JPH0692869A - Polymerizing carrier for releasing agent covalent - Google Patents

Abstract

PURPOSE: To provide a polymeric carrier which releases covalent bonded agents and is useful for in-vivo image formation as well as diagnosis and treatment. CONSTITUTION: This carrier contains the covalent bonded side chains through cuttrable linkers in arbitrary combinations and contains one set of the α-amino acids expressed by the formula (PG is an N-terminal protective group; AA is the α-amino acid; SG is a spacer group; CG is a conjugation group; AGENT is a chelate agent; (n) is 2 to 28; (m) is 2 to 18: (r) is 0, 1; (s) is 0, 1).These agents may be directly bonded to the side chains through the cuttable linkers or may be covalent bonded to the side chains through the cuttable linkers after the chemical modification of the side chains. Hydrazone, disulfide and ester bond may be made to exist at the polymeric carrier between the side chains of the α-amino acids and the agents. The selection of the covalent bond of the side chains in the polymeric carrier and the agents is determined by the functional groups in the side chains of the α-amino acids and the functional groups existing in the agents.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to chemically defined polymeric carriers that provide advantageous properties for in vivo imaging and therapy. The polymeric rejector contains a side chain covalently attached to (i) a diagnostic and therapeutic molecule and (ii) a chelator capable of binding a diagnostic or therapeutic radionuclide.
-Consisting of amino acids.

[0002]

BACKGROUND OF THE INVENTION Monoclonal antibodies have been developed that localize to cancerous tissues due to their high specificity and affinity for antigens on the surface of ulcer cells. This development has increased the potential for clinical application if the antibody could bind diagnostic and therapeutic agents. Due to the high specificity of the antibody,
Antibodies make candidates desirable as target molecules for the delivery of diagnostic or therapeutic agents to the cancer site.

Unfortunately, the direct attachment of such agents to antibodies weakens the immunoreactivity. Any derivatization of an antibody weakens its immunoreactivity. Therefore, an antibody that has multiple binding to a diagnostic or therapeutic agent is an antibody with low specificity. Currently, chelating agents linked to diagnostic and therapeutic radionuclides are directly linked to antibodies. This precludes the controlled release of radionuclide from the antibody.
Due to the large size of antibody molecules, it is difficult to determine the exact nature of the chelator's binding to the antibody using conventional detection techniques, and to determine the number of chelator bound to the antibody. This lack of accurate information causes problems in obtaining legal approval of chelating agents. Regulatory authorities require that all substances that are licensed must be accompanied by information that clearly identifies the structure of the substance to be introduced into the body. What is needed is an approach to derivatize target molecules, such as antibodies, with minimal sites for carrying large amounts of diagnostic and therapeutic agents.
There is also a need for an approach that can measure the nature of chelator binding to an antibody and the number of chelator bound to the antibody.

[Structure of Invention]

According to the present invention, a chemically defined polymeric carrier is provided. The present invention encompasses a series of α-amino acids containing any combination of side chains to which diagnostic / therapeutic and chelating agents can be covalently attached via a cleavable linker. These agents can be directly covalently attached to the side chain via a cleavable linker or can be covalently attached to the side chain via a cleavable linker after chemical modification of the side chain. The hydrazone, disulfide and ester linkages can be present in the polymeric carrier between the side chain of the α-amino acid and the agent in any combination. The choice of the particular covalent bond between the side chain in the polymeric carrier and the agent is determined by the functional group in the α-amino acid side chain and the reactive functional group in the agent. The α-amino acid having a side chain to which the agent is not covalently bound can act as a spacer that minimizes interactions between the macromolecules linked to the polymeric carrier. In addition, amino acids with charged or hydrophilic side chains that do not covalently bind the agent can impart increased solubility to the polymeric carrier.

Optional N-terminal protecting groups for the polymeric carrier include any of the standard amine protecting groups. C-terminal conjugating groups that are optional for linking the polymeric carrier to the target molecule include all conjugating groups known in the art. A spacer group is present on the polymeric carrier between the α-amino acid and the conjugating group to provide effective linkage of the polymeric target molecule. The spacer group provides any steric hindrance to ligation by any agent added to the C-terminus of the carrier. These spacer groups are terminal amino acids such as γ-aminobutyric acid (Aba). In the absence of a conjugating group, the spacer group, eg, Aba via its carboxy group, can link the polymeric carrier to the target molecule. The peptide that constitutes the polymeric carrier is produced from the α-amino acid by a conventional solution method or by solid phase peptide synthesis. These peptides are modified to carry derivatized diagnostic / therapeutic agents and chelating agents that bind diagnostic and therapeutic radionuclides. These agents can be released at the target site or after uptake by cells.

Many advantages result from the present invention. The polymeric carrier can carry the maximum number of agents while derivatizing the target molecule at the minimum number of sites. Thus, the biological activity of the target molecule is maintained at high levels even when linked to multiple agents. For example, the less binding in an antibody, the higher its specificity. The rate at which the agent can be released from the polymeric carrier linked to the target molecule is controlled by adjusting the nature of the covalent bonds in the polymeric carrier.
For example, the agents are released at various rates by adjusting the stability of the covalent bond or by using different types of covalent bonds in the polymeric carrier. This is especially important when the disease requires long-term diagnostic or therapeutic applications.

Multiple agents, which may be the same or different, are linked to the polymeric carrier. Not only can the same agent be released at different rates, but different agents can be released at different rates at the same target site. A polymeric carrier, together with its covalently attached agent, is a relatively small molecule compared to the target molecule. Therefore, prior to linking the polymeric carrier, the exact nature of the agent's binding to the target molecule can be pre-determined by conventional sensing techniques. Furthermore, by radiolabeling technology,
The exact number of polymeric carriers attached to the target molecule can be determined.

The present invention relates to chemically defined polymeric carriers that enhance diagnostic / therapeutic and chelator loading of target molecules. The polymeric carrier includes a set of 2 to about 18 α-amino acids in any combination that includes side chains covalently attached to the agent via a cleavable linker. The number of agents covalently attached to the polymeric carrier via a cleavable linker is from 2 to about 18.
This number is defined by the number of α-amino acid side chains in the polymeric carrier available for covalent attachment to the agent via the cleavable linker.

The term "polymeric carrier" as used in the present invention means a peptide carrier. Alpha-amino acids whose side chains are not covalently attached to the agent can function as spacers for the polymeric carrier. These spacers reduce any non-binding interactions between agents linked to modified α-amino acids. In addition to acting as a spacer, α-amino acids with charged or hydrophilic side chains that are not covalently attached to the agent can confer increased solubility on the polymeric carrier. The polymeric carrier optionally includes a protecting group at its N-terminus and an optionally conjugating group at its C-terminus. The conjugating group can itself link the polymeric carrier to the target molecule. A spacer group is placed between the α-amino acid and the conjugating group to assist in linking the polymeric carrier to the target molecule. The spacer group inhibits any steric hindrance to ligation by any group added from the C-terminus of the carrier. Furthermore, the spacer group, the terminal amino acid, can link the polymeric carrier to the target molecule without the presence of a conjugating group. This can occur by reaction of the carboxyl group of the terminal amino acid with the functional group on the target molecule to form covalent bonds such as ester and amide bonds.

In the polymeric carrier, increasing polarity,
Consequently, α-amino acids having side chains that increase water solubility are desirable. It is considered that the increase in water solubility further contributes to a decrease in hepatobiliary absorption of the radiolabeled polymeric carrier protein. Α with a side chain that increases water solubility
-Amino acids include amino acids having a charged side chain (lysine,
Arginine, histidine, cysteine, aspartic acid, glutamic acid, tyrosine, tyrosine-O-SO
_3- ) and amino acids having hydrophilic side chains (serine, threonine, asparagine, glutamine) are included.
Standard amine protecting groups can be used as the N-terminal protecting group of the polymeric carrier. Preferred embodiments of the present invention include acetyl, propionyl, phenylacylsulfonyl, substituted phenylacylsulfonyl and other hydrophilic protecting groups.

