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WO1997033626A1 - Nouveaux chelateurs et compositions preparees a l'aide de ceux-ci - Google Patents

Nouveaux chelateurs et compositions preparees a l'aide de ceux-ci Download PDF

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Publication number
WO1997033626A1
WO1997033626A1 PCT/US1997/004052 US9704052W WO9733626A1 WO 1997033626 A1 WO1997033626 A1 WO 1997033626A1 US 9704052 W US9704052 W US 9704052W WO 9733626 A1 WO9733626 A1 WO 9733626A1
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WIPO (PCT)
Prior art keywords
chelator
composition
nucleic acid
moiety
radionuclide
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PCT/US1997/004052
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English (en)
Inventor
Donald J. Hnatowich
Mary Rusckowski
George Mardirossian
Paul Winnard, Jr.
Fengchun Chang
Tong Qu
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University of Massachusetts Amherst
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University of Massachusetts Amherst
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Priority claimed from US08/614,078 external-priority patent/US5980861A/en
Application filed by University of Massachusetts Amherst filed Critical University of Massachusetts Amherst
Priority to AU24210/97A priority Critical patent/AU2421097A/en
Publication of WO1997033626A1 publication Critical patent/WO1997033626A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo

Definitions

  • this chelator has been radiolabeled prior to conjugation (i.e., preconjugation labeling) with macromolecular polymers such as proteins or polypeptides, which cannot withstand harsh conditions.
  • Preconjugation labeling can be a complex procedure with multiple intermediate purification steps. More importantly from the point of view of application of radionuclide-labeled macromolecules for diagnostics, imaging and therapeutics, preconjugation labeling can limit the usefulness of the product: the pre-conjugation radionuclide-chelator complex frequently has a short half-life, cannot be transported without necessary precautions for radioactivity, and can expose end-users to radioactivity during a number of complex synthetic steps, all required prior to end use with samples or patients.
  • Ram and Buchsbaum disclose the labeling of antibodies with chelators, however these require a specially modified phenylalanine residue n the chelator to accomplish conjugation.
  • Figure 9 shows size exclusion HPLC radiochromatograms of labeled PNA in buffer (A) and of several samples obtained at 2.5 hrs post administration of labeled PNA to a mouse which include a serum sample (B), the soluble fraction of a kidney homogenate (C), urine (D) and the 2.5 hrs urine sample (B), the 1.5 hr serum sample after adding complementary PNA immobilized on beads to extract radiolabeled PNA (C) and the 2.5 hrs urine sample after removing labeled PNA with complementary PNA immobilized on bead (E).
  • Figure 10 shows whole body radioactivity as a function of time (each of the experimental animals plotted as a different symbol) following injection of radiolabeled PNA.
  • Figure 14 shows the structures of N2S2-I, -2 and -3 N2S2 chelators, and synthesis of N2S2-I chelators and corresponding biocytin conjugates.
  • a patient may be in need of further categorization by clinical procedures well-known to medical practitioners of the art (or may have no further disease indications and appear to be in any or all respects normal).
  • a patient's diagnosis may alter during the course of disease progression, such as development of further disease symptoms, or remission of the disease, either spontaneously or during the course of a therapeutic regimen or treatment.
  • diagnosis does not preclude different earlier or later diagnoses for any particular patient or subject.
  • PNA Peptide nucleic acid
  • the invention further contemplates the use of nucleic acids, polynucleotides, and oligonucleotides that are alternatives to, or analogs of, naturally occuring deoxynucleic acid with its sugar-phosphate backbone, or synthetic oligo-and polynucleotide sugar- phosphate polymers. While native single-stranded phosphodiester DNA has been considered for in vitro or in vivo applications; these unmodified oligonucleotides are highly susceptible to degradation by nucleases (Wickstrom E. J Biochem Biophys Methods. 13:97-102, 1986; Cazenave C, Chevrier M., Ngugent T, Helene C. Nucleic Acids Res.
  • a nucleic acid molecule may be single-stranded or double-stranded, but preferably is single-stranded.
  • the nucleic acid is DNA, PNA phosphorothioate DNA, or RNA, and most preferably is PNA.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence and structure of backbone that occurs in nature, whether it is prepared by isolation from an organism or is chemically synthesized.
  • a nucleic acid may be chemically synthesized using a commercially available automated synthesizer and reagents, or custom made by a commercial supplier (for example, PerSeptive Biosystems, Framingham, MA).
  • a "chimeric nucleic acid” is a covalently linked first base sequence with a second base sequence of different chemical character, for example, a PNA strand covalently linked to a DNA or RNA strand.
  • an "isolated" nucleic acid molecule is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • an isolated tumor-specific nucleic acid molecule may contain less than about 5 kb, 1 kb, 0.5 kb, 0.1 kb or 50 bases of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (e.g. a human brain tumor).
  • an "isolated" nucleic acid molecule such as an RNA molecule, may be free of other cellular material.
  • the nucleic acid molecule may comprise only a portion of a coding region of a naturally occurring sequence.
  • an isolated nucleic acid molecule of the invention comprises a base nucleotide sequence with a specific linear order of the bases adenine, guanine, cystosine, thymine or uracil, or modified derivatives of these bases (e.g., methyladenine, and hydroxymethyluracil).
  • a nucleic acid molecule having a known nucleotide sequence can be isolated using standard molecular biology techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
  • mRNA can be isolated from cells (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al.
  • cDNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL).
  • reverse transcriptase e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL.
  • Synthetic oligonucleotide primers for PCR amplification can be designed based upon knowledge of the nucleotide sequence or fragments of the sequence, as will be appreciated by those with skill in the art, and are used to obtain and clone an isolated nucleic acid adjacent to the primer.
  • an isolated nucleic acid molecule useful in the compositions and methods of the invention is at least about 12 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of interest.
  • a nucleic acid is at least 15, 20, 30, 50, or 100 nucleotides in length.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60 % homologous to each other typically remain hybridized to each other.
  • nucleic acid molecules which are antisense thereto.
  • An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a “sense” nucleic acid, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.
  • Antisense constructs of the present invention by antagonizing the normal biological activity of tumor-specific or infectious DNA, can be used in the therapeutic context, both to deliver a therapeutic dose of radionuclide, and to inhibit expression of tumor-specific or infectious genetic information.
  • antisense nucleic acid can deliver a radionuclide complexed to a chelator covalently linked to the nucleic acid, to the target cells, tissue or sample.
  • antisense nucleic acids can be designed according to methods known in the art, e.g., according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence.
  • the term "coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence of interest.
  • noncoding region refers to 5' and 3' sequences which flank a coding region and are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • Antisense nucleic acid may be complementary to either or both of a coding region and an adjacent noncoding region.
  • the antisense nucleic acid molecule may be complementary to an entire coding region, but more preferably is an oligonucleotide which is antisense to only a portion of a coding or noncoding region.
  • an antisense oligonucleotide may be complementary to the region surrounding the translation start site of an mRNA.
  • An antisense oligonucleotide can be, for example, about 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and/or enzymatic reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., peptide nucleic acid or phosphorothioate nucleotides can be used.
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid).
  • Nucleic acids of the present invention are suitable molecules for the purposes of therapeutic "pretargeting," in which administration of a first composition, e.g., a pretargeting agent, to a subject causes the administered composition, agent, or material to become located or concentrated at a particular, preferably preselected, in vivo site.
  • a second composition e.g., a targeting agent
  • the molecular affinity of the pretargeted material for the in vivo site is, in a preferred embodiment, achieved by use of a specific antibody or antibody fragment, such as an antibody to a tumor marker.
  • a pretargeting agent can be a binding protein with engineered ligands, or an antisense nucleic acid of specific nucleotide sequence.
  • a second binding determinant of a pretargeting agent is a ligand with affinity for the subsequently administered "targeting" complex, agent or composition.
  • a pretargeting agent or complex carries a nucleic acid sequence
  • the targeting agent or complex carries a nucleic acid sequence complementary to the sequence of the pretargeting agent.
  • the pretargeting complex includes avidin or streptavidin
  • the targeting complex includes a biotin compound.
  • the pretargeting complex can include a biotin compound
  • the secondarily administered targeting complex includes avidin or streptavidin. It will be understood that other members of specific binding pairs, e.g., hormone/receptor, antibody/antigen, and the like, can be used as complementary pairs in targeting and pretargeting agents.
  • the pretargeting molecule or complex can facilitate the specific localization of the subsequently-administered targeting agent, which includes a functional moiety (e.g., a radionuclide) for therapeutic, diagnostic or imaging use.
  • a functional moiety e.g., a radionuclide
  • the advantage of pretargeting includes a greater localization ratio of specific to non-specific background of the targeted radionuclide for in vivo imaging and for therapy, leading to the use of lower doses of radioactive material, with enhanced contrast in imaging and fewer side effects in therapy.
  • Further improvement in targeting specificity is achieved by addition of "chase" regimens, e.g., by administration of compositions that can displace non-specifically bound pretargeting complexes, thereby enhancing image contrast or therapeutic index following delivery of the targeting radionuclide composition.
  • a “targeting" composition may in certain embodiments be employed alone or in combination with any of variety of different pretargeting compositions.
