is a spacer group that by preventing steric
hindrance by agents appended from the C-
terminal end of the carrier promotes efficient
attachment of the polymeric carrier to a
targeting molecule;
CG is a conjugation group useful for attachment
of the polymeric carrier to a targeting
: molecule;
AGENT is rom 2 to about 18 units of a
diagnostic or therapeutic agent, or a chelating
agent capable or bind~ng diagnostic the or
therapeutic radionuclides in the polymeric
carrier;
' ~
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- 15 -
~r~
q is o or 1;
r is o or 1; and
s is ~ or 1.
In general, attachment of radionuclide metals (e.g., M =
99mTc, ~86Re or ~38Re) to monoclonal antibodies using
bifunctional chelating agent A has been carried out by the
following procedure. Formation of M-chelate containing
active ester B followed by attachment to monoclonal
antibodies to give C, according to the following reaction
scheme:
~ O ~ a
~ U~
)~ 5/ \?~0
LC I I C~ Ecacl~c~
r OOC B
EOE refers to an ethoxy ethyl protecting group. COOTFP
refers to 2,3,5,6-tetra~luorophenyl ester. The a~ove
chelate is an N3S derivative, and N252 derivatives follow
a similar procedure. Between the chelate and the antibody
: ' ' . , '
:
is a carbon chain attached to the ~-amino group of the
antibody. After metabolism takes place in various organs,
~ha major metabolite D is retained in the gut an~ ki~ y
and is not excreted, according to the following:
N~Nf9 ~ ~ L
~ ~CO~ Lys-.~b , CO-~-L~s
E~}C EOC~ ~~
C D
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- 17 -
This retention interferes with imaging in the lower
abdominal area and gives a high dose to the kidney during
therapy. It was found that the presence of a cleavable
linker between the chelate and the antibody (compound F,
prepared from E) is metabolized and results in the
formation of G, which has no retention in the gut and low
retention in the kidney, according to the following
reaction scheme:
1~\
~coc \,~ ,
o
o
'`L~Il
~o
. ~
.
:: :
- 18 -^
,9
The observation that the cleavable linker is metabolized
ln the above reaction scheme leads to attaching the
hydroxy groups of the following compounds: -
~ ~f OE ~f ~
~S ~-- ~S Sr ~S Sr
~OE ~ O~
~ J E
to the aspartic and glutamie aeid side chains to form
ester linkages in the polymerie earriers.
The resulting polymerie earrier ean earry more than one
radionuclide metal/per attachment to the antibody or
fragment and offers the advantage of a metabolite that can
be removed via the renal system instead of being retained
in the gut.
Compound H belongs to N3S type ehelates and J and ~ belong
to the N2S2 ehelate system. The groups THP (tetrahy-
dropyranyl) and Acm (aeetamidomethyl) are used as sulfur
protecting groups.
,
The targeting molecule is any molecule that will serve to
dcliver the polymeric carrier -with a~tachad
diagnostic/therapeutic or chelating agents to a desired
target site (e.g., target cells) in vitro or in vivo.
Examples of targeting molecules include, but are not
limited to, steroids, cholesterol, lymphokines, and those
drugs and proteins that bind to a desired target site.
The targeting molecule may be a targeting protein, which
is capable of binding to a desired target site. The term
"protein" as used herein includes proteins, polypeptides,
and fragments thereof. The targeting protein may bind to
a receptor, substrate, antigenic determinant, or other
binding site on a target cell or other target site. The
targeting protein serves to deliver the agent attached
thereto by polymeric carrier to a desired target site ln
vlvo. Examples of targeting proteins include, but are not
limited to, antibodies and antibody fragments, hormones,
fibrinolytic enzymes, and biologic response modifiers. In
addition, other molecules that localize in a desired
target site in vivo although not strictly proteins, are
included within the definition of the term "targeting
prot~ins" as used herein. For example, certain
carbohydrates or glycoproteins may ~e used in the present
invention. The proteins may be modified, e.g., to produce
variants and fragments thereof, as long as the desired
biological property (i.e., the ability to bind to the
target site) is retained. The proteins may be modified by
using various genetic engineering or protein engineering
techniques.
Among the preferred targeting proteins are antibodies,
most preferably monoclonal antibodies. A number of
,
:,
- 20 -
monoclonal antibodies that bind to a specific type of cell
have been devcloped includ~ng monoclonal an~ibodies
specific for tumor-associated antigens in humans. Among
the many such monoclonal antibodies that may be used are
anti-T~C, or other interleukin-2 receptor antibodies;
9.2.27 and NR-ML-05, reactive with the 250 kilodalton
human melanoma-associated proteoglycan; and NR-LU-10,
reactive with a pancarcinoma glycoprotein. The antibody
employed in the present invention may be an intact (whole)
molecule, a fragment thereof, or a functional equivalent
thereof. Examples of antibody fragments are F(ab')2,
-Fab', Fab, and Fv fragments, which may be produced by
conventional methods or by genetic or protein engineering.
Proteins contain a variety of functional groups; e.g.,
carboxylic acid (COOH) or free amine (-NHz) groups, which
are available for reaction with a suitable protein
conjugation group on a polymeric carrier to bind the
polymeric carrier to the targeting protein. For example,
an active ester on the polymeric carrier reacts with
epsilon amine groups on lysine residues of proteins to
form amide bonds. Alternatively, a targeting molecule
and/or a polymeric carrler may be derivatized to expose or
attach additional reactive functional groups. The
deri~atization may involve attachment of any of a number
of linker molecules such às those available from Pierce
Chemioal Company, Rockford, Illinois. (See the Pierce
1986-87 General Catalog, pages 313-54.) Alternatively,
the derivatization may involve chemical treatment of the
protein (which may be an antibody). Procedures for
generation of free sulfhydryl groups on antibodies or
antibody fragments are al~o known. (See U.S Patent No.
;
- 21 - z ~ J ~ ~t~
4,659,839). Maleimide conjugation groups on polymeric
car~iers are reactiv2 with the sulfhydryl (thiol) groups.