The conjugating group is a chemically reactive functional group that reacts with the target molecule to attach the polymeric carrier. When the target molecule is a protein, the conjugating group is reactive under conditions that do not denature or otherwise adversely affect the protein. Therefore, the conjugating group is sufficiently reactive with the functional groups of the protein so that the reaction can be carried out in a substantially aqueous solution and needs to be forced by, for example, heating to elevated temperatures that can denature the protein. There is no. Non-limiting examples of conjugating groups include active esters, isothiocyanates, amines, hydrazines, maleimides or other Michael-type acceptors, thiols and activated halides. Among the preferred active esters are N-hydroxysuccinimidyl esters, sulphosxin imidyl esters, thiophenyl esters, 2,3,5,5.
6-Tetrafluorophenyl ester and 2,3
An example is 5,6-tetrafluorothiophenyl ester. The latter three preferred active esters may include groups that enhance water solubility at the p- (ie, 4) or o-position of the phenyl ring. Examples of such groups include:
CO ₂ H, SO ₃ ⁻ , PO ₃ ²⁻ , OPO ₃ ²⁻ , OS
Examples thereof include O ₃ ⁻ , N ⁺ R ₃ (wherein R represents H or an alkyl group) and O (CH ₂ CH ₂ O) _n CH ₃ groups.

The terminal amino acid used as a spacer group in the present invention includes aminocaproic acid, aminopentanoic acid, γ-aminobutyric acid, β-alanine, glycine and the like. Agents including hydrazides, R (CO) NHNH
₂ reacts with the α-amino acid side chain containing the aldehyde RCHO or the ketone R ₂ (CO) to form a polymeric carrier with a hydrazone bond having the formula:

[0013]

[Chemical 5]

[Wherein the α-amino acid in the polymeric carrier is 2 to about 18 units; PG is an N-terminal protecting group;
AA is an α-amino acid; SG is a spacer group that promotes effective ligation of the polymeric target molecule by inhibiting steric hindrance by the agent added from the C-terminus of the carrier; CG is The polymeric carrier is a conjugating group useful for linking to the target molecule; AGENT is a diagnostic or therapeutic agent or chelating agent capable of binding a diagnostic or therapeutic radionuclide 2 to about 18 units; R is H, CH ₃ , phenyl or phenyl substituted with electron donating and / or electron withdrawing groups; q is 0 or 1;
r is 0 or 1; and s is 0 or 1.]

Hydrazone formation is an effective method of linking certain therapeutic agents to monoclonal antibodies [King et al., Biochem.
25), p. 5774, 1986]. Recent work in the field of therapeutic immunoconjugates has presented hydrazone functionality as a potential cleavable linker between chemotherapeutic agents and monoclonal antibodies. Laguzza et al. [J. Med Chem (J. Me
d. Chem. ), 32, 548, 1989] demonstrated that Vinca, alkaloids can be conjugated to antibodies via a hydrazone bond and that the pH dependence of the drug could be studied. The hydrazone conjugation approach is useful for the purpose of delivering a drug conjugate to the ulcer site, where the conjugate formed via a serum stable but acid labile hydrazone linker is then exposed to the acidic environment of the ulcer. It was based on the assumption that it will be gradually released [Tannock et al., Cancer Research, Volume 49,
4373, 1989]. This conditional requirement necessitated the screening of several small molecule hydrazones for evaluation of stability in human serum and acetate buffer at pH 5.6.

The design of polymeric carrier systems with hydrazone linkages incorporates the results observed in small molecule studies. Peptides of known amino acid sequence are configured to carry a primary or secondary hydroxy group that can be oxidized to a carbonyl compound (the chain may carry two or more hydroxy amino acids, the primary or secondary). two).
The results of small molecule studies have revealed that hydrazones from aromatic aldehydes may be too stable to be useful. Hydrazones derived from aliphatic ketones have serum half-lives of 15-20 hours (derived from peptides from threonine and other amino acids containing secondary -OH groups). Hydrazones derived from aliphatic aldehydes (derived from 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 (derived from peptides from phenylserine and substituted phenylserines) have a serum half-life of 130 hours. By selecting an antibody or fragment thereof having a half-life in human serum similar to hydrazone,
Maximum delivery of antibody or fragments thereof to the ulcer is expected. After this, the release of therapeutic units can occur at a rate that depends on the half-life of the selected hydrazone in the acidic environment of the ulcer site. In order to prevent premature decomposition of the polymeric carrier, the polymeric carrier may be D-amino acid only or D-amino acid
And a mixture of L-amino acids.
An agent containing a thiol SH is reacted with a cysteine side chain to form a polymeric carrier having a disulfide bond having the formula:

[0017]

[Chemical 6]

[Wherein the α-amino acid in the polymeric carrier is 2 to about 18 units; PG is an N-terminal protecting group;
AA is an α-amino acid; SG is a spacer group that promotes effective ligation of the polymeric target molecule by inhibiting steric hindrance by the agent added from the C-terminus of the carrier; CG is The polymeric carrier is a conjugating group useful for linking to a target molecule; AGENT is 2 to about 18 units of a diagnostic or therapeutic agent or chelating agent capable of binding a diagnostic or therapeutic radionuclide; R is H or CH ₃
R'is H or CH ₃ ; q is 1 or 2;
R is 0 or 1; and s is 0 or 1
]].

The release rate of an agent linked to a polymeric carrier via a disulfide bond can be reduced by replacing hydrogen with an α-alkyl group (R, R '= CH ₃ ). An agent containing a hydroxy group is reacted with aspartic acid and glutamic acid side chains to form a polymeric carrier having an ester bond having the formula:

[0020]

[Chemical 7]

[Wherein the α-amino acid in the polymeric carrier is 2 to about 18 units; PG is an N-terminal protecting group;
AA is an α-amino acid; SG is a spacer group that promotes effective ligation of the polymeric target molecule by inhibiting steric hindrance by the agent added from the C-terminus of the carrier; CG is The polymeric carrier is a conjugating group useful for linking to a target molecule; AGENT is a diagnostic or therapeutic agent or chelating agent capable of binding a diagnostic or therapeutic radionuclide 2 to about 18 units; q is 0 or 1 R is 0 or 1; and s is 0 or 1.].

In general, a bifunctional chelating agent (A) is used to attach a radionuclide metal (eg, M =
Ligation of ^99m Tc, ¹⁸⁶ Re or ¹⁸⁸ Re) was performed according to the following procedure. Active ester (B)
The formation of (C) by the formation of an M-chelate containing ## STR1 ## followed by ligation to a monoclonal antibody is performed according to the following reaction scheme.

[0023]

[Chemical 8]

EOE represents an ethoxyethyl protecting group. C
OOTFP stands for 2,3,5,6-tetrafluorophenyl ester. The chelate is an N ₃ S derivative,
Also, the N ₂ S ₂ derivative follows a similar procedure. A carbon chain linked to the α-amino group of the antibody exists between the chelate and the antibody. After metabolism in various organs, the major metabolite (D) remains in the intestine and kidney and is not excreted as follows.

[0025]

[Chemical 9]

This residue inhibits imaging in the lower abdominal region and results in high renal dose during treatment. As shown in the following reaction scheme, the presence of a cleavable linker between the chelate and the antibody [compound (F) produced from (E)] is metabolized to form (G), which is formed in the intestine. It was found that it did not persist and that it remained in the kidney to a low degree.

[0027]

[Chemical 10]

With the observation that the cleavable linker is metabolized in the above reaction scheme, the hydroxy group of the next compound is linked to the aspartic acid and glutamic acid side chains to form an ester bond on the polymeric carrier.