  • pretargeting agents can be designed to have affinity for a certain specific type of targeted tumor or an infectious agent or infected cell, e.g., the pretargeting agents can be antibodies for different tumor-specific antigens of a tumor cell or cells. All of these pretargeting agents can share a common ligand, e.g., a specific nucleic acid sequence, for a targeting agent.
  • a variety of different tumor types and infectious diseases may be pretargeted, each with a specific pretargeting composition, then treated with the same general targeting composition, comprising for example, a complementary nucleic acidconjugated chelator which is complexed to a radionuclide.
  • a particular targeting agent of the invention can be useful in several medical regimens, and a single targeting agent can be used for a variety of conditions by use of appropriate pretargeting agents.
  • the term "polymer” is intended to include molecules formed by the chemical union of two or more combining subunits called monomers. Monomers are molecules or compounds which usually contain carbon and are of relatively low molecular weight and simple structure.
  • a monomer can be converted to an oligomer or to a polymer by combination with itself or other similar molecules or compounds.
  • An oligomer comprises at least two monomers.
  • An oligomer or a polymer may be composed of a single identical repeating subunit or multiple different repeating subunits (copolymers).
  • Polymers within the scope of this invention include substituted and unsubstituted oligopeptides, carbohydrates, polypeptides, oligonucleotides, polynucleotides, and polypeptide backbones substituted with purine and purimidine bases or base analogs (e.g. PNA).
  • compositions in which a chelator moiety is conjugated to a polymer or macromolecule can be conjugated to any suitably functionalized molecule.
  • a chelator moiety can be conjugated to small molecules, such as drugs, provided that the small molecule includes suitable reactive functionality, e.g., an amino group, for reaction with the chelator moiety.
  • peptide includes two or more amino acids covalently attached through a peptide bond.
  • Amino acids which can be used in peptide molecules include those naturally occurring amino acids found in proteins such as glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan.
  • the tripeptide moiety does not contain an aromatic residue.
  • amino acid further includes analogs, derivatives and congeners of naturally occurring or synthetic amino acids, one or more of which can be present in a peptide derivative.
  • amino acid analogs can have lengthened or shortened side chains or variant side chains with appropriate functional groups.
  • the present invention does not require modification of the side chain of the amino acids of the chelator.
  • the D and L stereoisomers of an amino acid when the structure of the amino acid admits of stereoisomeric forms.
  • peptide derivative further includes compounds which contain molecules which mimic a peptide backbone but are not amino acids (so-called peptidomimetics), such as benzodiazepine molecules (see e.g. James, G. L. et al.
  • an oligopeptide can be designed to interact with a cell membrane constituent (e.g., if comprised primarily of hydrophobic amino acids). Accordingly, in one embodiment, an oligopeptide comprises three or four peptide residues, and a polypeptide comprises four or more residues. Polymers comprising oligopeptides or peptide backbones may be covalently linked to other moieties or functionalitites, for example, to an amine group attached via a linking arm.
  • the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.
  • the substituents of a substituted alkyl may include substituted and unsubstituted forms of aminos, azidos, iminos, amidos, phosphoryls (including phosphonates and phosphinates), sulfonyls (including sulfates, sulfonamidos, sulfamoyls and sulfonates), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like.
  • Preferred alkyls are lower alkyls.
  • aryl refers to an aromatic ring moiety having zero to four heteroatoms, and which may be substituted or unsubstituted, and can be fused to other aromatic or non-aromatic rings. Exemplary aryls are phenyl, pyridyl, naphthyl, and the like.
  • aralkyl refers to an alkyl group substituted with one or more aryl moieties, e.g., benzyl.
  • antibody as used herein is intended to include fragments thereof which are also specifically reactive with one of the imaging, diagnostic and therapeutic targets.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies.
  • F(ab)2 fragments can be generated by treating an antibody with pepsin.
  • the resulting F(ab)2 fragment can be treated to reduce disulfide bridges to produce Fab fragments.
  • the term "antibody” is further intended to include single chain, bispecific and chimeric molecules.
  • the term “antibody” includes possible use both of monoclonal and polyclonal antibodies (Ab) directed against a target, according to the requirements of the application.
  • Polyclonal antibodies can be obtained by immunizing animals, for example rabbits or goats, with a purified form of the antigen of interest, or a fragment of the antigen containing at least one antigenic site. Conditions for obtaining optimal immunization of the animal, such as use of a particular immunization schedule, and using adjuvants e.g. Freund's adjuvant, or immunigenic substituents covalently attached to the antigen, e.g. keyhole limpet hemocyanin, to enhance the yield of antibody titers in serum, are well-known to those in the art,. Monoclonal antibodies are prepared by procedures well-known to the skilled artisan, involving obtaining clones of antibody- producing lymphocyte, i.e.
  • Many monoclonal and polyclonal antibody preparations are commercially available, and commercial service companies that offer expertise in purifying antigens, immunizing animals, maintaining and bleeding the animals, purifying sera and IgG fractions, or for selecting and fusing monoclonal antibody producing cell lines, are available.
  • Specific binding proteins with high affinities for targets can be made according to methods known to those in the art.
  • proteins that bind specific DNA sequences may be engineered (Ladner et al, U.S. Patent 5,096,815), and proteins that bind a variety of other targets, especially protein targets (Ladner et al, U.S. Patent 5,233,409; Ladner et al, U.S. Patent 5,403,484) may be engineered and used in the present invention for covalent linkage to a chelator moiety.
  • Antibodies and binding proteins can be incorporated into large scale diagnostic or assay protocols that require immobilizing the compositions of the present invention onto surfaces, for example in multi-well plate assays, or on beads for column purifications.
  • chelator refers to a moiety that is capable of binding a radionuclide, preferably through non-covalent interactions, e.g., through ionic interactions.
  • chelator moiety is also used herein to describe S-protected forms of moieties which, in an unprotected state, are capable of chelating a radionuclide. although the skilled artisan will recognize that such a protected moiety may not be capable of chelation until the protecting group is removed.
  • an S- acetyl mercaptoacetyltripeptide is sometimes referred to herein as a chelator moiety.
  • Chelator moieties suitable for use in the compositions and methods of the invention are preferably capable of binding to a radionuclide with a high affinity, e.g., a binding affinity sufficiently high to permit binding of a radionuclide, preferably under physiological conditions, e.g., in vivo.
  • a chelator moiety which comprises peptide residues e.g., an oligopeptide chelator.
  • nucleic acid-chelator refers to a compound comprising a nucleic acid, including PNA, covalently bound to a chelator moiety.
  • protein-chelator refers to a protein, including an antibody, covalently bound to a chelator moiety.
  • Chelators which bind to radionuclides are known in the art, see, e.g., M. Nicolini et al, eds., "Technetium and Rhenium in Chemistry and Nuclear Medicine," SGEditoriali, Padova (1995).
  • preferred chelators are capable of binding to radionuclides such as Tc(O) 3+ .
  • a chelator moiety will be a tetradentate chelator, i.e., will be capable of four-point binding to a radionuclide.
  • Exemplary tetradentate chelators include N 2 S2 and N 3 S chelators, as described in, e.g., A.R. Fritzberg, et al, J. Nucl Med. 23:592-598 (1982); S. Liu and D.S. Edwards, in M. Nicolini et al, eds., "Technetium and Rhenium in Chemistry and Nuclear Medicine," op. cit., pp. 383-393; and S. Vallabhajousula et al, J. Nucl. Med. 30:599-604 (1989).
  • N2S2 chelator can chelate a radionuclide through two nitrogen atoms (e.g., amido nitrogens, e.g., of a peptide backbone) and two sulfur atoms (e.g., of a mercaptoacetyl moiety), while N3S chelators can chelate to a radionuclide through three nitrogen atoms and one sulfur atom.
  • nitrogen atoms e.g., amido nitrogens, e.g., of a peptide backbone
  • sulfur atoms e.g., of a mercaptoacetyl moiety
  • preferred chelator moieties include amidothiols, including, e.g., mercaptoacetyloligopeptides, and more preferably, mercaptoacetyltripeptides, such as, e.g., mercaptoacetyltriglycine (MAG3), mercaptoacetyltriserine, and the like.
  • mercaptoacetyl-tripeptides can chelate radionuclides such as Tc(O) 3+ by coordination through the three amide nitrogens of the peptide backbone, and the terminal mercapto group.
  • chelator moieties which may find use in the present invention include cyclams, porphyrins, crown ethers, azacrown ethers, and the like.
  • a chelator moiety will preferably be capable of covalently bonding to a polymer, e.g., a protein or a nucleic acid, e.g., DNA, RNA, phosphorothioate, or PNA, or other polymer compound.
  • the tripeptide moiety does not contain an aromatic residue.
  • a mercaptoacetyltripeptide molecule can form an amide bond, e.g., through the C-terminal carboxyl moiety of the tripeptide, with a nitrogen atom of a peptide or a polymer (which can be derivatized if necessary to provide a suitable reactive moiety).
  • a mercaptoacetyltripeptide can form an ester bond to a polymer through an oxygen atom of the polymer.
  • the chelator moiety can be covalently linked to the polymer through covalent bonds to other functionalities of the chelator moiety.
  • a mercaptoacetyltripeptide which includes an aspartate residue can form an ester or amide bond to a polymer through the side-chain carboxylate of the aspartate residue.
