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WO1991004057A1 - Stable therapeutic radionuclide compositions and methods for preparation thereof - Google Patents

Stable therapeutic radionuclide compositions and methods for preparation thereof Download PDF

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Publication number
WO1991004057A1
WO1991004057A1 PCT/US1990/005313 US9005313W WO9104057A1 WO 1991004057 A1 WO1991004057 A1 WO 1991004057A1 US 9005313 W US9005313 W US 9005313W WO 9104057 A1 WO9104057 A1 WO 9104057A1
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WIPO (PCT)
Prior art keywords
therapeutic radionuclide
therapeutic
stabilizing agent
radionuclide
group
Prior art date
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PCT/US1990/005313
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English (en)
French (fr)
Inventor
Jean-Luc Vanderheyden
Alan R. Fritzberg
Joseph E. Bugaj
Fu-Min Su
Prasanna Venkatesan
Original Assignee
Neorx Corporation
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Publication of WO1991004057A1 publication Critical patent/WO1991004057A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
    • 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
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy

Definitions

  • the present invention relates generally to stable therapeutic radionuclide-labeled compositions, and to methods for preparation and stabilization thereof.
  • Radio-labeled compositions are important tools in medical diagnosis and treatment. Such compositions may be employed in a variety of techniques, including the
  • radionuclides are generally coupled to targeting agents to provide preferential binding to or absorption by the particular cells or tissue (s) of interest. Radionuclides are typically bound to a chelating agent, and the chelating agent is coupled to a targeting moiety to provide a radio-labeled
  • composition capable of binding selectively to a specified population of target cells or tissue (s).
  • Radionuclides such as 99m Tc, 131 I, 123 I, 117m Sn, 111 In, 113 ln, 97 Ru, 76 Br, 77 Br, 203 Pb, 18 F, 67 Ga, 89 Zr, and Cu have been proposed for use as diagnostic imaging agents.
  • 99m Tc is one particularly promising diagnostic imaging agent.
  • Technetium-99m is1 produced commercially in generators by eluting a saline solution through a matrix containing molybdenum. The metastable technetium isotope in such eluates is found in the chemically stable,
  • pertechnetate- 99m Tc has a valence state of +7, it will not complex with the most commonly used carriers for
  • 99m TcO 4 - is therefore commonly reduced to lower oxidation states, such as
  • Radionuclides such as 32 P and 131 I ave been used to treat malignancies such as polycythemia vera and metastatic thyroid carcinoma, respectively.
  • a variety of radionuclides may be useful for therapeutic applications, including alpha emitters, low, medium and high range beta emitters, and radionuclides which act through electron capture and/or internal conversion (auger electrons).
  • Sources of alpha emitters, such as 211 At, are relatively limited, while beta sources are far more plentiful. Numerous medium range beta sources, including
  • I therapeuti.c composi.ti.ons may be substantially reduced by attachment of para-iodophenyl moieties as taught in European Patent Application Publication 0 203 764.
  • Long range beta particle sources potentially suitable for therapeutic administration include 32 P, 90 Y and 188 Re.
  • 186 Re and 188 Re also emit gamma radiation at essentially the same energy as the gamma emission of ATc, which allows the biodistribution of rhenium radiopharmaceuticals to be readily monitored using conventional gamma camera instrumentation.
  • compositions comprising beta emitting radionuclides may undergo radiolysis during preparation and/or in vitro storage.
  • Radiolysis emissions from the radionuclide attack other constituents of the complex or compound, or other compounds in proximity, which results in inter- and intra-molecular decomposition.
  • Radiolytic decay poses serious safety concerns, since decomposition or destruction of the radionuclide chelate, the radionuclide chelate-targeting agent linkage, or the specificity conferring portion of the targeting agent results in non-targeted radioactivity.
  • Radioactivity which is not linked to a functional targeting agent will accumulate in non-target tissues, and decomposition of the radionuclide composition prior to or during administration dramatically decreases the targeting potential and thus increases the toxicity of the therapeutic radionuclide composition. It is thus important, particularly with respect to therapeutic radionuclide preparations intended for in vivo administration, to ensure that the radioactive moiety is stably linked to the targeting moiety, and the specificity of the targeting agent is preserved.
  • Rhenium and technetium share many physical properties, such as size, shape, dipole moment, formal charge, ionic mobility,