[0029]

[Chemical 11]

The resulting polymeric carrier can carry more than one radionuclide metal per ligation of antibody or fragment, taking advantage of the metabolites that can be eliminated via the renal system instead of remaining in the intestine. It can be provided. Compound (H) belongs to the N ₃ S type chelate, and (J) and (K) belong to the N ₂ S ₂ chelate system. The groups THP (tetrahydropyranyl) and Acm (acetamidomethyl) are used as sulfur protecting groups. A targeting molecule is any molecule that serves to deliver a polymeric carrier with a linked diagnostic / therapeutic or chelating agent to a desired target site (eg, target cell) in vitro or in vivo. Examples of target molecules include, but are not limited to, steroids, cholesterol, lymphokines, and agents and proteins that bind to the desired target site.

The target molecule may be a target protein capable of binding to a desired target site. As used herein, the term "protein" includes proteins, polypeptides and fragments thereof. The target protein may bind to a receptor, substrate, antigenic determinant or other binding site on the target cell or other target site. The target protein serves to deliver the agent linked by the polymeric carrier to a desired target site in the living body. Non-limiting examples of target proteins include antibodies and antibody fragments, hormones, fibrinolytic enzymes and biological response modifiers. In addition, other molecules that localize to the desired target site in vivo (though not strictly proteins) are also included within the definition of the term "target protein" as used herein. For example,
Certain carbohydrates or glycoproteins may also be used in the present invention. The protein can be modified, eg, to produce variants and fragments thereof, so long as the desired biological properties (ie, the ability to bind the target site) are retained. Proteins can also be modified using various genetic or protein engineering techniques.

Among the preferred target proteins are antibodies, most preferably monoclonal antibodies. Many monoclonal antibodies have been developed that bind to specific types of cells,
Included are monoclonal antibodies specific for human ulcer-associated antigen. Among the many monoclonal antibodies that can be used are anti-TAC, or other interleukin-2 receptor antibodies; 250 kilodalton human melanoma associated proteoglycans 9.2.2
7 and NR-ML-05; and NR-LU-10 having reactivity with pan-oncoglycoprotein. The antibody used in the present invention may be an intact (whole) molecule, a fragment thereof or a functional equivalent thereof. Examples of antibody fragments include F (ab ') ₂ , -Fab', F
There are ab and _Fv fragments, which may be produced by conventional methods or by genetic or protein engineering.

Proteins contain various functional groups, such as carboxylic acid (COOH) or free amine (-NMH ₂ ) groups, which groups react with the appropriate protein conjugating groups on the polymeric carrier to produce a polymeric carrier. Can be used to bind to a target protein. For example, the active ester of the polymeric carrier reacts with the ε-amino group of protein lysine residues to form an amide bond. Alternatively, the target molecule and / or the polymeric carrier can be derivatized to expose or link additional reactive functional groups. Derivatization can be accomplished using a number of linker molecules such as the Pierce Chemical Company (Pie).
rcChemical Company, Rockford, Illinois and may include any linkage of linker molecules (Pierce 1986-87. General Catalog, 313-54).
See page). Alternatively, derivatization may involve chemical treatment of the protein, which may be an antibody. Techniques for producing free sulfhydryl groups on antibodies or antibody fragments are also known (see US Pat. No. 4,659,839). The maleimide conjugating group in the polymeric carrier is reactive with the sulfhydryl (thiol) group. Alternatively, if the target molecule is a carbohydrate or glycoprotein,
Derivatization may involve chemical treatment of carbohydrates, such as periodate glycol cleavage of sugar moieties of glycoprotein antibodies (free aldehyde group formation). The free aldehyde groups of the antibody may react with the free amine or hydrazine conjugating groups of the polymeric carrier.

In the present invention, a therapeutic agent (eg, a drug, therapeutic radionuclide or toxin) is linked to a chemically defined polymeric carrier. Preferably, various therapeutic agents (which may be the same or different) are linked to the polymeric carrier. Exemplary therapeutic agents include toxins and pharmaceuticals. In the present invention, preferably the toxin holotoxin such as abrin, ricin, modeccin, Pseudomonas (Pseudomonas) exotoxin; Diphtheria (Duphtheria) toxin, pertussis toxin and Shiga toxin; and A chain or "A chain" molecules, such as ricin A , Abrin A chain, modesin A chain, enzyme part of pertussis toxin, enzyme part of Shiga toxin, gelonin, pokweed antiviral protein, saporin, yam barley toxin and snake venom peptide.

Exemplary drugs include daunomycin, adriamycin, vinblastine, doxorubicin, bleomycin, methotrexat.
e), 5-fluorouracil, 6-thioguanine, cyclavine, cyclophosphamide, and similar conventional chemotherapeutic agents [eg, "Cancer: Principles and Practices of Oncologies" (Canc.
er: Principles and Practic
es of Ohcology), 2nd edition, V.I. T. DeVita, Jr. , S. Hermann
lman), S.L. A. Rosenberg
rg), J. B. Lipincott Company (J.B.
Lippincott Co. ), Philadelphia,
PA, 1985, see chapter 14]. Yet another suitable pharmaceutical agent that may be used in the present invention is trichocecene (t
Rorithecene), Roridine (Ror
idin) A is particularly preferred. Laboratory medicines are also suitable for use in the present invention [eg NCI. Investor Relational Drags Pharmaceutical Data (NCI Investigative)
Drugs Pharmaceutical Dat
a) NIH Publication No. 1987. 882141, 1
Revised November 987].

In the present invention, the radiolabeled molecule is linked to a chemically defined polymeric carrier. Preferably,
A number of radiolabeled molecules, which may be the same or different, are linked to the polymeric carrier. Radionuclide metal chelates are one type of radiolabeled molecule that can be used. Many chelating compounds of various structures, as well as synthetic and radiolabeled methods for the preparation of radionuclide metal chelates are known. As an example, sulfur, nitrogen,
Chelating compounds containing various combinations of oxygen and phosphorus donor atoms can be used. The chelate compound is, for example, a total of 4 to 6 selected from nitrogen and sulfur atoms.
The number of donor atoms may be included. During the radiolabeling procedure, a bond is formed between the donor atom and the radionuclide metal, which produces a radionuclide metal chelate. The chelate compound can be incorporated into the polymeric carrier during the synthetic procedure. Alternatively, the chelating compound may be synthesized separately and then linked to the polymeric carrier.

One type of chelating compound that can be used contains two nitrogen and two sulfur donor atoms and therefore can be referred to as an "N ₂ S ₂ " chelating compound. Suitable N ₂ S ₂ chelate compounds are disclosed in US Pat. No. 4,897,255 with the title of the invention “Metal Radionuclide Labeling Proteins for Diagnostic and Therapeutic”, which is hereby incorporated by reference in its entirety. . An example of the N ₂ S ₂ chelate compound is as follows.

[0038]

[Chemical 12]

Wherein n is from 1 to about 4 (preferably 2); each R is independently selected from ═O and H ₂ ; T is an active ester or other reactive functional group (in the present invention). Useful for incorporating chelating compounds into polymeric carriers). Any suitable conventional sulfur protecting group may be attached to the sulfur donor atom of the compounds of this invention. Protecting groups may be added prior to or during the radiolabeling reaction,
Must be removable. Among the preferred sulfur protecting groups are Acm and hemithioacetal protecting groups (EO
E, THP), which can be displaced from the chelating compound during the radiolabeling reaction. The N ₂ S ₂ chelate compound is preferably radiolabeled after ligation to the polymeric carrier to produce a radionuclide metal chelate having the formula:

[0040]

[Chemical 13]

In the formula, P represents a polymeric carrier, M represents a radionuclide metal or an oxide thereof, and the other symbols are as described above. The radionuclide, which is not limited to the radionuclide metal, is ^99m effective for diagnosis.
Tc and therapeutically effective radionuclide ¹⁸⁸ Re,
¹⁸⁶ Re, ⁶⁷ Cu, ⁶⁴ Cu, ²¹² Pb, ²¹²
Bi and ¹⁰⁹ Pd are included. ¹⁸⁶ Re and
¹⁸⁸ Re is the radionuclide metal for use in the present invention.