  • n 1 or 2;
  • R] is, independently for each occurrence, selected from the group consisting of alkyl, cycloalkyl, aryl, aralkyl; and can be substituted or unsubstituted;
  • R 2 is, independently for each occurrence, hydrogen or a protecting group; and
  • R3 is -OH, or with the carbonyl moiety to which it is attached, forms an active ester or a linking group; or a salt or ester thereof.
  • the chelator moiety can be represented by formula I, and n is 1.
  • the chelator moiety can be represented by formula I, and n is 2.
  • the chelator moiety can be represented by formula II.
  • R is hydrogen.
  • R 2 is -C(O)-lower alkyl, more preferably acetyl.
  • R 3 is -OH.
  • R 3 is selected such that an active ester is formed; e.g., R3 is -N-hydroxysuccinimidyl.
  • R3 can also be a functionality linking the chelator moiety to a polymer; for example, where the chelator moiety is covalently linked to a polymer through an amide linkage, R 3 can be -N- polymer.
  • the term "protecting group” is known in the art and refers to a moiety which blocks reaction at a particular atom or reactive center.
  • a "protected” compound is a compound in which at least one atom is blocked by a protecing group.
  • an "S- protected” compound includes a sulfur atom that is blocked with a protecting group.
  • activated ester refers to a derivative of a carboxylate moiety that is suitable for use in a coupling reaction with a nucleophile, e.g., to produce an ester or amide bond.
  • a nucleophile e.g., to produce an ester or amide bond.
  • a variety of activated esters are known in the art, see, e.g., G.A. Grant, Ed., "Synthetic Peptides: A User's Guide", W.H. Freeman, New York (1992), Chp.3. and M. Bodansky, “Principles of Peptide Synthesis", 2nd ed., Spring-Verlag (1993).
  • an activated ester suitable for use in the present invention will be sufficiently stable to permit storage of the ester for a suitable period of time before coupling to a nucleic acid.
  • exemplary activated esters include, in addition to carboxylic esters, thioesters, acyl azides, anhydrides, mixed anhydrides, and the like.
  • a preferred activated ester is an N-hydroxysuccinimide (NHS) ester, which is readily available from inexpensive starting reagents, see, e.g., Example 2, infra.
  • Activated esters of the invention do not include isothiocyanate derivatives.
  • activating compound refers to a compound which, when esterified with a carboxylate, forms an activated ester.
  • activating alcohol refers to an alcohol or hydroxyl-containing compound which, when esterified with a carboxylate, forms an activated ester.
  • activating compounds include activating alcohols, thiols, acid halides, and the like.
  • Exemplary activated alcohols include N-hydroxysuccinimide, pentafluorophenol, HOBt, and the like. N-hydroxysuccinimide is particularly preferred.
  • Coupled reagent refers to a reagent capable of effecting or promoting coupling of, e.g., an amine and a carboxylate, to form an amide, or an alcohol and a carboxylate to form an ester.
  • Coupling reagents are known in the art, see, e.g., M. Bodansky, "Principles of Peptide Synthesis", 2nd ed., supra.
  • a preferred coupling reagent is dicyclohexylcarbodiimide (DCC).
  • mild conditions refers to reaction conditions that do not result in significant degradation or destruction of a nucleic acid-chelator- radionuclide or protein-chelator-radionuclide composition.
  • mild conditions generally include reaction in aqueous or aqueous-organic solution, at a pH range of about 5.5 to about 8.5, more preferably about 6 to about 8.
  • Mild conditions also generally feature temperatures less than 100° C, more preferably less than 80° C, and still more preferably less than 60° C.
  • Mild conditions include the absence of harsh reagents or high salt concentrations that would cause significant loss of structural integrity or secondary and tertiary structural features of polymeric molecules such as proteins.
  • an antibody-chelator composition may be unstable above a temperature of about 45° C due to denaturation of the antibody portion.
  • mild conditions for radiolabeling an antibody-chelator composition may include a temperature no greater than 45° C, more preferably less than 40° C.
  • the rate or efficiency of conjugation can be affected by changes in reaction conditions such as temperature or pH.
  • elevated temperature can result in more rapid conjugation of radionuclide to the chelator; however it will be appreciated by one of ordinary skill in the art that elevated temperatures can cause denaturation or degradation of certain polymers, such as proteins, and use of elevated temperature can be appropriate when use of such temperature will not denature or degrade the polymer.
  • polymers can be sensitive to extremes of pH.
  • the temperature for conjugation and/or radionuclide complexation is not less than about 20° C, 30° C, 40° C, 50 ° C, 60 ° C, 70 ° C, or 80° C. In certain embodiments, the temperature is not greater than about 80 ° C, 70 ° C, 60 ° C, 50 ° C, or 40 ° C.
  • the pH is in the range of pH 5.5 to 8.5.
  • the invention provides a composition
  • a composition comprising a polymer, e.g., a nucleic acid or peptide, in which the polymer is covalently linked to a chelator moiety, and a radionuclide is bound to the chelator.
  • a polymer e.g., a nucleic acid or peptide
  • a radionuclide is bound to the chelator.
  • the DNA, phosphorothioate, peptide nucleic acid or ribonucleic acid will be selected to be complementary to a sequence of interest, e.g., a diagnostic sequence or a sequence on a pretargeting agent. While the nucleic acid need not be perfectly complementary to the sequence of interest, in preferred embodiments, complementarity will be sufficient to permit hybridization to the sequence of interest, either in vitro or in vivo, while substantially excluding non-specific hybridization to other sequences. In preferred embodiments, the nucleic acid is perfectly complementary to a sequence of interest.
  • DNA or RNA which has been chemically modified to have a terminal amine group can be used to provide compositions of the invention.
  • a chelator moiety can be linked to a nucleic acid base, e.g., a purine, a pyrimidine or a modified base, of a nucleic acid moiety.
  • the ratio of chelator moiety to nucleic acid moiety is 1 :1, i.e., there is one chelator moiety bound to each oligonucleotide or oligopeptide nucleic acid moiety.
  • radionuclides can be associated with one nucleic acid chain by using a plurality of chelating moieties per nucleic acid; thus, several therapeutic or diagnostic radionuclides can be employed with a single nucleic acid moiety.
  • the chelator moieties can all be the same or can be different.
  • nucleic acid-chelator-radionuclide compositions of the invention can further comprise a group capable of specific bonding to a binding partner complement, which in a most preferred embodiment comprises the complementary strand of nucleic acid.
  • binding partners contemplated for use in the instant invention include biotin and biotin compounds, including biotin derivatives and analogs; ligands for the biotin compounds including avidin or streptavidin; an antigen and an antibody; a receptor and its ligand; an engineered binding protein and its target, and the like.
  • a nucleic acid- chelator-radionuclide composition can be bound, e.g., to a solid support, by contacting another binding moiety, e.g., through the complementary strand of nucleic acid.
  • a biotin binding group or a biotin compound such as biocytin conjugated to the chelator complex can be bound by avidin or streptavidin, and any of these entities can be bound to a solid surface, e.g., a bead or a 96-well plate.
  • Biotinylated polymers can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and the compositions can be immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • Exemplary methods for detecting such complexes include enzyme- linked assays, which rely on detecting an enzymatic activity associated with an enzyme activity linked to streptavidin or bound to streptavidin via an antibody.
  • a polypeptide can be chemically cross-linked or genetically fused with alkaline phosphatase, and the amount of bound polypeptide trapped in the complex can be assessed with a chromogenic substrate of the enzyme, e.g. paranitrophenylphosphate.
  • the addition of a binding group permits the ready isolation or purification of the nucleic acid complex from solution.
  • biotinylated compounds can be readily immobilized for studying the in vitro binding or other properties of the nucleic acid- chelator or nucleic acid-chelator-radionuclide compositions of the invention.
  • a chelator- protein composition can also be immobilized utilizing conjugation of biotin and streptavidin, or the like, and pretargeting and targeting compositions can be designed with high affinity by conjugation of one with a biotin compound and the other with avidin or streptavidin. Suitable methods and compositions will be apparent to the skilled artisan in light of the teachings herein.
  • nucleic acid e.g., DNA, RNA, phosphorothioate or PNA
  • protein, and chelator moiety will generally be guided according to at least some of the following criteria: 1) minimal non-specific binding in vitro or in vivo (e.g., little binding to serum proteins and the like); 2) ability to bind to specific nucleic acid sequences, e.g., diagnostic sequences, in vitro and/or in vivo; 3) stable complexation to a radionuclide; and 4) ease of synthesis.
  • a nucleic acid- chelator-radionuclide complex will bind to specific nucleic acid sequences, in vivo or in vitro, with relatively little non-specific binding, and the radionuclide will remain complexed under physiological conditions.
  • the ability of a nucleic acid to bind to a complement, the extent of non-specific binding, and the binding affinity of the radionuclide for the chelator moiety can be assessed according to methods known in the art or described herein.
  • the invention provides a composition
  • a composition comprising a nculeic acid (e.g., deoxyribonucleic acid, peptide nucleic acid, phosphorothioate or ribonucleic acid) and a chelator moiety covalently linked to the nucleic acid.
  • a nculeic acid e.g., deoxyribonucleic acid, peptide nucleic acid, phosphorothioate or ribonucleic acid
  • a chelator moiety covalently linked to the nucleic acid.