Alternatively, when the targeting molecule is a
carbohydrate or glycoprotein, derivatization may involve
chemical treatment of the carbohydrate; e.g., glycol
cleavage of the sugar moiety of a glycoprotein antibody
with periodate to generate free aldehyde groups. The free
aldehyde groups on the antibody may be reacted with free
amine or hydrazine conjugation groups on polymeric
carriers.
In the present invention, therapeutic agents (e.g., a
drug, therapeutic radionuclide or toxin) are attached to
the chemically defined polymeric carrier. Preferably,
multiple therapeutic agents (which may be the same or
different) are attached to the polymeric carrier.
Exemplary therapeutic agents include toxins and drugs.
Within the present invention, preferred toxins include
holotoxins, such as abrin, ricin, modecin, Pseudomonas
exotoxin A; Di~htheria toxin, pertussis toxin and Shiga
toxin; and A chain or "A chain-like" molecules, such as
ricin A, abrin A chain, modeccin A chain, the enzymatic
portion of pertussis toxin, the enæymatic portion of Shiga
2S toxin, gelonin, pokeweed antiviral protein, saporin,
barley toxin, and snake venom peptides.
Exemplary drugs include daunomycin, adriamycin,
vinblastine, doxorubicin, bleomycin, methotrexate,
5-fluorouracil, 6-thioguanine, cytarabine,
cyclophosphamide, and similar conventional ~-
chemotherapeutics (for example, see Cancer: Principles and
E~ =L~8~ r~ , 2d ed., V.T. DeVita, Jr., S.
'
.
, .
., .; . ~: .
.
-
- 22 ~
Hellman, S.A. Rosenberg, J.B. Lippincott Co.,
rhiladelphia, PA, 1985, Chapter 14). Yet other preferred
drugs that can be used with the present invention belong
to the tricothecene family, with Roridin A particularly
preferred. Experimental drugs may also be suitable for
used within the present invention (see, e.g., NCI
Investiqational Druqs Pharmaceutical Data 1987, NIH
Publication No. 882141, Revised November 1987).
In the present invention, radiolabeled molecules are
attached to the chemically defined polymeric carrier.
Preferably, a multiple number of radiolabeled molecules
(which may be the same or different) are attached to the
polymeric carrier.
Radionuclide metal chelates are one type of radiolabeled
molecule that may be employed. Many chelating compounds of
various structures, as well as methods for the synthesis
and radiolabeling thereof to produce radionuclide metal
chelates, are known. Chelating compounds comprising
various combinations of sulfur, nitrogen, oxygen, and
phosphorous donor atoms may be used, for example. The
chelating compound may, for example, comprise a total of
from four to six donor atoms seIected from nitrogen and
sulfur atoms. During the radiolabeling procedure, bonds
form between the donor atoms and the radionuclide metal,
thereby producing a radionuclide metal chelate. Chelating
compound(s) may be incorporated into the polymeric carrier
during the synthesis procedure. Alternatively, the
chelating compound(s) may be synthesized separately and
subsequently attached~ to the polymeric carrier.
~ ~ :
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.
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~q~q~
One type of chelating compound that may be employed
comprises two nitrogen and two sulfur donor atoms and thus
may be designated an "N2Si" chelating co~pound. Suitable
N~S, chelating compounds are described in U.S. Patent
number 4,897,255, entitled "Metal Radionuclide Labeled
Proteins for Diagnosis and Therapy", which is hereby
incorporated by reference in its entirety. One example of
20 an N2S~ chelating compound is as follows:
~~C--C-'~
H~ H~
- C' 'C~
~C~ ~.
T T
wherein n is from 1 to about 4 (preferably 2); each R
independently is selected from =O and H2; T represents a
sulfur protecting group; and Z represents an active ester
or other reactive functional group (which in the present
invention may be useful for incorporating the chelating
compound into the polymeric carFier).
Any suitable conventional sulfur protecting group(s) may
be attached to the sulfur donor atoms of the compounds of
the present invention. The protecting groups should be
removable, either prior to or during the radiolabeling
reaction. Among the preferred sulfur protecting groups are
.
Acm and hemithioacetal protecting groups (EOE, THP), which
,: '
. . . , ~ : .
- 24 -
~q~
are displaceable from the chelating compound during the
radiolabeling reaction.
The N2S2 chelating compound advantageously is radiolabeled
after attachment to the polymeric carrier to produce a
radionuclide metal chelate of the formula:
I
~\ I ~ / ~
S 5
wherein P represents the polymeric carrier, M represents
a radionuclide metal or oxide thereof, and the other
symbols are as described above.
Radionuclide metals include, but are not limited to, the
diagnostically effective radionuclide 99~Tc, and the
therapeutically effective radionuclides ~a8Re, l86Re, 67Cu,
6~Cu, 2l2Pb, 2l2Bi, and l09pd. ~86Re and ~88Re are radionuclide
metals for use in the present invention.
Methods for preparing these isotopes are known.
Molybdenum/technetium generators for producing 99Tc are
commercially available. Procedures for producing ~86Re
include the procedures described by Deutsch et al., lNucl.
. ~, , .. : ~ . ,
.~ , ,. .; ....... . .
- : ' :-: . .i ,
I ~,
,
Med. Biol., Vol. 13:4:465-477, 1986) and Vanderheyden et
al., (Inorqanic ChemistrY, Vol. 24:1666-1673, 1985), and
222Bi mathGds for production of '3~Ae have been desc~ibed by
Blachot et al. (Intl._ J of Applied Radiation and
Isotopes, Vol. 20:467-470, 1969) and by Klofutar et al.
(J. of RadioanalYtical Chem., Vol. 5:3-10, 1970).
Production of I~Pd is described in Fawwaz et al., J. Nucl.
Med. (1984), 25:796. Production of 2l2Pb and n2Bi is
described in Gansow et al., Amer. Chem. Soc. Svm~. Ser.
(1984), 241:215-217, and Xozah et al. Proc. Nat~1. Acad.
sci . USA (January 1986) 83:474-478.