Methods for producing these isotopes are known. Molybdenum / technetium generators for producing ⁹⁹ Tc are commercially available. ^The operation for producing ¹⁸⁶ Re is described in Deutsch, et al. [Nucl.Med.Biol., Vol. 13, 4].
No., pp. 465-477, 1986] and Vanderheiden et al. [Inorganic Chem.
(Istry), 24, 1666-1673, 198.
5 years], and the ²²² Bi process for the production of ¹⁸⁸ Re is described in Brachoto (Blacho).
t) etc. [Intle J. Of Applied Radiation and Isotope (Intl. J. of
Applied Radiation and Iso
top), vol. 20, pp. 467-470, 1969]
And Kiofutar et al. [J. Of Radeio Analytical Chem (J. of Radi
oanalytical Chem. ) Volume 5, 3-1
0, 1970]. ¹⁰⁹ Pd
Are produced by Fawaz et al., J. J. Nucl Med., (1984), 25
Vol. 7, page 796. ²¹² Pb and
²²² Bi is produced by Gansow et al., Amer Chem Suk Simp Siri (Amer. Chem.
Soc Symp. Ser. ) (1984), 241
Vol. 215-217 and Kozah et al., Proc. Natru Acad. Sai USA (Proc. Na.
t'1. Acad, Sci, USA) (1986, 1)
Mon) 83, pp. 474-478.

The radiolabeling reaction (for this N ₂ S ₂ compound and other chelating compounds described below) is carried out using conventional methods. Additional chelating compounds, including carboxylic acid substituents, for improved biodistribution properties have the title of the invention "Radionuclides Metal Chelates for Radiolabeling of Proteins", pending US patent application Ser. / 367,
No. 502, which is incorporated herein by reference. Examples of such chelating compounds include:

[0044]

[Chemical 14]

[0045]

[Chemical 15]

In the formula, the symbols T and Z are as described above for the other N ₂ S ₂ chelate compounds. Another type of chelating compound that may be used include one sulfur and three nitrogen supply atoms, so may be referred to as "N _{3 S"} chelating compound. Suitable N ₃ S chelating compounds are disclosed in European Patent Application Publication No. 284,071 and pending US patent application Ser. Protein and glycoprotein ”).
The entire of these applications are hereby incorporated by reference. N ₃ S
Examples of chelating compounds include, but are not limited to,
The following seven compounds are included, where "T" indicates a sulfur protecting group and "COOTFP" indicates a 2,3,5,6-tetrafluorophenyl ester group.

[0047]

[Chemical 16]

The COOTFP active ester can also be substituted with other chemically reactive functional groups. Other chelating compounds may have different combinations of donor atoms.
Such compounds include, among others, the N ₂ S ₄ , N ₂ disclosed in pending US patent application Ser. No. 07 / 201,134 under the title “Improved Chelating Speed Wheel Metal Radiation Nuclide Chelating Compounds”. ₂ S ₃ and N ₃ S ₃ chelate compounds are included and are incorporated herein by reference in their entirety. Further, the N ₂ S ₂ and N ₃ S chelate compounds described above include varying numbers of substituents such as carboxylic acid groups and 0 to 3 oxygen atoms (═O) linked to the carbon atoms of the chelate core. You may

In the present invention, the chelating compound includes or is linked to a cleavable linker. Many linkers are known that are cleavable under defined conditions (eg, acidic pH, reducing conditions or in the presence of enzymes such as proteases). Thus, the chelate can be released from the polymeric carrier under desired conditions. Chelating compounds that include, but are not limited to, cleavable linkers are disclosed in pending US patent application Ser. No. 07 / 457,480 having the title "Radiolabeled Protein for Diagnostic and Therapeutic Applications". The entire contents of this application are incorporated herein by reference. U.S. Patent Application No. 07 / 457,480, N ₂ S ₂ and N _{3 S} chelating compounds, including linkers of defined structure which chemically reactive functional groups is in the terminal are disclosed. The bond can be cleaved with an ester group present in a special coordination. Non-limiting examples of such chelate compounds include:

[0050]

[Chemical 17]

[0051]

[Chemical 18]

Where T represents a sulfur protecting group and Z represents a chemically reactive group (eg, active ester) that may be used to incorporate the chelating compound into the polymeric carrier in accordance with the present invention. Other examples of radiolabeled molecules linked to polymeric carriers according to the present invention include radiohalogenated molecules. An example of a molecule having a radiohalogen attached to the m- or p-position of the phenyl ring is U.S. Pat. No. 4,855,153 having the title "Radiohalogenated Protein" of the invention.
, Which is incorporated herein by reference in its entirety. These compounds can be represented by the formula:

[0053]

[Chemical 19]

Where ^* X is iodine, a radioisotope of bromine or astatine; Ar is an aromatic or heteroaromatic ring; and R is the order in which they are generated by hydroxy or amino substitution of the ring. It is a chemical bond or substituent containing 1 to 12 linear carbon atoms that does not activate Ar towards electronic substitution. The bond or substituent is linked to a chemically reactive functional group useful in the invention for incorporating the compound (or its non-radioactively labeled precursor) into a polymeric carrier. ^* I-paraiodophenyl compound (where
^* 1 is a radioisotope of iodine) is an organometallic group Sn (n-Bu) ₃ or SnMe _{3 in a} haloaromatic compound.
Can be prepared using an operation that generally includes the substitution of The radioisotope of halogen is then replaced with an organometallic group by halodemetallation. An example of a radiohalogenated molecule that can be produced using such a procedure is represented by the formula:

[0055]

[Chemical 20]

Here, n represents an integer of 0 to 3, Z represents a reactive functional group, and ^* X represents a radioactive isotope of halogen. Additional radiohalogenated molecules that can be used in the present invention are described in US Pat. No. 4,876,081, which is incorporated herein by reference in its entirety. Radiohalogenated molecules include vinyl groups.

The radiolabeled polymeric carrier targeting molecules of the present invention are useful in diagnostic and therapeutic procedures for both in vitro assays and in vivo medical procedures. The radiolabeled polymeric carrier molecule may be administered intravenously, intradermally, intralymphaticly, topically or by any other suitable means depending on such factors as the type of target site. Dosages include, for example, the type of radionuclide (eg, whether it is a diagnostic or therapeutic radionuclide), the route of administration, the type of target site, the affinity of the target molecule for the target site of interest and the normal tissue and target molecule. Depending on factors such as cross-reactivity with any.

An appropriate dose can be established by a conventional method, and a doctor skilled in the field of the present invention can determine an appropriate dose for a patient. A diagnostically effective dose is generally about 5-35 mCi, typically about 10-30 mCi.
Ci / 70 kg body weight. A therapeutically effective dose is generally about 20-300 mCi. For diagnosis, the biodistribution of the diagnostic radionuclide is detected using a conventional non-invasive procedure (eg gamma camera), which determines the presence or absence of the target site of interest (eg ulcer). .

Agents that increase urine pH may also be administered to the patient in order to make the ester in the polymeric carrier molecules of the present invention more susceptible to cleavage in the kidney. Such agents include, for example, salts of ascorbic acid (eg sodium ascorbate) or bicarbonates (eg sodium bicarbonate), which are administered intravenously. Raising the urine pH to basic levels promotes the cleavage of the ester in the conjugate or catabolite, which is localized in the kidney.
This enhances clearance of the released radionuclide metal chelate from the body. Administration of such agents to promote cleavage of the ester linker in vivo is disclosed in US patent application Ser. No. 07 / 251,900,
This application is inserted here for reference. The therapeutic radiolabeled polymeric carrier antibody of the present invention or its catabolite is localized in the intestine at a relatively low level, whereby the dose can be increased. The reason is that the intestinal tissue is exposed to radiation to a lesser extent. The clarity and accuracy of the diagnostic image is also improved by reducing the localization of the radiolabeled polymeric carrier antibody or its catabolite in normal tissue.