  • the chelator moiety is a mercaptoacetyl oligopeptide, more preferably a tripeptide, i.e., a tripeptide covalently linked (preferably at the amine terminus) to a mercaptoacetyl moiety, i.e., -C(O)-CH 2 SR, wherein R is hydrogen or a protecting group.
  • the mercapto group is protected, preferably as a lower alkyl thioester, e.g., -S-C(O)-lower alkyl, prior to chelation with the radionuclide.
  • a preferred protecting group for the mercapto group can be represented by the formula -C(O)-lower alkyl; in more preferred embodiments, the lower alkyl is a Cj-C 3 alkyl, and in a most preferred embodiment, the lower alkyl is a methyl group (e.g., the protecting group is an acetyl group).
  • the protecting group can prevent undesired side reactions at the sulfur atom, e.g., oxidation, during synthesis or storage of the nucleic acid-chelator compound.
  • Protecting groups for sulfur are known (see, e.g., T. Greene and P. Wuts, "Protective Groups in Organic Synthesis," J. Wiley, (1991)).
  • the chelator moiety is an amidothiol chelator.
  • kits are useful for the synthesis of radiolabeled compositions under mild conditions suitable for a variety of polymers. Further, radionuclides may be conjugated shortly prior to medical application, for formulation with pharmaceutically acceptable carriers (see infra).
  • the invention provides a kit comprising an antibody-chelator or a binding protein-chelator in a container, wherein the chelator comprises a sulfur atom protected by a protecting group of the formula -C(O)-lower alkyl (more preferably an acetyl group), and instructions for complexing the antibody-chelator or binding protein- chelator with a radionuclide under mild conditions.
  • the invention provides libraries of chelator compounds, libraries of nucleic acid-chelators, and libraries of nucleic acid-chelator-radionuclides.
  • libraries can be synthesized according to methods for combinatorial synthesis (see infra).
  • Libraries of chelators, polymer-chelator (e.g., nucleic acid-chelator or protein- chelator) compounds, and polymer-chelator-radionuclides (e.g.. nucleic acid-chelator- radionuclide or protein-chelator-radionuclide) are useful for rapidly screening for compounds with desired properties, e.g., low non-specific binding, selected lipophilicity, high or low affinity for radionuclides, and the like.
  • the invention provides a method of synthesizing an activated ester of an S-protected mercaptoacetyl amino acid comprising the steps of (a) reacting an amino acid with an activated ester of an S-protected thioglycolic acid under conditions such that an S-protected mercaptoacetylamino acid and an activating alcohol are formed; and (b) reacting the S-protected mercaptoacetylamino acid and the activating alcohol with a coupling reagent under conditions such that an activated ester of an S-protected mercaptoacetyl amino acid is formed.
  • groups other than mercaptoacetyl groups e.g., substituted mercaptoacetyl groups
  • a chelator moiety e.g., by use of ⁇ - substituted thioglycolic acids to derivatize a tripeptide, thereby forming a substituted mercaptoacetyltripeptide.
  • the amino acid is preferably contacted with the activated ester of S- acetylthioglycolic in solution.
  • the solvent is a polar aprotic solvent, although any solvent capable of solubilizing the reactants without causing or participating in undesired side reactions can be used.
  • exemplary solvents include dimethylformamide (DMF), dichloromethane, dimethylacetamide, dioxane, tetrahydrofuran, ether, dimethoxyethane, and the like, or mixtures thereof. Reaction times will generally be in the range of 0.25 - 24 hours; progress of the reaction can be monitored by standard techniques, e.g., HPLC, thin-layer chromatography, NMR spectroscopy, and the like.
  • the reactions can be performed at temperatures ranging from about 0°C to about 100°C, more preferably about 10°C to about 60°C, and more preferably about 15°C to about 40°C.
  • the reactions can also be performed under anhydrous conditions and inert atmosphere, e.g., of nitrogen or argon.
  • the method provides advantages over known methods of synthesis of S- protected meraptoacetyl peptides.
  • the synthesis requires only two steps, which can be performed in one pot, preferably without isolation or purification of intermediates.
  • the inventive method is simple and rapid, and can provide high yields of the desired compounds.
  • the starting materials e.g., the NHS ester of SATA
  • Activated esters of S-protected mercaptoacetyl peptides can be used as protected chelator moieties for synthesis of radionuclide-labeled molecules, e.g., polymers such as nucleic acids (including peptide nucleic acids), antibodies, polypeptides, carbohydrates, hormones, and the like.
  • covalent attachment of the chelator moiety to the target molecule can be easily and selectively achieved through methods known in the art for coupling of molecules with activated esters.
  • the chelator moiety is covalently attached to the target via an amide linkage; hence, a molecule with a free amine group can be suitable as a target.
  • the chelator moiety is attached to a polymer through a group such as an amide, an ester, or a thioester.
  • the chelator moiety is covalently bonded to a polymer through a moiety other than a thiourea.
  • Other functionalities suitable for covalent attachment of a chelator moiety to a targeting molecule will be apparent to the skilled artisan.
  • the invention provides a method of synthesizing a polymer- chelator-radionuclide complex.
  • the method includes the steps of contacting a polymer- chelator compound (e.g., polymers including nucleic acids (preferably PNA), proteins or polypeptides (e.g., antibodies) or carbohydrates) with a radionuclide under mild conditions, and allowing a polymer-chelator-radionuclide complex to form.
  • a polymer- chelator compound e.g., polymers including nucleic acids (preferably PNA), proteins or polypeptides (e.g., antibodies) or carbohydrates
  • a radionuclide e.g., a radionuclide
  • the polymer is a nucleic acid, e.g., a DNA, an RNA, a phosphorothioate or a PNA, more preferably a peptide nucleic acid.
  • the chelator moiety is a tetradentate chelator, more preferably an amidothiol, yet more preferably an oligopeptide-thiol, and still more preferably a tripeptide-thiol.
  • a preferred embodiment of a chelator moiety is mercaptoacetyltriglycyl.
  • the efficiency of labeling of the chelator-polymer with radionuclide is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80%.
  • the radionuclide is technetium-99m.
  • the contacting step for radio-labeling occurs under conditions suitable for formation of a polymer-chelator-radionuclide complex.
  • the radionuclide can be supplied in the form of a chelate, e.g., a tartrate or glucoheptonate complex, which can undergo transchelation with the macromolecule- chelator to form a macromolecule-chelator-radionuclide complex.
  • the radionuclide can be supplied in a form which requires further treatment, e.g., reduction, to a chelatable form.
  • the method includes the step of contacting the radionuclide with a reducing agent to provide the radionuclide in a chelatable form.
  • pertechnetate can be reduced to Tc(V), e.g., Tc(O)3 + , which is then complexed by the chelator moiety.
  • Preferred reducing agents include SnCl 2 and other reducing agents capable of reducing an oxidized radionuclide to a chelatable oxidation state, preferably under mild conditions. It will be appreciated that the mild conditions of chelation, e.g., of temperature and pH, and the absence of harsh denaturants, can enable the user to complex a chelator- polymer to a radionuclide without significant denaturation and loss of structure and function of that polymer.
  • postconjugation labeling can result in decreased radiation exposure of the personnel involved in clinical and research laboraties and in shipping and handling, and the economy of decreased radionuclide procurement and waste.
  • the invention provides a variety of custom-designed chelators that are suitable for various in vivo target organs, tissues, and compartments.
  • the invention provides methods for synthesizing libraries of chelator compounds. In yet another aspect, the invention provides methods for synthesizing libraries of polymer-chelator compounds. In still another aspect, the invention provides methods for synthesizing libraries of polymer-chelator-radionuclide complexes.
  • combinatorial libraries are well known in the art and has been reviewed (see, e.g., E.M. Gordon et al, J. Med. Chem. 37:1385-1401 (1994)).
  • the subject invention contemplates methods for synthesis of combinatorial libraries of chelator compounds, polymer-chelator compounds, and polymer-chelator-radionuclide complexes.
  • Such libraries can be synthesized according to a variety of methods.
  • Soluble compound libraries can be screened by affinity chromatography, followed by identification of the isolated compounds by conventional techniques (e.g., mass spectrometry, NMR, and the like).
  • Immobilized compounds can be also be screened by methods known in the art. Where libraries of immobilized compounds are screened to determine the ability of a chelator moiety to chelate a radionuclide, the immobilized compounds can be contacted with a radionuclide under complexing conditions, and the presence or absence of radioactivity can be measured to determine the chelating ability of the chelator moiety.
  • a method for screening chelator compounds by conjugation with biocytin, followed by chelation with a radionuclide and immobilization on straptavidin, is described in the Examples, infra.
  • Combinatorial libraries of compounds can also be synthesized with "tags" to encode the identity of each member of the library (see, e.g., W.C. Still et al, PCT Publication No. WO 94/08051 ).
  • this method features the use of inert, but readily detectable, tags, that are attached to the solid support or to the compounds.
  • tags that are attached to the solid support or to the compounds.
  • an active compound is detected (e.g., by one of ⁇ he techniques described above)
  • the identity of the compound is determined by identification of the unique accompanying tag.
  • This tagging method permits the synthesis of large libraries of compounds which can be identified at very low levels.
  • the invention provides a method of forming a polymer- chelator-radionuclide complex, comprising the steps of (a) contacting a polymer-chelator compound with a radionuclide under mild conditions; and (b) allowing a polymer- chelator-radionuclide complex to form.