~he radiolabeling reaction (for this N2S2 compound and the
other chelating compounds described below) is conducted
using conventional procedures.
Additional N2S2 chelating compounds comprising carboxylic
acid substituent(s) for improved biodistribution
- properties are described in copending U.S. Patent
Application Serial number 07/367,502, entitled
"Radionuclide Metal Chelates for the Radiolabeling
of Proteins", which is hereby incorporated by
reference.
Examples of such chelating compounds are as follows:
: .
~ r~ ~r:
OCC ~ ) ~ 0CC I ~ C_0~.
:
' ' ', : :.. - ,.,.
~, :
'~ ~
c~z n~~
~o
-~C~s~ S)~C~ ac~ J~co~
T ~ ` `
.
wherein the symbols T and Z are as described above for the
other N2S2 chelating compounds.
:
: Another type of chelating compound that may be employed
: comprises one sulfur and three nitrogen donor atoms and
~hus may be designated an :"N3S" chelating compound
~: 30 Suitable;N3S chelatin~ compounds ~include those describedin European patent application~publication number 284,071
and copending U.5. Patent App~l--tion Ser-al numbcr
`
.i . . . . . . .
:
,
~r.~
07/172,004, both entitled "Metal-Radionuclide-Labeled
Proteins and Glycoproteins for Diagnosis and Therapy",
which are hereby incorporated by reference in their
entirety. Examples of N3S chelating compounds include but
are nct limited to the following seven compounds, wherein
"T" represents a sulfur protecting group and "COOTFP"
represents a 2,3,5,6-tetrafluorophenyl ester group:
r~,Ro
I ~N ~
o~( ~R'
S l
R~
B
C~p~und 1 8 a c~2~am
Cq13pound 1 ~2-COOEIR ,
Co~p~und ~ C~2~4~tCil2-C2411
Co~pour~d 4 C~2~2~~CR2~0H C_~12~4'
Cq~pound S E~C'12~2-C445~ ,4F~
Cq~pound 6 C~2~2~2~0
C~pound 7 H E~ C~2~
The COOTFP active ester may be replaced by other
chemically reactive functional groups.
Other chelating compounds may have different combinations
of donor atoms. Such compounds include among others, the
N2S4, N2S3, and N3S3 chelating compounds described in
copending U.S. Patent Application Serial no. 07/201,134,
entitled "Metal Radionuclide Chelating Compounds for
Improved Chelation Kinetics", which is hereby incorporated
by reference in its entirety. In addition, the N2S~ and N3S
.
- , ~ ' ~,.
': . : . : ,
i,9
- 28 -
compounds presented above may comprise varying numbers of
Oubstituents such as carboxylic acid groups and from 0 to
3 oxyyen atoms (=0) attached to carbon atoms of the
chelate core.
In the present invention, the chelating compounds
comprise, or are attached to, cleavable linkers. ~ number
of linkers that are cleavable under defined conditions
(e.g., at acidic pH, under reducing conditions, or in the
presence of an enzyme such as a protease) are known. The
chelates therefore may be released from the pol~neric
carrier under the desired conditions.
Suitable chelating compounds comprising a cleavable link
age include but are not limited to those descri~ed in
copending U.S. Patent Application Serial no. 07t457,480,
entitled "~adiolabeled Proteins for Diagnostic and
Therapeutic Vse", which is hereby incorporated by
reference in its entirety. The U.S.S.N. 07/457,480
application discloses N2S2 and N3S chelating compounds
comprising a linker of defined structure that terminates
in a chemically reactive functional group. The linkage is
cleavable at an es~er group positioned in a particular
orientation therein. Examples of such chelating compounds
include, but are not limited to, the following:
- , :
.
,
'
s
- 29
O O
\~0~
}~ ~H C'--~ ~
E!OOC-( )~Q 5 Lo
S S I I
I I 'c~~
-
Z
~' ~0 ~ -:
'
wherein T represents a sulfur protecting group and Z
represents a chemically reactive group (e.g., an active
ester) which may be used to incorporate the chelating
compound into the polymeric carrier in accordance with the
present invention.
Other examples of radiolabeled molecules that may be
attached to the polymeric carrier in accordance with the
present invention include radiohalogenated molecules.
Examples of molecules that bind radiohalogens at the meta
or para position on a phenyl ring are described in U.S.
Patent No. ~,855,153, en'itled "R~diohalogenated
. , .
' ,: ' ,
. : : -
: . :. : ,: ;
: ~: : , : :
.-
: ~
-
- 30 -
Proteins", which is hereby incorporated by reference in
its entirety. These compounds may be represented by the
following formula:
*X - Ar - R
wherein
*X is a radioisotope of iodine, bromine, fluorine,
or astatine;
Ar is an aromatic or heteroaromatic ring; and
R is a chemical bond or a substituent containing 1
to 12 straight-chain carbon atoms that does not
activate Ar toward electrophilic substitution on the
order produced by hydroxy or amino substitution of
the ring. The bond or substituent has attached
thereto a chemically reactive functional group
useful in the present invention for incorporation of
the compound (or a non-radiolabeled precursor
thereof) onto the polymeric carrier.
*I-paraiodophenyl compounds (in which *I
represents a radioisotope of iodine) may be
prepared using procedures that generally involve
substituting the organometallic group Sn(n-Bu)3 or
SnMe~ on a haloaromatic compound. A radioisotope of
a halogen then is substituted for the organometallic
group by halodemetalization. Examples of
radiohalogenated molecules that may be prepared
using such a procedure are represented by the
following formulas:
~ X ~ ( c~7 jn
o
~X ~ C~ )n ~'~
,,~; . . . . . ...
~,~
'
- 31 - ~ ~s~
wherein n represents an integer from 0 to 3, Z represents
a re~ctive functional group, and *v represents a
radioisotope of a halogen.
Additional radiohalogenated molecules that may be used in
the present invention are described in U.S. Patent No.
4,876,081, which is hereby incorporated by reference in `~!
its entirety. The radiohalogenated molecules comprise a
vinyl group.