[0060]

The above disclosure generally describes the present invention. The invention can be more fully understood by reference to the following specific examples. These examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Example 1 Three peptide carriers containing 6,12 and 18 α-amino acids with different side chain hydroxy groups were synthesized to demonstrate the use of hydrazones in covalent attachment to agents. The essential peptide is Merrifield
d) [G. Barany and R.A. B. Merrifield, "The Peptide Analysis, Syntheses and Biology" (The Peptide
s. Analysis, Synthesis and
Biology), E.I. Gross and J. Mainhoffer (Academic press, New York, pages 1-284 (1980)].

In this example, the peptide (1), N-
Acetyl-L-seryl-L-aspartyl (β-Otc
e) -Synthesis of -L-seryl-L-threonyl-L-aspartyl- (β-Otce) -L-threonyl-γ-aminobutyric acid. This is followed by oxidation of the hydroxy amino acid side chain to a carbonyl group. These carbonyl groups are then condensed with the hydrazide groups of the agent. The formation of active ester at the C-terminus of the peptide is then carried out. This allows the peptide or polymeric carrier with the bound agent to be conjugated to the antibody. The general procedure for the synthesis of the polymeric carrier with the attached agent and the conjugation of the polymeric carrier to the antibody is illustrated in Scheme 1. Step 2 illustrates a particular polymeric support synthesis and a particular conjugation procedure.

The above compound was prepared as described in Applied Biosystems 430.
Using its specific protocol with A-synthesizer and N-methylpyrrolidone as the coupling solvent [User's Manual Model 430A (User's
manual. Medel 430A) Synthesizer, Applied Biosystems Incorporated, Foster City. CA], using a PAM resin linked to the first C-terminal amino acid [J. M. Stewart and J. Young
g), "Solid phase peptide synthesis (Solid phase peptide synthesis)
sis), Pierce Chemical Company (Pier)
Ce Chemical Company, Rockford, Illinois (1984)
)], As the C-terminal carboxylate.

Preferred protecting groups are Ser (o-benzyl), Thr (o-benzyl), Glu (o-t-butyl), Glu (o-benzyl), Asp (o-t-butyl) and Tyr (Br). -Cb _s) [G. Balanyi and R.M. B. Merrifield, "The Peptide Analysis Syntheses and Biology", E.I. Gross and J. Main Hofua, Editor, Academic Press, New York, pages 1-284 (1980)]. Another preferred protecting group for glutamic acid and aspartic acid (and other carboxyl bearing side chains) is trichloroethyl ester trichloroethoxycarbonyl (Tce) for tyrosine. The presence of this protecting group at the carboxyl of the Asp and Glu residues provides protection via the sequence of side chain derivatization, ligation of the agent and final activation of the terminal carboxyl group for conjugation to the target molecule. After activation, the trichloroethyl group becomes Zn
-HOAc or Zn-THF-phosphate buffer can be used to remove [R. B. Woodward (Wo
Edward), K.S. Heusler,
J. Gosteli, P.G. Naegeri (Na
egeli), W. Oppolzer,
R. Ramage, S.M. Langanatan (R
anganathan) and H .; Vorbruggen, J. et al. J. Amer. Chem. Soc., 88, 85
2 (1989) and M.S. F. Somelhack (So
mmelhack) and G.M. E. Heinson
nshn), J. Amel Chem Suk, Volume 94, 51
39 (1972)]. After initial deprotection with trichloroacetic acid, the N-terminal residue is acylated with acetic anhydride and finally cleaved from the resin with HF.

Cleavage of the peptide from the resin is achieved by
m) and Merrifield low-high HF cleavage procedures [J.
P. Tam, W. F. Heath and R.H.
B. Merrifield, "SN _2. Deprotection of Synthetic Peptides with Low Concentration of HF. In Dimethyl Sulfide (SN ₂ depletion of sy
nthetic peptides with low
concentration of HF in d
immedial sulphide: evidence and application in peptide synthesis (evidence and application)
in peptide synthesis), J.
Am Chem Suk, 105, 6442 (1983)
)] (Method A) or with HF: anisole: dimethyl sulfide: p-thiocresol 10:
Achieved in 1: 1: 2 (volume) at 5 ° -0 ° C. in 1 hour. After cleavage, the organic scavenger and extracted 3 times from the resin with ether and the peptide 20~40% HOAc _/ H 2 O 5
Extracted twice with ml volume. After lyophilization, the peptide was loaded onto a semi-preparative Vydec LC4 reverse phase column with 100% H ₂ O-
0.1% TFA to _{40% H 2 O-0.1%} TFA + 6
Purified using a gradient of _{0% CH 3 CN-0.1%} TFA. Peptides are analyzed by FAB mass spectrometry for correct amino acid composition and molecular weight [T. D. Lee, “Method of Protein Microcharacterization” (Metho)
ds of Protein Microcharac
teriation), J. E. Shivuri (Shiv
ely), editor, The Hiyumana Press (The Hu
mana Press, Clifton
n), New Jersey, p. 403 (1986)].

It is necessary to prepare the corresponding C-14 labeled peptide with an N-terminal residual acetyl for the purpose of defining the stoichiometry of the linkage of the peptide and the modified peptide containing the therapeutic molecule. The N-terminal residue after the initial deprotection of the N-terminal Boc group is acetylated with labeled acetic anhydride. As an example, 10 mg of peptide is converted into C-14-acetic anhydride (1 mC
i, 11.3 mCi / mmol), N-acetylated,
This is added to the resin and shaken for 2.5 hours. A 5-fold molar excess of diisopropylethylamine was added and N-acetylation was continued for 30 minutes. The acetylation is completed by washing the peptide resin sealed in a 1 square inch polypropylene bag several times with methylene chloride, 5% diisopropylethylamine / methylene chloride and finally 4% 10% cold acetic anhydride / methylene chloride / bag. Excess labeled anhydride was washed from the resin with successive washings with methylene chloride, dimethylformamide, isopropanol, methylene chloride, methanol. The resin was dried overnight before deprotection as described above. The N-terminal residue or all residues are in the D-coordinate to avoid potential proteolysis of the peptide or polymeric carrier linked to the target molecule while present in serum. Changes in the coordination of the peptide backbone do not modify the release rate of the therapeutic molecule linked to the side chain. However, this change may reduce the immunogenicity of the peptide backbone of these carriers.

The next step involves the Moffatt oxidation of peptide (1) to the corresponding carbonyl compound (2). Peptide oxidation is performed with DMSO, DCC, pyridine trifluoroacetate and benzene or toluene. Mahat oxidation is preferred for compound (2). The reason is that this procedure does not result in over-oxidation of hydroxy compounds. [For general methods, see A. F. Cook and J.C. G.
Moffatt, J. Amel Chem Suk, 89, 2697 (1967) and K.K. E.
Pfitzner and J. G. Mohat, J. Amel Chem Suk, 87, 5661 (1965)].