  • the polymer-chelator compound is selected from the group consisting of protein-chelators and nucleic acid- chelators.
  • the radionuclide is technetium-99m.
  • the technetium-99m is provided in the form of a pertechnetate.
  • the method comprises further contacting the pertechnetate with a reducing agent.
  • Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • lozenges using a flavored basis, usually sucrose and acacia or tragacanth
  • Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate, for example, to tumors or infections of the genito-urinary tract.
  • Dosage forms for the topical or transdermal administration of compositions of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to a composition of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the present therapeutic inventions to the body.
  • dosage forms can be made by dissolving or dispersing the composition in the proper medium.
  • Abso ⁇ tion enhancers can also be used to increase the flux of the composition across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the composition in a polymer matrix or gel.
  • Devices, including patches, which transdermally deliver a composition by iontophoresis or other electrically-assisted methods can also be employed in the present invention, including, for example, the devices described in U.S. Patent Nos. 4,708,716 and 5,372,579.
  • Ophthalmic formulations eye ointments, powders, solutions, drops, sprays and the like, are also contemplated as being within the scope of this invention, and are suitable for exigencies of pathological situations such as ophthalmic tumors or infections.
  • compositions of this invention suitable for parenteral administration comprise one or more composition of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged abso ⁇ tion of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay abso ⁇ tion such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
  • the compounds of this invention for a patient when used as an imaging agent, are preferred to be administered in the range of 0.1 milliCuries per kg of body weight to about 10 milliCuries per kilogram of body weight per day, more preferably from about 1 milliCurie per kg to about 4 milliCuries per kg.
  • the effective daily dose of a therapeutic compositions may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • composition While it may be possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).
  • compositions can be administered with medical devices known in the art.
  • a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941 ,880, 4,790,824, or 4,596,556.
  • a needleless hypodermic injection device such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941 ,880, 4,790,824, or 4,596,556.
  • Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4.,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No.
  • the compounds of the invention can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier excludes many highly hydrophilic compounds.
  • the therapeutic compounds of the invention cross the BBB (if desired)
  • they can be formulated, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331.
  • the liposomes may comprise one or more moieties which are selectively transported into specific cells or organs ("targeting moieties"), thus providing targeted drug delivery (see, e.g., V.V. Ranade (1989) J Clin. Pharmacol. 29:685).
  • Targeting moieties may be used to pretarget (supra) a tumor or site of infection, prior to delivery of the radionuclide therapeutic complex.
  • exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al); mannosides (Umezawa et al, (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P.G. Bloeman et al. (1995) FEBSLett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. ( 1995) Am. J.
  • the therapeutic compounds of the invention are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety. In a most preferred embodiment, the therapeutic compounds in the liposomes are delivered by bolus injection to a site proximal to the tumor or infection.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by including in the composition an agent which delays abso ⁇ tion, for example, aluminum monostearate or gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the therapeutic compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by inco ⁇ orating the therapeutic compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient (i.e., the therapeutic compound) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • a "therapeutically effective dosage” preferably inhibits tumor growth or pathogen infection by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects.
  • the ability of a compound to inhibit cancer or infectious disease can be evaluated in an animal model system that may be predictive of efficacy in human tumors and infectious diseases. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit an activity in vitro by assays well-known to the skilled practitioner.
  • a therapeutically effective amount of a therapeutic compound can decrease tumor size, prevent or delay death of infected tissues or organs, decrease fever and white cell count, improve CD4 cell count, or otherwise ameliorate symptoms in a subject.
  • One of ordinary skill in the art would be able to determine a therapeutically effective amount based on such factors as the subject's size measured by mass or surface area, the severity -40-
  • the nucleic acid moieties of the subject compositions are preferably chemically synthesized.
  • DNA and RNA are also available from biological sources, for example, produced in reasonably high yields from a recombinant organism in which the gene for the DNA is cloned, or in which the RNA is transcribed by a high activity promoter of a cloned gene. In this manner either the sense strand or the antisense strand is obtained, according to the objectives of the skilled artisan practicing this invention.
  • the source of the nucleic acid is from chemical synthesis or a biological sample
  • the material is preferably single stranded, and purification methods known to the skilled artisan, for example acrylamide gel electrophoresis or column chromatography for example on a P4 column (BioRad, Melville, NY), are used to obtain isolated nucleic acids for covalent coupling to the chelator of the invention.
  • Covalent linkage of the chelator molecule can be achieved by coupling, e.g., via an amine attached to the terminal phosphate group through a 6-member methylene carbon spacer. Attachment of a biotin compound moiety, if appropriate (J. Nuclear Med.
  • nucleic acid 36: 2306-23144 can be achieved, e.g., directly or through a 15-member amide-polyether to the terminal phosphate.
  • Other entities that would enhance the therapeutic function of the nucleic acid are similarly linked to a terminus of the nucleic acid, for example a member of the cytokine class of proteins such as epidermal growth factor, platelet-derived growth factor, and nerve growth factor.
  • MAG2F enables the user to custom design properties to improve a desired activity as -41 -
  • the invention provides a method for imaging a tumor, comprising the steps of administering to a patient a radionuclide-chelator-peptide nucleic acid composition in a pharmaceutically acceptable carrier, and detecting a radioactive signal to detect a tumor.
  • the nucleic acid sequence of the composition used for this method is complementary to a tumor-specific sequence that is uniquely expressed in the tumor. As described herein, such a sequence may be in an oncogene, a mutated tumor suppressor gene, a form of a gene that confers predisposition to a cancer, or a gene for a protein marker that appears on a tumor.
  • the base sequence of the nucleic acid is complementary to that of a nucleic acid of a pretargeted composition.
  • Examples of these are well-known to those skilled in the art, and are given here for the pu ⁇ oses of illustration, without limiting the present invention to these examples.
  • the nestin protein which is expressed in normal mammalian fetal development, is expressed on tumors of the central nervous system, including most forms of brain cancer (McKay, D.G. Ronal ⁇ /.S". Patent No. 5,338,839, 8/16/94). It is also expressed on melanomas and on metastasized melanomas (V.A. Florenes, R. Holm, O. Myklebost, U. Lendahl, O.
  • the methods of the invention include administration of, for example, PNA of a sequence complementary to the mRNA of the nestin protein, preferably to the unique portion of the human nestin gene, to target the PNA-chelator-radionuclide composition to the brain tumor, or to the metastasized melanoma cells, and localize the radioactivity to these sites.
  • the base sequence of the nucleic acid is complementary to that of a nucleic acid of a pretargeted composition.
  • the preferred site of delivery is within the central nervous system or directly to the brain via spinal injection or fine needle delivery, and the tumors may be imaged with equipment standard at a clinical nuclear medicine facility, for example, a whole body scanner (Siemans).
  • the whole body scanner is equipped with computing equipment and software, such as a Harris Computer Center with multiple terminals permitting programming and the use of data reduction programs.
  • the radioactivity accumulating a specific sites in the body can be converted to images of the tumor with the associated software.
  • Methods and routes for detection of nestin DNA sequence information for brain cancer can be applied to diagnostics and imaging of meningiomas, another pathological condition associated with the nervous system, using data available on the identification and characterization of genes differentially expressed in mengiomas (M. Mu ⁇ hy, M.J. Pykett, P. Harnish, K.D. Zang, D.L. George, Cell Growth Differ 4:115, 1991).
  • Wilm's tumor A.J. Buckler, K.M. Call, T.M. Glaser, D.A. Haber, D.E. Housman, C.Y. Ito, J. Pelletier, Rose, E.A. Rose, U.S. Patent No. 5,350,840,
  • Wilm's tumor can be cured surgically in 95% of cases, but an imaging agent is appropriate for monitoring remission, and for detection and imaging of newly appearing tumors.
  • inventive compositions and methods for diagnosis, imaging and therapeutic applications are tools to be used in combination with standard medical compositions and methods, such as surgery, chemotherapy, and radiation therapy for treating cancer.
  • the inventions of this application while described as diagnostic or therapeutic, may in fact serve both pu ⁇ oses, for example a therapeutic dose of radionuclide also serves as a tool to monitor the course of therapy. For example, accumulation of radiolabel at a site of a tumor or an infection can be monitored for maintenance of size and shape over the course of the therapy without necessity of a separate dosage merely for diagnostic imaging.
  • the known half-life of the radionuclide permits calculation of a schedule by which the labeling density will decline from its initial value. A greater decrease in size of the tumor or infected site indicates effectiveness of the treatment, i.e., a decline in label density at a rate faster than that of the half-life of the radionuclide.
  • compositions and methods of the current invention are useful for diagnostics, imaging and for therapeutic agents for infectious diseases of humans, animals and plants.
  • infectious disease is meant to include disorders caused by one or more species of bacteria, viruses, fungi, and protozoans, which are disease- producing organisms are collecively referred to as "pathogens.”
  • pathogens are exemplified, but not limited to, Mycobacterium tuberculosis, M. leprae, Pseudomonas aeruginosa, Shigella dysenteria, Salmonella typhi, S. paratyphi,
  • Streptococcus hemolyticus Hemophilus pneumoniae, Escherichia coli serotype 0157, Chlamydia species, Helicobacter species; HIV- 1,-2, and -3, HSV-I and -II, non- A non- B, non-C hepatitis virus, pox viruses, rabies viruses, Aspergillus species, Entamoeba histolytica, Giardia species, Erwinia carotovora, cauliflower mosaic virus, and Newcastle disease virus.