The radiolabeled polymeric carrier targeting molecules of
the present invention have use in diagnostic and
therapeutic procedures, both for in vitro assays and for
in vivo medical procedures. The radiolabeled polymeric
carrier molecules may be administered intravenously,
intraperitoneally, intralymphatically, locally, or by
other suitable means, depending on such factors as the
type of target site. The amount to be administered will
vary according to such factors as the type of radionuclide
(e.g., whether it is a diagnostic or therapeutic
radionuclide), the route of administration, the type of
target site(s), the affinity of the targeting molecule for
the target site of interest, and any cross-reactivity of
the targeting molecule with normal tissues~
Appropriate dosages may be established by conventional
procedures and a physician skilled in the fieId to which
this invention pertains will be able to determine a
suitable dosage for a patient. A d~agnostically effective
dose is generally from about; 5 to about 35 mCi
and typically from about 10 to about 30 mCi per 70 kg body
weight. A therapeutically effective dose is generally
from about 20 mCi to about 300 mCi. For diagnosis,-
.
- , . . .
.. -, : , .~, .
- . , .
, " : ~
. . : ,,
- 32 - ~?~7~
conventional non-invasive procedures (e.g., gamma cameras)
are used to detect the biodistribution of the diagnostic
radionuclide, thereby determining the presence or absence
of the target sites of interest (e.g., tumors).
To render the ester in the polymeric carrier molecules of
the present invention more susceptible to cleavase in the
kidneys, an agent that raises urine pH may also be
administered to the patient. Such agents include, for
example, a salt of ascorbate (e.g., sodium ascorba~e) or
a bicarbonate salt (e.g., sodium bicarbonate), which may
be administered intravenously. ~aising the urine pH to a
basic level promotes cleavage of the ester in conjugates
or catabolites thereof localized in the kidneys. Clearance
of the released radionuclide metal chelates ~rom the body
is thereby enhanced. Administration of such agents to
promote cleavage of ester linXers in VlVO is described in
U.S. Patent Application Serial No. 07/251,900, which is
hereby incorporated by reference.
The comparatively low intestinal localizatisn of the
therapeutic radiolabeled polymeric carrier antibodies of
the present invention or catabolites thereof permits
increased dosages, since intestinal tissues are exposed to
less radiation. The clarity and accuracy of diagnostic
images also is improved by the reduced localization of
radiolabeled polymeric carrier antibodies or catabolites
thereof in normal tissues.
The above disclosure generally describes the present
invention. A mor~ complete understanding can be obtained
by reference to the following specific examples which are
: ., . ~ . .
,
' '
- 33 - 2~
provided for purposes of illustration only, and are not
intended to lir.it the scope of the invention.
EXAMPLE 1
Three peptide carriers containing 6, 12, and 18 ~-amino
acids with different side chain hydroxyl groups are
synthesized to show the use of hydrazones in covalently
linking to agents. The requisite peptides are synthesized
using the solid phase methodology of Merrifield (G. Barany
and R.B; Merrifield, "The Peptides. Analysis, Synthesis
and ~iology" E. Gross and J. Meinho~er, Editors, Academic
Press, New York, pages 1-284 (1980).
.
This example first involves the synthesis of peptide 1,
N-Acetyl-L-seryl-L-aspartyl(~-Otce)-L-seryl-L-threonyl-
L-aspartyl-(~-Otce)-L-threronyl-~-aminobutyric acid. This
is followed by the oxidation of the hydroxyl amino acid
side chain groups to carbonyl groups. These carbonyl
groups are then condensed with the hydrazide groups on the
agents. Next, there is formation of the active ester on
the C-terminal of the peptide. This enables the peptide
or polymeric carrier with attached agents to conjugate
with the antibody. The general procedure for the
synthesis of a polymeric carrier with attached agents and
the conjugation of the polymeri~ carrier to an antibody is
illustrated in Figure 1. Figure 2 lllustrates a specific
polymeric carrier synthesis and a specific conjugation
procedure.
The above compounds are synthesized as C-terminal
carboxylates using PAM resin attached to the first C-
terminal amino acid (J.~. Stewart and J. Young, "Solid
- ~ , : ! `
~: , ` , '. , '
.
~' ~
~ 34 ~ 2~
phase peptide synthesis", Pierce Chemical Company,
P~ockford, Illinois, 1984)) on an Applied Biosystems ~30
synthesizer using its specific protocols with N-
methylpyrrolidone as a coupling solvent (User's manual.
Model 430A synthesizer. Applied Biosystems, Inc., Foster
City, CA)).
The preferred protecting groups are Ser (0-benzyl), Thr
(O-benzyl), Glu(0-t butyl), Glu(0-benzyl), Asp(0-t-butyl)
and Tyr(Br-Cbz) (G. Barany an~ R.B. Merrifield, "The
Peptides. Analysis, Synthesis and Biology" E. Gross and
J. Meinhofer, Editors, Academic Press, New York, pages
1-284 (1980)). The other preferred protecting group for
gluta~ic and aspartic acids (and other carboxyl bearing
side chains) is the trichloroethyl ester
trichloroethoxycarbonyl for tyrosine (Tce). The presence
of th~s protecting group on the carboxyl of Asp and Glu
residues offer protection through the sequence of
derivatization of the side chain, attachment of the agents
and the final activation of the terminai carboxyl group
for conjugation to the targeting molecule. After the
activation, the trichloroethyl group(s) can be removed by
the using 2n-HOAc or Zn-THF-phosphate buffer (R. B .
Woodward, K. Heusler, J. Gosteli, P. Naegeli, W. Oppolzer,
Z5 R. Ramage, S. Ranganathan and H. Vorbruggen, J. Amer.
Chem. Soc., 88, 852 (1989) and M.F. Sommelhack and G. E.
Heinsohn, J. Amer. Chem Soc., 94, 5139 (1972)). After
first deblocking by trifluoroacetic acid, the N-terminal
residue is acetylated using acetic anhydride and finally
cleaved ~rom this resin using HF (See Figure 1).
The clea~age of peptides from the resin are accomplished
using the low-high HF cleavage procedure of Tam and
,. ' ' . . ! .