The next step is the preparation (modification) of the therapeutic molecule by converting the alcohol group to a hydrazide and then condensing it with a modified peptide. The therapeutic molecules of interest are Verrucarin A and Roridin (Ro).
ridin) A, which belong to the trichocecene group of antibiotics [B. B. Jarvis
And A. Acierto, "Trichothecen
e Mycotoxosis: Pathy Physiolog
I. Effects. ", Vol. R. Beasley edition, CRC press (Pres)
s), Boca Raton, EL.
1989, pp. 73-105]. These compounds with broad spectrum biological activity are the most potent synthetic inhibitors containing C, H and O. These compounds exhibit their inhibitory action by interacting with EF2 on the ribosome [C. S. McLaughlin,
M. H. Vaughen, I. M. Campbell, I.C. M. Wei
And B. S. Hansen, "Mycotoxins in Human and Animal Health"
d Animal Health), J. Am. V. Mederiks, Editor, Pathotox Publishers
s), pp.263-273, 1977]. The structures of these compounds are shown below. Bercalin A (6) and Roridin (8) were converted to the corresponding succinyl hydrazide derivatives according to the following published procedure [(6), (9)
To (7) and (8) to (10)]. R. O. Kollah, "The Chemistry and Biology of Macrocyclic Trichocenes" (The Chem5ry and Biol)
oxy of Macrocyclic Tricho
Thences), Ph. D Theme, University of Maryland (University of Maryland),
1989; Zeng, "Studies in Chemical and Biological Structures of Macrocyclic Trichocenes"
d Biological Structures o
fMacrotric Trichothecen
es), Ph. D Theme, University of Merriland, 1989
Year; V. M. Vrudhula, T .;
M. Comezoglu and A. Srinivasan, Abstract No. MEDI50, ACS National Meeting (National Meeting)
ng), Boston ,. April 1990].

Similar procedures can be used to convert other molecules of therapeutic interest containing alcohol functional groups to hydrazides for conjugation to defined polymers (2) (succinyl moiety). After adding). In a similar way,
Molecules of therapeutic interest containing carboxylic acid functional groups can be linked to defined polymers via hydrazide formation.

[0069]

[Chemical 21]

The next operation is the preparation of hydrazone (3) from compound (2) and bercarin A hydrazide (7). To a solution of the peptide (1 mmol) in isopropanol, vercarin A hydrazide (7:10 mmol) 5
Mmol is added and the solution is left at room temperature for several hours.
The formation of the hydrazone was followed by chromatography and crystallized or isolated by C-18 column chromatography. The product is characterized by NMR and FAB mass spectrometry. Hydrazones derived from peptide (4) and loridin A derivatives (9) and (10) are similarly prepared. The next synthesis is the production of active ester (4) of peptide (3) (see step 3). Add 3 to the DMF solution of peptide (3) from the above reaction.
Equivalents of 2,3,5,6-tetrafluorophenol and 3 equivalents of DCC are added and the solution is allowed to stand at room temperature for 10-1
Stir for 2 hours. The precipitated dicyclohexylurea is filtered off and the residue is chromatographed to isolate the product. The product was dissolved in phosphate buffer containing 10% tetrahydrofuran and trichloroethyl groups were added to M.P. F. Somelhack and G.M. E. Heinson [J. Amel Chem. Soku. 94, 5139 (1972
Removal) to produce a peptide or polymeric carrier (4) containing an active ester for ligation to therapeutic and target molecules.

The final step is to convert the active ester (4) to NR-
That is, it is conjugated with LU-10 to produce a conjugate (5). NR recognizing pan cancer antigen with active ester
Condensate with the LU-10 murine monoclonal antibody.
Other proteins or fragments may replace the NR-LU-10 antibody. pH to an antibody solution of pH 9-9.5
A solution of 9.3 of the active ester in 250 mM bicarbonate buffer is added, gently stirred to mix, and incubated for 30 minutes at room temperature to conjugate the peptide carrier to the antibody. The conjugate is purified on a column containing the anion exchanger DEAE-Sephadex or QAE-Sephadex. All of the above reactions are shown in Scheme 2.

Similarly, a long-chain peptide, N-acetyl- [L-seryl-L-aspartyl (β-Otce)-
L-seryl-L-threonyl-L-aspartyl- (β
-Otce) -L-threonyl] ₂ -γ-aminobutyric acid,
Peptide (11) and N-acetyl-L-seryl-L
A conjugate is prepared from -aspartyl (β-Otce) -L-seryl-L-threonyl-L-aspartyl (β-Otce) -L-threonyl-γ-aminobutyric acid, peptide (12). Follow the general procedure to evaluate the stability of hydrazones. Prior to the evaluation of the conjugate, peptide (1),
The hydrazones derived from (11) and (12) are converted to the free acids (13)-(15) (see step 3). For human serum stability experiments, the hydrazone to be studied is cultured at a concentration of 1 mg / ml in fresh human serum at 37 ° C. Aliquots (100 μl) are diluted with an equal volume of acetonitrile at different time points (2-150 hours). The suspension is centrifuged and the centrifuge is subjected to HPLC
Is analyzed for the presence of therapeutic agent released by. Similarly, compounds are tested for stability at pH 5.6.

Example 2 This example involves conjugation of a bifunctional chelating ligand to a defined peptide or polymeric conjugate of a carrier to an antibody followed by radiolabelling. The procedure of this example is based on N-acetyl-L-tyrosyl (O-CO-CH _{2
-CCl 3) -L-Asp- (β} -OtBu) -Glu
(Γ-OtBu) Gly-Glu (γ-OtBu) -γ
-Ara-PAM resin (16), synthesis of peptide (17) by removal of the protecting groups from the carboxyl groups in Asp and glutamyl residues using trifluoroacetic acid,
Synthesis of (25) by condensation of the bifunctional chelate, S-ethoxymethylmercapto-acetylglycylglycylserine-trichloroethyl ester (24), production of (27) by cleavage of this peptide from the resin, deprotection (( 28)) and chelate by radiolabelling (2
Production of 9), followed by conjugation to antibody to produce conjugate (30) (see step 4).

[0074] required peptide, N- acetyl -L- tyrosyl _{(O-CO-CH 2 -CCl} 3) -L-Asp (β
-OtBu) -Glu (γ-OtBu) -Gly-Gl
u (γ-OtBu) -γ-Aba-PAM resin (16)
Is a solid field method of Merrifield [G. Balanyi and R.M.
B. Merrifield, “The Peptide, Analysis, Synthesis and Biology” E. Gross and J. Main Hotshua, Editor, Academic Press,
New York, pages 1-284 (1980)]. The protecting group at each step is N- used in the synthesis of (1).
It is 9-fluorenylmethoxycarbonyl (Fmoc) rather than the tBoc group. This method uses Glu and As
It protects the protecting group of p (and other amino acid residues bearing the carboxyl side chain). Fmoc in each continuous process
Removal of the protecting groups is done with aqueous piperidine. Acylation is achieved according to the procedure disclosed above.

Preparation of C-14 radiolabelled peptide with N-terminal residue acetyl. There is a need to produce the corresponding C-14 labeled peptides for the purpose of determining the stoichiometry of the linkage of the peptide containing the therapeutic molecule and the modified peptide. N-
The N-terminal residue after the initial deprotection of the terminal Fmoc group is acetylated with labeled acetic anhydride. As an example, 10 mg of the peptide was added to C-14 acetic anhydride (1 mCi, 11.3 m
N-acetylated with Ci / mmol), added and shaken with resin for 2.5 hours. A 5-fold molar excess of diisopropylethylamine is added and N-acetylation is continued for 30 minutes. Peptide resin sealed in 1 square inch polypropylene bag several times with methylene chloride, 5
The acetylation is completed by rinsing with 4 ml / bag of% diisopropylethylamine / methylene chloride and finally with 10% cold acetic anhydride / methylene chloride. Excess labeled anhydride is washed from the resin with successive washes of methylene chloride, dimethylformamide, isopropanol, methylene chloride, methanol, and the resin is dried overnight prior to removal of the t-Boc protecting group.

To avoid potential proteolytic degradation of the peptide carrier linked to the biological macromolecule while in serum, the N-terminal residue or all residues are D-coordinated. . Changes in the coordination of the peptide backbone do not modify the release rate of the therapeutic molecule linked to the side chain. This change may also reduce the immunogenicity of the peptide backbone of these carriers.