  • Obtaining unique sequences from these organisms by screening available data bases and by performing hybridizations in vitro are commonly known to those skilled in the art (see, e.g., Ladner et al, U.S. Patent No.
  • nucleotide sequences of interest can be complementary to tumor-specific mRNA (or to a unique sequence from a pathogen), so that the nucleic acid of the composition is doubly functional as a "homing" or delivery molecule for the target tumor or infected cells, but also may possess a therapeutic function in that it forms a stable complex with the naturally occuring mRNA, or with genomic DNA to inhibit transcription.
  • the specific activity of a radionuclide pertains to the ratio of molecules of composition bearing technetium-99m or equivalent (supra) to the total number of molecules of the composition.
  • the high coupling efficiencies described in the examples below indicate that the specific activity of the radionuclide is sufficiently high for use as a therapeutic, diagnostic, or imaging composition.
  • the chelator N-[N-[N-[(benzoylthio)acetyl]glycyl]glycyl]glycine (benzoyl MAG3) nas b een use successfully to radiolabel proteins and other molecules with tecnnetium-99m and radiorhenium.
  • the sulfur in this mercaptotripeptide chelator has previously been protected by a benzoyl leaving group which requires extreme alkaline pH or boiling temperatures for deprotection.
  • the benzoyl-protected chelator is generally deprotected and radiolabeled prior to conjugation (i.e.
  • the radiolabel in these complexes was found to be similarly stable to transchelation to cysteine. However, in contrast to SHNH-DNA, little serum protein binding of the labeled MAG3-DNA was observed. Since binding to proteins in serum and in tissue as a major drawback in the use of oligonucleotides labeled with technetium-99m via the chelator SHNH, the MAG3 chelator provides certain advantages for radiolabeling these biomolecules.
  • Example 4 describes radiolabeling by transchelation from technetium-99m-tartrate, and in this example, a yield of 84 + 6% from 5 different experiments and specific activity of 70 microCuries per microgram is obtained within 15 min at room temperature.
  • Example 5 establishes that the radionuclide is quantitively associated with DNA, that DNA thus labeled can bind its complementary nucleic acid strand, and that an attached biotin moiety is capable of binding avidin.
  • Example 6 uses cysteine challenge to show that the radionuclide-chelator complexes with each of SHNH-DNA and MAG3-DNA conjugates are stable.
  • Example 7 shows that the MAG3 chelate of technetium-99m on DNA has less nonspecific binding to serum protein than the SHNH chelate.
  • Example 1 Synthesis of acetyl-MAG3 As shown schematically in Fig. 1, acetyl- MAG3 was synthesized by reacting triglycine with S-acetylthioglycolic acid N- hydroxysuccinimide ester (SATA) for 15 min at room temperature. S-acetylthioglycolic acid N-hydroxysuccinimide ester (SATA) was obtained from Sigma Chemical Co., St. Louis, MO and was used without further purification. To 0.97 milliliters of a 0.225 M NaOH was added 50 milligrams of triglycine (264 micromol) and 10 microliters of a freshly-prepared 50 mM EDTA.
  • SATA S-acetylthioglycolic acid N-hydroxysuccinimide ester
  • Example 2 Synthesis of NHS acetyl-MAG3. Without purification, NHS-MAG3 is prepared directly from the reaction of Example 1, usually in 15 hrs but in as little as 1-2 hrs by dicyclohexylcarbodiimide (DCC)-mediated coupling to acetyl-MAG3 of the in situ-generated N-hydroxysuccinimide.
  • DCC dicyclohexylcarbodiimide
  • DCC is obtained from from Sigma Chemical Co. (St. Louis, MO) and used without further purification.
  • a solution of 60 milligrams (290 micromol) of DCC in 3.6 milliliters of dry DMF is added rapidly to the stirred triglycine/SATA solution (apparent pH of about 5.0).
  • the NHS-acetyl-MAG3 water/DMF solution is evaporated to near-dryness in 15-30 min on a rotary flash evaporator (Rotavapur-R, Buchi, Switzerland) and then lyophilized to dryness within 1 hr on a lyophilizer (Virtis, Garden NY). After drying in this fashion, the NHS-acetyl-MAG3 can be stored indefinitely at room temperatures in a dessicator When using the dry, powdered NHS-acetyl-MAG3 for conjugation, an arbitrary value of 50% by weight was assumed for its purity.
  • the chemical shifts for S-acetyl MAG3 were 2.36 (s,3H), SCOCH3; 3.66 (s,2H), COCH 2 S; 3.72-3.78 (m, 6H), NCH 2 CO); 8.15-8.36 (m,3H), NHCO.
  • the chemical shifts for NHS-acetyl-MAG3 were 2.38 (s,3H), SCOCH3; 2.80 (s,4H), succinimidyl; 3.68(s,2H), COCH 2 S; 3.70-3.80 (m, 6H), NCH 2 CO); 8.20-8.38 (m,3H), NHCO.
  • the uncorrected melting points were determined (Mel-Temp, Laboratory Devices, Cambridge, MA) to be 210-212 °C (dec.) for acetyl-protected MAG3 and 148- 151 °C for NHS-acetyl-MAG3.
  • Reagent-grade avidin (Molecular Probes, Eugene, OR), disodium ethylenetriaminetetraacetic acid (EDTA) (Aldrich Chemical Co., Milwaukee, WIS), D-biotin, dicyclohexylcarbodiimide (DCC), L-cysteine, dimethylformamide (DMF), sodium glucoheptonate, sodium tartrate, tricine, triglycine, streptavidin was obtained from Sigma Chemical Co., St. Louis, MO. and was used without further purification.
  • the base sequences were 5'-biotin-TA ATA CGA CTC ACT ATA GGG AG-amine-3' and its complement, as described previously
  • DNA 100 - 1000 microgram was prepared at a concentration of 2 milligrams/milliliters in 0.25 M NaHCO 3 -l M NaCl-1 mM EDTA, pH 8.5.
  • the DNA solution was heated to 60-70 °C for 5-10 min to dissociate any DNA duplexes and immediately plunged into ice water.
  • the DMF/water solution of NHS-acetyl-MAG3 was then added to the stirred DNA solution to a MAG3 : DNA molar ratio estimated to be 20 : 1. This solution was incubated at room temperature for 15 min in the dark.
  • the conjugation of the same oligonucleotides with SHNH was achieved by reacting the NHS derivative of SHNH with the DNA primary amine as has been previously described (Hnatowich D.J., Winnard P. Jr., Virzi F, Fogarasi M, Sano T, Smith CL, Cantor CR, Rusckowski M., J Nucl Med 36:2306-2314, 1995) and was similar to that described above for MAG3 conjugation.
  • the DMF solution of NHS-SHNH was added, while vortexing, to the DNA solution until a final molar ratio of SHNH : DNA of 25 : 1 was reached. The solution was incubated at room temperature in the dark for 1 hr.
  • the labeled DNA was purified on a 0.7 x 20 cm gel filtration column of Sephadex G-25 using sterile 0.25 M N ⁇ Acetate, pH 5.2, or saline, as eluant. Radioactivity and absorbency at 260 nm were used to ' identify and quantitate peak fractions. Preparations were routinely analyzed by size exclusion HPLC using a single 1 x 30 cm Superdex 200 column (Pharmacia, Piscataway, NJ). The recovery was routinely recorded. Control labeling was performed in which the native, unconjugated DNA was subjected to the identical labeling procedure to assess the extent of nonspecific labeling.
  • Hybridization was accomplished by incubating 0.4 microgram of the labeled DNA in saline with a 4-fold molar excess of the complementary DNA-avidin preparation. After 1 hr at room temperature, the unpurified solution was analyzed by HPLC. The identical study was performed with the biotin-saturated complementary DNA-avidin preparation.
  • Figure 2 presents several radiochromatograms obtained by size exclusion HPLC analysis.
  • Panel A is that of the radiolabeled DNA itself.
  • Panel B is the result of adding the labeled DNA to biotin saturated avidin. In this case, the absence of a shift to higher molecular weight suggests the absence of nonspecific binding of the labeled DNA to avidin.
  • Panel C is the result of adding the labeled DNA to unsaturated avidin. In this case, the pronounced shift in radioactivity to higher molecular weight is the result of binding of the DNA to avidin through its biotin moieties.
  • the shift indicates that the radiolabel is on the DNA as expected and that the conjugation and labeling procedures did not affect the biotin moiety in its affinity for avidin. The partial shift is most likely explained by assuming that approximately half the DNA molecules were obtained without the biotin group attached.
  • Example 6 Cysteine challenge.
  • the stability of technetium-99m-MAG3-DNA to cysteine transchelation compared to that of technetium-99m-SHNH-DNA was evaluated at one cysteine concentration and after 1 hr in 37°C 25 milliM N ⁇ Acetate, pH 7.0 buffer. Both labeled DNAs were added at a final concentration of 0.4 mM to a solution of 1-cysteine in 25 mM N ⁇ Acetate, pH 7.0 such that the cysteine : DNA molar ratio was 650 : 1 (Hnatowich DJ, Virzi F, Fogarasi M, Winnard P. Jr.. Rusckowski M, Nucl Med. Biol 1994; 21 : 1035-1044). After an incubation period of 1 hr in a 37 °C water bath, samples were removed for size exclusion HPLC analysis. The area under the DNA and cysteine peaks in the radiochromatographic profiles were evaluated and compared between the two labeling methods.