'' . ' ~.
., . , ' ' ",
. ' .
~ '
::
/. ' ~ I ' '
,
- 35 -
,9
Merrifield tJ.P. Tam, W.F. Heath and R.B. Merrifield, "SN2
deprotection o synthetic peptid2i, with low concentraticn
of HF in dimethyl sulfide: evidence and application in
peptide synthesis." J. Amer. Chem. Soc., 105, 6442
(1983~) (method A) or in 10:1:1:2 (by volume) of
HF:anisole:dimethylsulfide:p-thiocresol for 1 hour at 5
to OC. After cleavage, the organic scavengers are
extracted from the resin 3 times with ether and the
peptides extracted twice with 5 mL volume of 20-40%
HOAc/H20. After lyophilization, the peptides are purified
on a semi-preparative Vydec LC4 reversed phase column
using a gradient of 100% H~O-0.1% TFA to 40~ H2O-0.1%
TFA+60% CH3CN-0.1% TFA. They are analyzed for correct
amino acid composition and molecular weight by FAB mass
spectrometry ( T.D. Lee, "Methods of Protein Micro-
characteri2ation" J.E. Shively, editor. The Humana
Press, Clifton, New ~ersey, p. 403 (1986).
,.
It is necessary to prepare the corresponding C-14 labeled
peptides at the N-terminal residue acetyl for the pùrpose
of determining the stoichiometry of at~achment of peptides
and modified peptides containing therapeutic molecules.
The N-terminal residue after the first deblocking of the
N-terminal Boc group is acetylated using labeled acetic
anhydride. As an example, 10 mg of the peptide is N-
acetylated with C-14-acetic anhydride (1 ~Ci, 11.3
mCi/mmol) which is added and shaken for 2.5 houris with the
resin. A 5-fold molar excess of diisopropylethylamine was
added and N-acetylation was continued for 30 minutes.
Peptide resin sealed in 1 inch square polypropylene bags
was washed several times with 4 mL/bag of methylene
chloride, 5% diisopropylethylamine~ methylene chloride and
.. j , . .
' ', ' .
, . ., :.. ,:
, .~ .
.
- 36 ~
finally with 10% cold acetic anhydride/methylene chloride
to complctc thc acrtylatioll. Excess labelled anhydride was
washed from the resin by consecutive rinses of methylene
chloride, dimethyl formamide, isopropanol, methylene
chloride, methanol and the resin was dried overnight prior
to deblocking by the procedures described above.
To avoid the potential proteolytic degradation of the
peptide or polymeric carrier attached to the targeting
molecules while in the serum, the N-terminal residue or
all the residues are in the D-configuration. The change
in the configuration of the peptide backbone will not
alter the rate of the release of the therapeutic molecules
attached to the side chain. However, this change may
diminish the immunogenicity of the peptide backbone of
these carriers.
The next step involves MoÆfatt oxidation of peptide 1 to
the corresponding carbonyl compound 2. The oxidation of
the peptide is carried out using DMS0, DCC, pyridine
trifluoroacetate, and benzene or toluene. Moffatt
oxidation is preferred for compound 2 since the procedure
does not result in over oxidation of the hydroxyl
compound. (For general methods see, A.F. Cook and J.G.
Moffatt, J. Amer. Chem. Soc., 89, 2697 (1967) and K. E.
Pfitz~er and J.G. Moffatt, J. Amer. Chem Soc., 87, 5661
(1965)).
The nPxt step is preparation (modification) of therapeutic
molecules by changing their alcohol groups to hydrazides
and-then condensing them with modified peptides. The
therapeutic molecules of interest are Verrucarin A and
Roridin A which belong to the trichothecene group of
'
:
- 37 -
antiboiotics (B.B. Jarvis and A. Acierto in "Trichothecene
Mycotoxicosis: Pathyophysiological ~ffects" Vol. l. V. A.
Beasley, Editor, CRC Press, Boca Raton, FL, 1989. pp.
73-105). These compounds with broad spectrum biological
activities are the most potent synthesis inhibitors
containing C, H and 0. They exert their inhibition by
interacting with the EF2 on the ribosomes (C.S.
McLaughlin, M.H. Vaughen, I.M. Campbell, I.M. Wei and B.S.
~ansen, "Mycotoxins in Human and Animal Health", J.V.
Rodericks, Editor. Pathotox Publishers, 1977, pp 263-
273). The structures of these compounds are shown below.
Verrucarin A t6) and Roridin A (8) were converted to the
corresponding succinyl hydrazide derivatives (7 from 6,9
and 10 from 8) according to published procedures (R. O.
Xollah, "The Chemistry and Biology of Macrocyclic
Trichothecenes", Ph.D. Thesis, University of Maryland,
1989; M. Zeng, "Studies in Chemical and Biological
Structures of Macrocyclic Trichothecenes", Ph.D. Thesis,
University of Maryland, 1989; V.M. Vrudhula, T.M.
Comezoglu and A. Srinivasan, Abstract No. MEDI 50, ACS
National Meetingj Boston, MA, April 1990).
A similar strategy can be used to convert other molecules
of therapeutic intrest containing alcohol function to
hydrazide (after incorporating a succinyl moiety) for
attachment to the defined polymer 2. In a similar manner,
molecules of therapeutic interest containing a carboxylic
acid ~unction can be attached to the defined polymer via
the formation of a hydrazide.
-: .
.: . , ~ : ~
;: :
~ ~ .
-- 38 --
;~r~
Y ~ H V~rucarin A 6 H
Y = Y' = 0~ Rondin A
Y = CO-( CH2)2-CONHNX2 7
Verrucarin A-~succinyl hydra2~de Y = CO-( CH~ CONHNH; R' = H 9
(E~ondin A-~'-O-succinyl hydrazide)
Y=H; Y' =CO (CH~)~-CO?1~YH, 10
(Roridir~ A- I~'-O-succinyl hydrazie-)
Compou~d 6 ~ Compound 1
- Compoul~d5 ~ Compound 9,Compound 10
¦ In F~a~lIo 2, Mae~ocyele O CO ~CH2~
The next procedure is preparation of hydrazone 3 from
compound 2 and Verrucarin A hydrazide 7. To a solution of
the peptide (1 mmol) in isopropanol 5 mmol of Verrucarin
A hydrazide (7; 10 mmol) is added and the solution is
~; allowed to stand at room temperatures for several hours.