N-acetyl-L-Tyr-L-Asp-
Glu-Gly-Glu-γ-Aba PAM resin (1
Synthesis of 7). General Procedures (G. Balanyi and R. B. Merifield, "The Peptides. Analysis, Synthesis and Biology", E. Gross and J. Mayhoffua, Editor, Academic Press, New York, 1-284 (1980). The peptide still linked to the resin is deprotected (conversion of t-butyl ester of glu and asp residues) to -COOH according to (Y.

S-Ethoxyethylmercaptoacetylglycylglycylserine trichloroethyl ester (2
In the synthesis of 4) (see step 5), Nt-Boc-serine-O-benzyl-trichloroethyl ester (1
8) Synthesis is included. Nt-Boc-serine-in methylene chloride containing 5 mmol triethylamine
To a solution of O-benzyl ester (5 mmol), N, N
-Add 5 mmol of dicyclohexylcarbodiimide,
The solution was then stirred overnight at room temperature. The precipitated dicyclohexylurea was filtered and the filtrate washed with 1% HCl and water. The organic layer was dried over anhydrous Na ₂ SO ₄ ,
Then, evaporation gives trichloroethyl ester, which is purified on a silica gel column.

Serine trichloroethyl ester trifluoroacetate (19) is produced as follows. A solution of 4 mmol of compound (18) above in 50 ml of glacial acetic acid containing 200 mg of palladium on charcoal was hydrogenated at 60 psi on a Paar apparatus for 10-12 hours. The catalyst is filtered off on Celite and the solvent removed in vacuo to give Nt-Boc serine trichloroethyl ester as an oil. This was dried overnight and used without further purification. Oil at room temperature for 3 hours 50% trifluoroacetic acid-C
The Boc group was removed by stirring with 10 ml of H ₂ Cl ₂ . The mixture was evaporated to dryness, co-evaporated several times with methylene chloride and dried to give (19). This compound was homogeneous by TLC and used in the next step without further purification.

S- (1-ethoxyethyl) mercaptoacetic acid (20) is prepared as follows. Mercaptoacetic acid (1) in 125 ml of dichloromethane containing p-toluenesulfonic acid monohydrate (0.24 g, 1.26 mmol).
A solution of 7.4 ml (250 mmol) was cooled to -18 to -25 ° C with stirring. Dichloromethane 12
Ethyl vinyl ether in 5 ml (23.9 ml, 25
0 mmol) was added dropwise to the cold solution over 90 minutes. -1
Stirring was continued for another 30 minutes at a temperature maintained in the range of 8 to -25 ° C. Next, pH 7 phosphate buffer 200m
1 was added and the reaction mixture was warmed with stirring for 10-15 minutes. The mixture is then mixed with tenyl acetate 9
Poured into a flask containing 00 ml and 200 ml of water. The layers were separated and the aqueous portion was extracted twice with ethyl acetate. The organic layers were combined, washed with brine and dried (MgSO _4). When the solvent was removed, 31.4 g of S- (1-ethoxyethyl) mercaptoacetic acid (20) remained as a colorless oil (yield 77%).

¹ H NMR (CDCl ₃ ) 1.15
(T, J = 7.0 Hz, 3H), 1.52 (d, J =
6.4 Hz, 3H), 3.36 (s, 2H), 3.60
(M, 2H), 4.84 (q, J = 6.4Hz, 1
H), 11.65 (s, 1H). This was used without further purification.

Succinimidyl S- (1-ethoxyethyl) mercaptoacetate (21) is prepared according to the following procedure. S- (1-ethoxyethyl) mercaptoacetic acid (5.76 g, 35.1 mmol) and N-hydroxysuccinimide (4.85) in 100 ml of anhydrous THF.
g, 42.1 mmol) was prepared. To this was added a solution of 1,3-dicyclohexylcarbodiimide (8.70 g, 42.1 mmol) in 65 ml anhydrous THF. The mixture was stirred at room temperature for 2 hours or until TLC analysis indicated complete formation of the succinimide ester. Then the mixture was filtered and the filtrate was concentrated in vacuo to a viscous residue. The residue was dissolved in ethyl acetate, water,
Washed with brine and dried (MgSO _4). Removal of solvent left crude succinimidyl ester as an oil, which was purified by flash chromatography on silica gel using ethyl acetate-hexane as column eluent. As colorless oil, S- (1-ethoxyethyl) mercaptoacetic acid succinimidyl ester 5.1
g (yield 56%) was obtained.

¹ H NMR (CDCl ₃ ) 1.21
(T, J = 7.0 Hz, 3 H), 1.58 (d, J =
6.4 Hz, 3H), 2.83 (s, 4H), 3.60
(M, 4H), 4.88 (q, J = 6.4Hz, 1
H).

The synthesis of (22) is as follows. Solid N
aHCO ₃ (1.09 g, 13.0 mmol) in water 10
A solution of glycylglycine (1.22 g, 9.3 mmol) in ml was added. After gas evolution has stopped, CH ₃ CN
Product in 12 ml (2.66 g, 10.2 mmol)
Was added to the reaction mixture. The mixture was stirred at room temperature for 22 hours then evaporated in vacuo. The residue was purified by flash chromatography on silica gel _{(85: 10: 5 CH 3} CN: H 2 O: HOAc) as NebaCho oil (22) was obtained 2.2 g (86%).

¹ H NMR (DMSO) 8.26
(T, 1H), 8.08 (t, 1H), 4.80 (q,
1H), 3.73 (m, 4H), 3.52 (m, 2)
H), 3.24 (s, 2H), 1.43 (d, 3H),
1.10 (t, 3H).

Details of the synthesis of (23) are as follows.
1,3-dicyclohexylcarbodiimide (0.66
g, 3.2 mmol) in 10 ml of CH ₃ CN (2
2) (0.81 g, 2.9 mmol) and N-hydroxysuccinimide (0.37 g, 3.2 mmol) was added to the stirred solution. After stirring for 2 hours, the mixture was filtered and the filtrate was evaporated in vacuo. The residue was flash chromatographed on silica gel (96: 4 EtOA).
c: HOAc) as a viscous oil (2
0.80g (73%) of 3) was obtained.

¹ H NMR (DMSO) 8.54
(T, 1H), 8.29 (t, 1H), 4.80 (q,
1H), 4.27 (d, 2H), 3.78 (d, 2)
H), 3.53 (m, 2H), 3.24 (s, 2H),
2.81 (s, 4H), 1.43 (d, 3H), 1.0
9 (t, 3H).

S-Ethoxyethylmercaptoacetylglycylglycylserine trichloroethyl ester (2
The synthesis of 4) is performed as follows (see process formula 5). Triethylamine (2 mmol) was added to a solution of (19) (1.7 mmol) and (23) (1.7 mmol) in 5 ml of anhydrous dimethylformamide. After stirring for 2.5 hours at room temperature, the mixture was evaporated in vacuo. The residue obtained was taken up in ethyl acetate (20 ml), washed with water, saturated sodium chloride, dried over sodium sulphate, filtered and evaporated. The residue was purified on C-18 column to give pure (24). This was used for condensation.

Glu and As in (24) and (17)
Condensation of p with one COOH residue is carried out as follows. Solid-state cleavage of peptides from resins is accomplished by the Tam- and Merrifield low-high HF cleavage procedure [J. P. Tam, W.
F. Heath and R.H. B. Merrifield, “SN ₂ ·
Deprotection of Synthetic Peptide
With Row Concentration Of HF In Dimethyl Sulfide: Evidence And Application In Peptide Syntheses ", J. Am. Am Chem Suk, 105, 6442 (1983)
)] (Method A) or with HF: anisole: dimethyl sulfide: p-thiocresol 10:
Achieved in 1: 1: 2 (volume) at 5 ° -0 ° C. in 1 hour. After cleavage, the organic scavenger and extracted 3 times from the resin with ether, and the peptide 20~40% HOAc _{/ H} 2 O
Extracted twice with a volume of 5 ml. After lyophilization, _{100% H} 2 O a peptide semipreparative Vydec LC4 reversed phase column
-0.1% TFA to _{40% H 2 O-0.1%} TFA +
Purified using a gradient of _{60% CH 3 CN-0.1%} TFA. Peptides are analyzed by FAB mass spectrometry for correct amino acid composition and molecular weight [T. D. Lee, "Method of Protein Microcharacterization", J. Am. E. The Sibley Editor, The Hiyumana Press Clifton, New Jersey, p. 403 (1986)].