  • Figure 3 presents radiochromatograms obtained by size exclusion HPLC analysis of the same single-stranded phosphodiester DNA labeled with technetium-99m via both SHNH and MAG3.
  • panel A presents the radiochromatograms of the radiolabeled DNAs themselves.
  • Panel B is the result of incubating the radiolabeled DNAs at 37°C with cysteine at a 650 molar excess for 1 hr. Under the conditions of this analysis, radiolabeled cysteine appears as a peak in fraction 75 (Hnatowich DJ, Virzi F, Fogarasi M, Winnard P. Jr., Rusckowski M., Nucl Med. Biol 1994; 21 : 1035- 1044).
  • Example 7 Serum Incubation Studies. Labeled MAG3 -DNA was incubated at a concentration of 10 micrograms/milliliters in fresh human serum at 37 °C. Samples were periodically removed over 24 hrs for size exclusion HPLC analysis.
  • the last panel (C) in Fig. 3 shows the result of analyzing serum samples into which the labeled DNA were added.
  • the technetium-99m radiolabel is primarily on serum proteins as shown by the shift to higher molecular weight (earlier fractions). This property has been observed previously for DNAs radiolabeled with technetium-99m via the SHNH chelator (Hnatowich D.J., Winnard P. Jr., Virzi F, Fogarasi M.
  • This chelator binds technetium-99m stably as the free chelate for kidney function investigations (Fritzberg AR., Kasina S., Eshima D., Johnson D.L, J Nucl Med 27: 11 1-1 16; 1986) and when conjugated to proteins such as antibodies, (Fritzberg A.R., Berninger R.W., Hadley S.W. et al, Pharmaceutical Res. 5: 325-334; 1988. No evidence of nonspecific protein binding through this chelate has been reported.
  • acetyl group for protection can improve the MAG3 labeling method both by simplifying both the synthesis and deprotection/labeling.
  • the ideal protecting group will be stable to indefinite storage of the conjugated molecule and would hydrolyze under mild conditions only at the point of labeling with technetium- 99m.
  • the acetyl group appears to satisfy these requirements.
  • the acetyl protected NHS-MAG3 may be prepared in a one-pot, two-step synthesis not requiring intermediate purification steps. Conjugation to an amine-deriviatized single-stranded DNA was successful.
  • MAG3 is a useful chelator for DNA since its synthesis has been simplified and the labeling procedures used in this investigation have provided adequate labeling efficiencies and specific activities. Furthermore, unlike the SHNH chelate. the MAG3 chelate of technetium-99m shows a limited tendency to bind nonspecifically to proteins. -54-
  • Example 8 PNA Labeling. Two complementary 15-base single-stranded PNAs were synthesized by PerSeptive Biosystems, Framingham, MA. One strand was derivatized with a primary amine on the amino terminus (i.e. 5' equivalent) end via a 17-member ethylene-ether linkage. The complementary sequence was prepared with a biotin group on this end via the same linker.
  • the base sequences were NH2- (CH 2 )2 ⁇ (CH2)2 ⁇ CH 2 CONH(CH2)2 ⁇ (CH2) OCH 2 CO-TGT-ACG-TCA-CAA-CTA-CONH 2 and biotin- (CH 2 )2 ⁇ (CH 2 )2 ⁇ CH 2 CONH (CH 2 )2O(CH 2 )OCH 2 CO-TAG-TTG-TGA-CGT-ACA-CONH2.
  • the melting temperature i.e., the temperature at which half the base pairs have dissociated
  • the melting temperature i.e., the temperature at which half the base pairs have dissociated
  • the calculated molecular masses were 4336 and 4634 Da respectively and were observed by mass spectrometry to be 4340 and 4635 Da.
  • Streptavidin- conjugated magnetic polystyrene beads 1 micron in size (BioMag, PerSeptive Biosystems, Framingham, MA), were stored wet at refrigerator temperatures as recommended by the manufacturer. The capacity of the beads for biotin was reported by the manufacturer to be 1.5 nanogram of biotin per milligram of beads.
  • the desired volume of the 4 mg/ml water solution of the amine-derivatized single stranded PNA was made 0.36 M NaHCO 3 , 1.4 M NaCl, and 1.4 milliM DTPA, pH 9.3.
  • the NHS-acetyl-MAG3 was dissolved in dry DMF at a concentration of 20 milligrams/milliliters.
  • a volume of the DMF solution representing a molar ratio of MAG3 to PNA of approximately 20: 1 was added to the PNA solution during vortexing.
  • the solution (now containing no more than 10% DMF) was incubated at room temperature for 1 hr.
  • Each preparation of radiolabeled PNA was analyzed by size exclusion high performance liquid chromatography (HPLC) using a single 30 cm Superose 12 column (Pharmacia, Piscataway NJ) with both in-line radioactivity and UV detection and 0.05 M phosphate, pH 7 eluant. Recovery of radioactivity was routinely determined. Confirmation of labeling was established by HPLC analysis before and after adding the sample to streptavidin-conjugated magnetic beads to which the biotinylated complementary PNA was bound (see below). Loss of radioactivity from solution was due to binding by hybridization of the labeled PNA to the beads.
  • Example 9 Rate of hybridization.
  • Complementary PNA was bound to streptavidin on magnetic beads through its biotin moiety.
  • the suspension of beads was rinsed three times with a washing buffer consisting of 20 M tris, 2 M sodium chloride, 1 mM EDTA, and 0.1% tween 20 , adjusted to pH 7.0 and six additional times with a 1 :1 dilution in water of this buffer.
  • the beads were manipulated for washing by using a magnetic separator (MPC, Dynal, A.S., Lake Success, NY). Following the last wash, the beads were incubated for 30 min with biotinylated complementary PNA at 6 micrograms of PNA per milligrams of beads (i.e. 100 % of saturation) in the washing buffer. The beads were then washed five additional times with the diluted washing buffer.
  • the rate of hybridization of the labeled PNA to its complement under the conditions of this study was determined at room temperature by adding 1 microgram of labeled PNA to 300 microliters of complementary PNA attached to beads and suspended at a 1 mg/ml concentration in 10 mM tris, 1 M sodium chloride, 0.5 mM EDTA, 0.05% Tween 20, adjusted to pH 7.0 buffer. Samples were removed for analysis periodically over 24 hrs. The beads in each sample were separated magnetically from the solution, washed five times in the washing buffer and counted in a Na ⁇ (Tl) well counter. As a control, the identical study was repeated with beads without the complementary PNA.
  • Figure 6 shows the percentage of labeled PNA bound to complementary PNA on beads as a function of time with early time points separated by 10 min. Under the conditions of this study, hybridization occurs rapidly and is essentially completed within an hour, and mostly completed within the first 10 minutes. The extent of nonspecific binding of labeled PNA to the beads is minimal, as shown by the control study in which identical beads without PNA were used. These data show that PNA molecules form double-stranded DNA complexes rapidly with complementary strands.
  • Figure 7 presents radiochromatographic profiles for labeled PNA after 1 and 24 hrs of incubation in 37°C human serum and after 24 hrs in saline. Multiple peaks are again apparent for the labeled PNA in Fig. 7 A. In serum, minimal binding of the label to serum proteins is apparent (Figs. 7B and 7C). The radioactivity ratios among the triplet PNA peaks have been consistently observed to changes in serum in favor of the peak eluting in fraction 83. One peak, eluting in fraction 105 in the Figure, is probably the result of catabolism. These general features were also observed during incubations in mouse serum and in human serum. A change in the radioactivity profile also occurs during incubation in room temperature saline (Fig. 7D)
  • Table 1 shows the mean biodistribution (in percentage injected dose/milligrams of tissue) obtained in normal mice at times of 2.5 hrs and 24 hrs post intraperitoneal administration of technetium-99m-labeled-MAG3-PNA.
  • the data are the mean of values from five experimental animals, with standard deviation in parenthesis.
  • oligomers For use in radiopharmaceutical applications, oligomers must possess certain essential properties. Since diagnostic applications require only tracer quantities of drug, toxicity is unlikely to be an issue. Among other considerations, suitable stability of the oligomer in vivo is also essential. In addition, the pharmacokinetic properties must be suitable for the intended application. For example, the oligomer should clear through the kidneys in a time frame consistent with the application to provide a favorable target/nontarget ratio. For use as radiopharmaceuticals, it must be possible to radiolabel with imagable radionuclides such as technetium-99m and that the label be suitably stable in vivo. Finally, the labeled oligomer must be capable of hybridization in vivo with its complement in the target.
  • Peptide nucleic acids are synthetic oligomers in which the sugar and phosphate backbone of oligonucleotides have been replaced with a polyamide linkage. Not only does this substitution provide an oligomer resistant to nuclease and protease attack, but the absence of charge improves the binding affinity of PNA-DNA heteroduplexes.
  • radiolabeling with technetium-99m was achieved by means of an acetyl-protected MAG3 chelator.