The formation of hydrazone is followed chromatographically
and is isolated either~by crystallization or by C-18
; column chromatography. The product is characterized by NMR
and FAB mass spectrometry.
The hydrazone derived ~from peptide 4 and ~Roridin A
derivativ~s g and lo are prepared~in a simi.ar manner.
.
.
.
., . , . ~ ..
~, :. . . ~: ,
- 39 - ~ ~ 9
The next synthesis is the preparation of active ester 4 of
peptide 3 (See Eisure 3). To a solution of the peptide ~
in DMF, from the above reaction, 3 equivalents of 2,3,5,6-
tetrafluorophenol and 3 equivalents of DCC are added and
the solution is stirred at room temperature for 10-12
hours. The precipitated dicyclohexylurea is removed by
- filtration and the residue is chromatographed to isolate
the product.
The product is dissolved in a phosphate buffer containing
10~ tetrahydrofuran and the trichloroethyl groups are
removed according to the procedure of M.F. Sommelhack and
G.E. Heinsohn (J. Amer. Chem. Soc., 94 5139 (1972)) to
yield the peptide or polymeric carrier 4, containing the
therapeutic molecules and an active ester for attachment
to the targeting molecule.
The last step is the conjugation of the active ester 4 to
the NR-LU-10 to give the conjugate 5. The active ester is
condensed with NR-LU-10 murine monoclonal antibody, which
recognizes a pancarcinoma antigen. Other proteins or
fragments may be substituted for the NR-LU-10 antibody.
To a solution of the antibody at pH 9-9.5, a solution of
the active ester in 250 mM bicarbonate buffer at pH 9.3 is
added and gently agitated to mix and incubated at room
temperature for 30 minutes to allow conjugation of the
peptide carrier to the antibody. The conjugate is
purified in a column containing an anion exchanger DEAE-
sephadex or QAE-sephadex. All of the above reactions are
shown in Figure 2.
In a similar manner, conjugates are prepared from longer
chain peptides, N-Acetyl-[L-seryl-L-aspartyl(~-Otce)-L-
, . . . .
.
- 40 - Z ~ ~ ~.x~,~
seryl-L-threonyl-L-aspartyl~ Otce)-L-threonyl]2-~-
aminobutyric acid, peptide 11 and N-Acetyl-L-seryi-L-
aspartyl(~-Otce)-L-seryl-L-threonyl-L-aspartyl ~-Otce)L-
threonyl-~-aminobutyric acid, peptide 12.
General procedure for the evaluation of stability of
hydrazones follows. Prior to the evaluation of conjugates
the hydrazones derived from peptides 1, 11, and 12 are
converted to the free acids 13-15 (see Figure 3). For the
experiments in human serum stability, the hydrazone under
investigation is incubated in fresh human serum at 37C at
a concentration of 1 mg/mL. Aliquots (100 ~L) at different
time points (2-150 hours) are diluted with equal volumes
of acetonitrile. The suspension is centrifuged and the
centrifugate is analyzed by HPLC for the presence of the
therapeutic drug released. In a similar manner, the
compounds are tested for their stability at pH 5.6.
,.
,
.
:
,
~ 41 ~
r ~9
EXAMPLE 2
This example covers the attachment of bifunctional chelate
ligands to the defined peptide or polymeric conjugation of
the carrier to the antibody followed by radiolabeling .
The procedures in this example are the synthesis of
N-Acetyl-L-tyrosyl (O-CO-CH2-CCl3)-L-Asp(~-OtBu)-Glu(~-
OtBu)Gly-Glu(~-OtBu)-~-Aba-PAM resin 16, the removal of
protecting groups from the carboxyl groups in ~sp and
glutamyl residues using trifluoroacetic acid to
synthesize, peptide 17, the condensation of bifunctional
chelate, S-ethoxyethylmercapto-acetylglycylglycylserine -
trichloroethyl ester 24 to give 25, the cleavage of this
peptide from the resin to give 26, the activation of the
terminal carboxylic acid to give 27, the deprotection (to
2~), and the radiolabeling to give the chelate 29,
followed by con~ugation to the antibody to yield the
conjugate 30 (see Figure 4).
The requisite peptide, N-Acetyl-L-tyrosyl (O-CO-CH2-CCl3) -
L-Asp(~-OtBu)-Glu(~-OtBu)-Gly-Glu(~-OtBu)-~-Aba-PAMresin
16, is synthesized using solid phase methodology of
Merrifield (G. Barany and R. B. Merrified, "The Peptides.
Analysis, Synthesis and Biology" E. Gross and J.
Meinhofer, Editors, Academic Press, New York, pages 1-284
(1980)). The protecting group in each step is 9-
flourenylmethoxycarbonyl (Fmoc) rather than the N-tBoc
group used in the synthesis of 1. This methodology
conserves the protecting group of the Glu and Asp (and
other amino acid residues bearing a carboxyl side chain).
Removal of the Fmoc protecting group in each successive
~ . ~
: . .
-
.
- 42 -
step is accomplished by using aqueous piperdine.
Acetylati^n is accomplished according to the procedure
described earlier.
Preparation of C-14 labeled peptides at the N-terminal
residue acetyl. It is necessary to prepare the
corresponding C-14 labeled peptides for the purpose of
determining the stoichiometry of attachment of peptides
and modified peptides containing therapeutic molecules.
The N-terminal residue after the first deblocking of the
N-terminal Fmoc group is acetylated using labeled acetic
anhydride. As an example, 10 mg of the peptide is N-
acetylatéd with C-14 acetic anhydride ~1 mCi, 11.3
mCi/mmol) which is added and shaken for 2.5 hours with the
resin. A 5-fold molar excess of diisopropylethylamine is
added and N-acetylation was continued for 30 minutes.