Production of the active ester (27) of (26) is as follows. To a solution of the peptide (26) in DMF from the above reaction was added 3 equivalents of 2,3,5,6-tetrafluorophenol and 2 equivalents of DCC and the solution was stirred at room temperature for 10-12 hours. The precipitated dicyclohexylurea was filtered off and the residue was chromatographed to isolate the product (27). The product (27) was dissolved in phosphate buffer containing 10% tetrahydrofuran and the trichloroethyl group was added to M.P. F. Somerhack and G.I. E. It is removed according to the procedure of Heinson (J. Amel Chem Suk, Vol. 94, page 5139). Filter and chromatograph the residue to isolate the product (27). The product (27) was dissolved in phosphate buffer containing 10% tetrahydrofuran and the trichloroethyl group was added to M.P. F. Somerhack and G.I. E. Heinson [J. Amel Chem Suk, Volume 94, 5139
Page (1976)] to give a peptide carrier (28) containing a chelator capable of forming a complex with a metabolically safe radionuclide and an active ester for linking to a target molecule.

The radiolabeling procedure with ¹⁸⁶ Re is as follows. A peptide containing a chelator radiolabeled with ¹⁸⁶ Re according to the following procedure. The sodium perrhenate generated from the W / Re generator is converted into citric acid (
The preferred complexing agent for ¹⁸⁶ Re), a reducing agent (usually SnCl ₂ ). The obtained ¹⁸⁶ Re-citrate exchange complex was heated with the chelate compound (28) at 75 to 100 ° C. for 10 to 15 minutes, and then transferred to an ice bath at 0 ° C. for a few minutes to make ¹⁸⁶ R- on the side chain. A peptide (29) containing the complex is obtained. 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 add antibody (whole or fragment) and incubate at room temperature for 10-15 minutes to complete conjugation to antibody. The conjugate thus produced was prepared under aseptic conditions under an anion exchanger column (DEA
Purify using E-Sephadex or QAE Sephadex).

In a similar approach, peptides (31) and (32) were synthesized by solid phase engineering and antibody conjugates (37) and (38) were prepared. The intermediate in the case of synthesizing these oligomers is shown in Process Formula 6. It will be apparent to those skilled in the art that many changes and modifications can be made to the invention described in detail herein without departing from its spirit or scope.

Step 1 shows the general procedure for the synthesis of the polymeric carrier with the linked agent and the conjugation of the polymeric carrier to the antibody. Step 2 illustrates the procedure for synthesizing the polymeric carrier with the linked therapeutic agent and conjugating the polymeric carrier to the antibody. Step 3 illustrates removal of the protecting group from the amino acid side chain of the polymeric carrier.
Steps 4, 5 and 7 are reaction schemes showing operations for synthesizing a polymeric carrier having a chelating agent linked thereto and conjugating the polymeric carrier to an antibody. Step 6 is a reaction scheme showing the operation of synthesizing the chelating agent.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication location A61K 49/02 A 9164-4C (72) Inventor Diana Eyeblickner Washington, USA 98037 Lynwood Meadow Daylord 17111

Claims (14)

[Claims] 1. A side chain in which the agent is directly covalently bound via a cleavable linker or which is covalently bound via a cleavable linker after chemical modification of the side chain in any combination. And containing the formula [Wherein PG is an N-terminal protecting group; AA is an α-amino acid; SG is a polymeric target molecule by inhibiting steric hindrance by an agent added from the C-terminal of the carrier. A spacer group that facilitates effective ligation; CG
Is a conjugating group useful for linking the polymeric carrier to the target molecule; AGENT is a diagnostic or therapeutic agent or a chelating agent capable of binding a diagnostic or therapeutic radionuclide; n is 2 to about 18 M is 2 to about 18; r
Is 0 or 1; and s is 0 or 1]. A chemically defined polymeric carrier comprising a set of α-amino acids. 2. An agent having side chains which are not covalently attached and which acts as a spacer to minimize interactions between the agents which are covalently attached to the polymeric carrier. -The polymeric carrier of claim 1 further comprising an amino acid. 3. The agent further comprises at least one α-amino acid which has non-covalently attached charged or hydrophilic side chains and which imparts increased solubility to the polymeric carrier. Polymeric carrier of. 4. An α-amino acid having a charged or hydrophilic side chain, which is a group consisting of serine, threonine, lysine, arginine, histidine, cysteine, aspartic acid, glutamic acid, asparagine, glutamine, tyrosine and tyrosine-O—SO ₃ ^—. The polymeric carrier of claim 3 selected from 5. The polymeric carrier of claim 1, wherein the N-terminal protecting group PG is selected from the group consisting of acetyl, propionyl, phenacylsulfonyl and substituted phenacylsulfonyl. 6. The spacer group SG is aminocaproic acid,
The polymeric carrier of claim 1 selected from the group consisting of aminopentanoic acid, γ-aminobutyric acid, β-alanine and glycine. 7. The polymeric carrier of claim 1 wherein the CG conjugating group is selected from the group consisting of active esters, isothiocyanates, amines, hydrazines, maleimides or other Michael type acceptors, thiols and activated halides. 8. The polymeric carrier according to claim 1, wherein all α-amino acids are in the L-coordinate. 9. The polymeric carrier according to claim 1, wherein all the α-amino acids are D-coordinated. 10. The polymeric carrier of claim 1, wherein the α-amino acid is any combination of L and D coordinations. 11. The α-amino acid AA is covalently attached to the agent via a hydrazone bond and has the formula: [Wherein PG is an N-terminal protecting group; AA is an α-amino acid; SG is a polymeric target molecule by inhibiting steric hindrance by an agent added from the C-terminal of the carrier. A spacer group that facilitates effective ligation; CG
Is a conjugating group useful for linking a polymeric carrier to a target molecule; AGENT is a diagnostic or therapeutic agent or a chelating agent capable of binding a diagnostic or therapeutic radionuclide; R is H, CH ₃ , phenyl Or phenyl substituted with an electron donating and / or electron withdrawing group; q is 0 or 1; r is 0 or 1; and s is 0 or 1] Item 1. The polymeric carrier according to item 1. 12. The α-amino acid AA is covalently attached to the agent via a disulfide bond and has the formula: [Wherein PG is an N-terminal protecting group; AA is an α-amino acid; SG is a polymeric target molecule by inhibiting steric hindrance by an agent added from the C-terminal of the carrier. A spacer group that facilitates effective ligation; CG
Is a conjugating group useful for linking a polymeric carrier to a target molecule; AGENT is a diagnostic or therapeutic agent or a chelating agent capable of binding a diagnostic or therapeutic radionuclide; R is H or CH ₃ . R'is H or CH ₃
And q is 1 or 2; r is 0 or 1; and s is 0 or 1.]. 13. The α-amino acid AA is covalently attached to the agent via an ester bond and has the formula: [Wherein PG is an N-terminal protecting group; AA is an α-amino acid; SG is a polymeric target molecule by inhibiting steric hindrance by an agent added from the C-terminal of the carrier. A spacer group that facilitates effective ligation; CG
Is a conjugating group useful for linking the polymeric carrier to the target molecule; AGENT is a diagnostic or therapeutic agent or a chelating agent capable of binding a diagnostic or therapeutic radionuclide; q is 0 or 1; r is 0 or 1; and s is 0 or 1]. 14. The polymeric carrier according to claim 1, wherein the α-amino acid is covalently bonded to the agent via a hydrazone bond, a disulfide bond, an ester bond and any combination thereof.