  • This labeling strategy was developed to avoid "nonspecific" serum protein binding observed for DNA labeled using a hydrazino nicotinamide (SHNH) chelator.
  • SHNH hydrazino nicotinamide
  • Similar properties for technetium-99m in vitro and in vivo are seen in animals when labeled to two IgG antibodies by MAG3 and SHNH chelators (data not shown).
  • MAG3 respectable labeling efficiencies and specific activities were achieved for PNA.
  • the stability of the label in 37°C serum was acceptable with minimal activity present on either higher or lower molecular weight species (Fig. 7).
  • a test of suitability for medical use of oligomers may be in vivo hybridization. Only in the left thigh were the implanted beads first bound with the complementary PNA. Following intraperitoneal administration of radiolabeled PNA, increased accumulation of label occurred in the left thigh due to hybridization, with the left/right radioactivity ratio increasing with time between 2 and 23 hrs. Apart from radioactivity in the left thigh, the whole body image show radioactivity only in bladder and kidneys.
  • This strategy was applied to a mouse model for infection and a mouse tumor model by in which the "pretargeted" material circulates in the study animal by passive diffusion, and localization of radiolabeled complementary PNA is targeted by in vivo hybridization.
  • the pretargeting material chosen for this model was PNA- streptavidin, prepared by addition of streptavidin to biotin-conjugated PNA.
  • the complementary strand PNA, derivatized with a primary amine was conjugated with acetyl S-protected NHS-MAG3 and radiolabeled with 99m Tc as described in Examples, supra. Sufficient time provided between the two administrations so that the earlier administered composition localizes to the target, and non-localized material can be cleared from circulation and normal tissues.
  • pre-targeting molecule, agent, composition or compound refers to a first-administered non-radioactive molecule.
  • targeting molecule, agent, composition or compound used in the context of pre ⁇ targeting strategies, refers here to a second administered composition in which a targeting moiety (e.g., a member of a specific binding pair, e.g., a nucleic acid sequence, an antibody, a biotin compound, or the like), derivatized with a chelator which complexes a radionuclide, is a ligand of (e.g., can bind to) the first administered agent.
  • a targeting moiety e.g., a member of a specific binding pair, e.g., a nucleic acid sequence, an antibody, a biotin compound, or the like
  • a chelator which complexes a radionuclide
  • the targeting agent delivers the radioactive agent specifically, e.g., to a tumor or infectious agent or infected cell.
  • the strategy localizes the activity of the radioactivity to the targeted tumor or infection by virtue of the interaction between the targeting agent and the pre-targeting agent.
  • the targeting agents described herein for example, PNA- MAG3- 99m Tc having a given base sequence, are applicable to wide variety of pretargeting compositions, e.g., which comprise a PNA sequence complementary to a targeting agent but differ in affinity for, e.g., a particular tumor type or infectious disease antigen.
  • useful molecules can comprise a nucleic acid covalently linked to a protein ligand, or antibody, specific for a tumor or infection, such that the antibody is a pre-targeting agent; then the complementary sequence of nucleic acid, covalently linked to a chelator and a radionuclide, comprises the targeting agent as defined herein.
  • Affinities of single- stranded DNA for the complementary strand can approach that of biotin to streptavidin ( Egholm, M., Kim, S.K., Norden, B., Nielsen, P.K., Nature 365: 566-568, 1993).
  • Example 8 Materials and methods for Examples 13 and 14 are given in previous Examples (supra), and the sequence and structure of the 15-base PNA strands is given in Example 8.
  • the strand for pretargeting is shown in Example 8 derivatized with a biotin group on the amino terminus end (i.e. 5' equivalent) through a 17-member ethylene-ether linkage.
  • the complementary 15 base sequence, used here for radiolabeling through covalent linkage to MAG3 contains a primary amine group on this end from the same 17- -64-
  • Example 14 Pretargeting and Targeting with PNA-MAG3- 99ra Tc in a Mouse Tumor Model.
  • the LSI 74T tumor was obtained from American Type Culture Collection and was grown in minimal essential medium (Gibco. Grand Island, NY). Cells were removed from the culture flask by trypsinization and then washed in the culture medium. Swiss male nu nu mice (Taconic Labs., Germantown, NY) were injected subcutaneously in the flank of the left thigh with 10 ⁇ cells in 0.1 ml of medium.
  • tissue radioactivity levels were found to be significantly higher in most tissues in study animals compared to control animals. This increase is related to the presence in study mice of PNA-streptavidin in concentrations sufficient to bind and retain the complementary radiolabeled PNA of the second injection.
  • Example 15 Synthesis of a library of novel amidothiol chelators for radionuclides, and conjugation to biocytin.
  • a combinatorial chemistry approach to identify one or more amidothiol bifunctional chelators with properties complementary or superior to that of MAG3 is accomplished by synthesis of a library of approximately 70 chelators using a novel synthetic route. Each is labeled with technetium-99m (99m c 3 ⁇ use ful properties (charge, lipophilicity and maximum specific activity) of each labeled chelator is detemined.
  • the NHS ester is prepared for conjugation to the biotin compound biocytin.
  • the method of synthesis of acetyl-MAG3 presented herein can be applied to the synthesis of chelators based on commercially available oligopeptides, e.g., tripeptides.
  • Analytical methods as described above, are used to ascertain successful reactions, verify chemical identity of product, and to measure activities, and are applied to each novel chelator.
  • a sample of the NHS ester of each chelator is conjugated to biocytin. a biotin compound.
  • Conjugated biocytin derivatives of the chelators are prepared (Figs.13, 14 and 15) and radiolabeled by methods of Examples above.
  • Radiochemical purity was estimated by Sep-Pak (Millipore) chromatography in which the C-18 column was first equilibrated in 0.001 M HCI solution and, after adding the sample, eluted with this solution. As in the case of "m ⁇ c-MAG3, radiolabeled pertechnetate appears in this fraction. Thereafter, the column was eluted with 50% ethanol to obtain the labeled chelator. Depending upon the chelator, between 40-100% of the radioactivity was present in the ethanol solution.
  • the chelators were also radiolabeled by transchelation from glucoheptonate.
  • the labeling procedure was essentially identical except that 9.4 microliters of a 50 mg/ml solution of fresh sodium glucoheptonate (Sigma) in 1 M NaHCO3, 0.25 mM NH4Acetate, pH 9 was added prior to the addition of pertechnetate. As determined by Sep-Pak analysis, labeling efficiency varied between 30 and 90% depending upon chelator.
  • Radiolabeling of the biotin conjugates was performed in the sub-microgram scale, and only indirect labeling with glucoheptonate was used.
  • the reaction mixture was evaporated to dryness under vacuum at 50°C and the dried residue dissolved in 200 microliters of DMF.
  • 0.018 mmole (2.3 mg) of NHS in 40 microliters DMF and 0.02 mmole DCC in 100 microliters DMF and the mixture was stirred for 3.5 hrs at room temperature.
  • the reaction was again monitored by SG-TLC using acetonitrile/triethylamine 50/1 (v/v) as solvent.
  • the dicylohexylurea precipitate was removed by filtration, and the yield estimated by TLC was about 50%.
  • the NHS-phenyl-MAG2 was used immediately without purification.
  • Radiolabeling of the biotin conjugate was accomplished by adding in order: the conjugate in DMF/bicarbonate buffer, pH 9.3, sodium tartrate, 9 rn Tc-pertechnetate eluant and stannous ion. After incubating the reaction for 15 min at room temperature, the labeling yield was determined to be 93% by Sep-Pak analysis. Analysis by HPLC (Superdex) showed three radioactivity peaks at retention times of 45, 51 and 63 min. The main UV peak was at 45 min.
  • R 4 is CH and H for each compound respectively.
  • Preliminary stability studies in serum have been performed with the phenyl- MAG2-biotin- 9 m Tc. Very little change in the HPLC profile was seen over 24 hrs at 37 °C in serum, and a high molecular weight peak signifying serum protein binding was seen. It is believed that the increased lipophilicity introduced by the addition of a phenylalanine is responsible for the increased protein binding.
  • Bifunctional chelators with desired properties can be evaluated as conjugates to antibodies, PNA and peptides radiolabeled with chelated m Tcby the methods described herein, or other methods known in the art.
  • desired properties e.g., stability to cysteine challenge, lipophilicity, and ability to achieve high specific activities
  • PNA and peptides radiolabeled with chelated m Tc can be evaluated as conjugates to antibodies, PNA and peptides radiolabeled with chelated m Tcby the methods described herein, or other methods known in the art.
  • a variety of chelators can be rapidly synthesized and tested to determine optimal characteristics.

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Abstract

Nouveaux chélateurs et procédés de synthèse et d'utilisation de ceux-ci. Les compositions, procédés et nécessaires décrits permettent de complexer des radiocucléides pour applications médicales à des conjugués-chélateurs dans des conditions peu rigoureuses.
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EP1045675A4 (fr) * 1997-11-03 2004-07-07 Boston Scient Ltd Stent radioactif in situ
WO2000036136A1 (fr) * 1998-12-14 2000-06-22 Palatin Technologies, Inc. Bibliotheque combinatoire de metallopeptides et applications correspondantes
US7385025B2 (en) 2000-12-19 2008-06-10 Palatin Technologies, Inc. Metallopeptide compounds

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