Peptide resin sealed in 1 inch square polypropylene bags
was washed several time~ with 4 mL/bag of methylene
chloride, 5~ diisopropylethylamine/methylene chloride and
finally with 10% cold acetic anhydride/methylene chloride
to complete the acetylation. Excess labelled anhydride is
washed from the resin by consecutive rinses of methylene
chloride, dimethyl formamide, isopropanol, methylene
chloride, methanol and the resin was dried overnight prior
to removal of the t-Boc protecting groups.
To avoid the potential proteolytic degradation of the
peptide carrier attached to the biological macromolecules
while in the serum, the N-terminal residue or all the
residues are in the D-configuration. The change in the
configuration of the peptide back bone will not alter the
rate of the release of the therapeutic molecules attached
- ,
. . :
- 43 - ~ ,
to the side chain. This change may also diminish the
immunoganicity of the peptide backbone GL ~hese carrieLs.
The synthesis of N-Acetyl-L-Tyr-L-Asp-Glu-Gly-Glu-~-Aba
PAM resin 17. The peptide still attached to the resin is
deblocked using trifluoroacetic acid (conversion of tbutyl
esters of glu and asp residues) to -COOH according to the
general procedure (G. Barany and R.B. Merrifield, "The
Peptides. Analysis, Synthesis and Biology" E. Gross and J.
Meinhofer, Editors, Academic Press, New York, pages 1-284
(1980)).
The synthesis of S-ethoxyethylmercaptoacetyl-
glycylglycylserinetrichloroethyl ester 24 ~see Figure 5)
involves first the synthesis of N-t-Boc-Serine-O-benzyl-
trichloroethyl ester 18. To a solution of N-t-Boc-
serine-o-benzyl ether (5 mmol) in methylene chloride
containing 5 mmol of triethylamine, 5 mmol of N,
N-dicyclohexylcarbodiimide was added and the solution was
stirred at room temperature overnight. The precipitated
dicyclohexylurea was filtered and the filtrate was washed
with 1~ HC1 and water. The organic layer was dried over
anhydrous Na2SO4, and evaporated to give the trichloroethyl
ester, which WâS purified over a silica gel column.
Serine trichloroethyl ester trifluoroacetate 19 is
prepared in the following manner. A solution of 4 mmoI of
the above compound 1~, in 50 mL of glacial acetic acid
containing 200 mg of palladium~ on charcoal was
hydrogenated at 60 psi in a Paar apparatus for 10-12
hours. The catalyst WâS removed by filtration over celite
and the solvent was removed in vacuo to give N-t-Boc
:; , ,:, ~ . . .
, : :
- 44 - 2~
serine trichloroethyl ester, an oil, which was dried
overnight and used without lur~her pur~ficdLion. ~he oil
was stirred with 10 mL 50% trifluoroacetic acid-CH2Cl2 for
3 hours at room temperature to remove the Boc group. The
mixture was evaporated to dryness, coevaporated several
times wlth methylene chloride and dried to give 19. The
compound was homoge~eous by TLC and was used in the next
step without further purification.
S-(l-ethoxylethyl) mercaptoacetic acid 20 is prepared
according to the following. A solution of mercaptoacetic
acid (17.4 mL, 250 mmol) in 125 mL of dichloromethane
containlng p-toluenesulfonic acid monohydrate (0.24 g,
1.26 mmol) was cooled to -18 to -250C with stirring. Ethyl
vinyl ether (23.9 mL, 250 mmol) in 125 mL of
dichloromethane was added dropwise to the cold solution
over a period of 90 minutes. The stirring was continued
for an additional 30 minutes with the temperature
maintained in the -18 to -25C range. Then 200 mL of pH7
phosphate bu~fer was added, and the reaction mixture was
allowed to warm with stirring for lo to 15 minutes The
mixture was then poured into a flask containing 900 mL of
theyl acetate and 200 mL of water. Layers were separated
and the aqueous portion extracted twice with ethyl
acetate. The organic layers were combined, washed with
brine and dried (MgS04). Removal of the solvent left 31.4
g of S~ ethoxyethyl) mercaptoacetic acid 20 as a
colorl2ss oil (77% yield): IH N~ ~CDC13) 1.15 (t, J=7.0Hz,
3H), 1.52 (d, J=6.4Hz, 3H), 3.36 (s, 2~), 3.60 (m, 2H),
4.84 (q, J=6.4Hz, lH), 11.65 (s, lH). The material was
used without further purification.
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Succinimidyl S-(l-ethoxyethyl) mercaptoacetate 21 is
preparcd -ccording to the ~ollowing procedure. A solution
of S~ ethoxyethyl) mercaptoacetic acid (5.76 g, 35.1
mmol) and N-hydroxysuccinimide (4.85 g, 42.1 mmol) was
prepared in 100 mL of anhydrous THF. To this was added a
solution of 1,3-dicyclohexylcarbodiimide (8.70 g, 42.1
mmol) in 65 mL of anhydrous THF. The mixture was stirred
at room temperature for 2 hours or until TLC analysis
indicated complete formation of the succinimidyl ester.
The mixture was then filtered, and the filtrate was
concentrated in vacuo to a viscous residue. T~e residue
was dissolved in ethyl acetate, washed with water, brine,
and dried (MgS04). Removal of the solvent left the crude
succinimidyl ester as an oil, which was further purified
by flash chromatography on silica gel, using ethyl
acetate-hexanes as the column eluent, to give 5.1 g of
S-(l-ethoxyethyl) mercaptoacetic acid succinimidyl ester
as a colorless oiI (56~ yield): IH NMR (CDC13) 1.21
(t, J=7.0Hz, 3H), 1.58 (d, J=6.4Hz, 3H), 2.83 (s, 4H),
3.60 (m, 4H), 4.88 (q, J=6.4Hz, lH).
The synthesis of 22 is as follows. Solid NaHCO3 (1.09 g,
13.0 mmol) was added to a solution of glycylglycine (1.22
g, 9.3 mmol) in 10 mL of water. After gas evolution
ceased, a solution of (2.66 g, 10.2 mmol) in 12 mL of
CH3CN was added to the reaction mixture. The mixture was
stirred at room temperature for ~2 h, then evaporated in
vacuo. The residue was purified by flash chromatography
on silica gel (85:10:5 CH3CN:H~0:HOAc) to yield 2.2 g (86%)
of 22 as a viscous oil. IH NMR (DMSO) 8.26 (t, lH), 8.08
(t, lH), 4.~30 (q, lH), 3.73 (m, 4H), 3.52 (m, 2H),
3.24 (s, 2H), 1.43 (d, 3H), 1.10 (t, 3H~.
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The following are details on the synthesis of 23. 1,3
Dicyclohexylcarbodiimide (0.66 g, 3.2 mmol) was added co
a stirring solution of 22 t0.81 g, 2.9 mmol) and N-
hydroxysuccinimide (0.37 g, 3.2 mmol) in 10 mL of CH3CN.
After stirrlng for 2 h, the mixture was filtered and the
filtrate was evaporated in vacuo. The residue was
purified by flash chromatography on silica gel (96:4
EtOAc:HOAc) to yield 0.80 g (73~) of 23 as a viscous oil.
IH NMR ~DMSO) 8.54 ~t, lH), 8.29 (t, lH), 4.80 (q, lH),
~.27 (d, ~H), 3.78 (d, 2H), 3.53 (m, 2H), 3.24 (s, 2H),
2.81 (s, 4H), 1.43 (d, 3H), 1.09 (t, 3H).
The synthesis of S-ethoxyethylmercaptoacetylglycyl
glycylserinetrichloroethyl ester 24 is prepared in the
following manner (see ~igure 5). Triethyl-amine (2 mmol)
was added to a solution of 19 (1.7 mmol) ànd 23 (1.7 mmol)
is 5 mL of anhydrous dimethylformamide. After stirring
f or 2.5 hours at room temperature the mixture was
evaporated in vacuo. The resulting residue was taken up
in ethyl acetate (20 mL) and washed with watPr, saturated
sodium chloride and dried over sodium sulfate, filtered
and evaporated. The residue was purified over a C-18
column to give pure ~4, which was used in the
condensation.
The condensation of 24 with the -COOH residues of Glu and
Asp in 17 occurs in the follo~ing manner. Solid cleavage
of peptides from the resin is accomplished using the
low-high HF cleavage procedure of Tam and Merrifield (J.P.
Tam, ~.F. Heath and R.B. Merrifield, I'SN2 deprotection of
synthetic peptides with low concentration of HJ in
dimethyl sulfide: avidence and application in peptide
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synthesis." J. Amer. Chem. Soc., 105, 6442 (1983)) (Method
A) or in 10~ 2 (by volume ) of HF:anisole:
dimethylsulfide:p-thiocresol for 1 hour at 5-0C. After
cleavage, the organic scavengers are extracted from the
resin 3 times with ether and the peptides extracted twice
with S mL volume of 20-40% HOAc/H20. After
lyophilization, the peptides are puri~ied on a
semi-preparative Vydec LC4 reversed phase column using a
gradient of 100% H20-0.1~ TFA to 40~ H20-0.1% TFA+60%
CH3CN-0.1% TFA. They are analyzed for correct amino acid
composition and molecular weight by FAB mass spectrometry
(Ref: T.D. Lee, "Methods of Protein Microcharacterization"
J.E. Shively, editor. ~he Humana Press, Clifton, New
Jersey, p. 403 (1986)).
The following is the preparation of the active ester 27 of
26. To a solution of the peptide 26 in DMF, ~rom the above
reaction, 3 equivalents of 2,3,5,6tetrafluorophenol and 3
equivalents of DCC are added and the solution i5 stirred
at room temperature for 10-12 hours. The precipitated
dicyclohexylurea is removed by -filtration and the residue
is chromatographed to isolate the product, 27.
The product 27 is dissolved in a phosphate buffer
containing 10~ tetrahydrofuran and the trichloroethyl
groups are removed according to the procedure of M. F.
Sommelhack and G.E. Heinsohn (J. Amer. Chem Soc., 94, 5139
filtration and the residue is chromatographed to isolate
the product, 27.
The product 27 is dissolved in a phosphate buffer
containing 10~ tetrahydrofuran and the trichloroethyl
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groups are removed according to the procedure of M. F.
Sommelh3ck and G.E. Heinsohn tJ. Amer. Chem Soc., 94, 5139
(197Z)) to yield the peptide carrier 28, containing the
chelator capable of forming metabolically stable complexes
with radionuclides and an active ester for attachment to
the targeting molecule.
The following is the radiolabeling procedure with ~6Re.
The peptide containing the chelator radiolabeled with ~86Re
according to the following procedure. Sodium perrhenate
produced from a W/Re generator is combined with citric
acid (a preferred complexing agent for ~86Re), a reducing
agent (usually SnC12). The resulting ~8~e -citrate
exchange complex is heated with the chelating compound 28
at 75-100C for 10-15 minutes and then transferred to a
ooc ice bath for a few minutes to obtain the peptide 29
containing l86Re-complexes on the side chain.
"
The above solution containing the chelate is removed from
the ice bath, 2.0 mL of 250 mM sodium bicarbonate buffer
(pH 9-10) is added and the vial is agitated to mix.
Immediately, the antibody (whole or fragments) is added
and incubated at room temperature for 10-15 minutes to
complete the conjugation to the antibody. The conjugate so
produced is purified using an anion exchange column
~DEAE-sephadeX or QAE-sephadex) prepared under aseptic
conditions to yield 30.
In a similar approach, peptides 31 and 32 are synthesized
by solid phase procedure and the antibody conjugates 37
- and 38 prepared. The intermediates in the case of these
oligomer syntheses are shown in Figure 6.
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The invention now being fully described, it will be
~pparcnt to one of ordinary skill in thc art that ~.~ny
changes and modif ications can be made without departing
from the spirit or scope of the invention.
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