  • chelating agents which have been developed and/or used for 99m Tc are also suitable for use with rhenium radionuclides. Little is known, however, about the properties of radioactive rhenium therapeutic compositions, since research involving rhenium radionuclides is still at a relatively preliminary stage.
  • Perrhenate (ReO 4 -) which is formed as a result of unstable rhenium complexes in lower oxidation states moving to higher oxidation states, is the primary decomposition product of rhenium radionuclide
  • radiopharmaceuticals The most relevant of the chemical differences may be that rhenium complexes are
  • Rhenium compositions are therefore more difficult to reduce than their technetium analogs, and reduced rhenium radiopharmaceuticals tend to be reoxidized back to perrhenate more readily than analogous technetium radiopharmaceuticals are re-oxidized back to pertechnetate.
  • radionuclide preparations was essential to provide
  • Stabilization of therapeutic radionuclide compositions is a recurrent challenge in the field of therapeutic radionuclide conjugates. Stabilization of radioactive compositions is generally more difficult to achieve without loss of desired properties than
  • Therapeutic radionuclide compositions behave differently from and are generally less stable than diagnostic radionuclide compositions.
  • radionuclide and conjugating the radionuclide to the targeting moiety. Additionally, significant resources are devoted to preparing patients so they may undergo
  • therapeutic radionuclide compositions would permit more centralized, controlled preparation of therapeutic
  • radionuclide compositions and thereby provide greater access to therapeutic compositions having higher purity levels.
  • the methods of the present invention provide stabilization of therapeutic radionuclide compositions by addition of an effective amount of a stabilizing agent. Effective in vitro stabilization of therapeutic
  • radionuclide compositions is critical when the composition is intended for in vivo patient administration.
  • Purity levels of radionuclide compositions are typically measured as the ratio of radionuclide bound to targeting moiety to the total radionuclide in the preparation.
  • Limitations relating to the purity of radionuclide preparations which can be administered to humans are strictly observed. In general, the preparation is at least 90% pure, and it is preferably at least 95% pure. High purity levels are, of course, preferable, and purity levels below about 90% are generally considered unsuitable for in vivo human
  • Stabilizers for use with therapeutic radio-labeled compounds desirably possess the following characteristics: they are toxicologically acceptable under conditions of use; they are suitable for in vivo administration; they do not interfere with delivery of the compound to the target cells or tissue (s); and they stabilize the product for reasonable periods during preparation and storage prior to use.
  • One of the important benefits of the stabilizing agents employed in the present invention is that they permit in vitro storage of prepared, purified radio-labeled compositions for at least several hours and up to several days, while maintaining acceptable purity levels.
  • therapeutic radionuclide composition means a radionuclide having therapeutic properties complexed or otherwise associated with a targeting moiety.
  • compositions of the present invention preferably
  • therapeutic radionuclides which exhibit redox chemistry, such as rhenium radionuclides, and/or are prone to decomposition as a result of radiolysis products such as peroxides and free radicals such as 131 I.
  • therapeutic radionuclide compositions of the present invention include therapeutic radionuclides which exhibit redox chemistry, such as rhenium radionuclides, and/or are prone to decomposition as a result of radiolysis products such as peroxides and free radicals such as 131 I.
  • beta radiation emitting isotopes such as 186 Re, 188 Re, and the like, which may be complexed or associated with various other constituents, such as chelating agents and targeting moieties.
  • Therapeutic radio-immunoconjugates comprising a therapeutic
  • radionuclide bound to a chelating agent which is in turn linked to a proteinaceous targeting moiety
  • a chelating agent which is in turn linked to a proteinaceous targeting moiety
  • the rhenium radionuclides, 186 Re and 188 Re, which emit both beta and gamma radiation, are preferred for use in stable therapeutic compositions according to the present invention.
  • a stabilizing agent is introduced to the
  • Suitable stabilizing agents include
  • antioxidants maintain the radionuclide in its reduced state, while challenging agents form complexes with unbound or loosely bound metals that may facilitate oxidation, e.g. Fe +3 /Fe +2 .
  • Enhanced stability has been observed when a stabilizing agent is contained in the eluate used during purification of the therapeutic preparation.
  • Ascorbic acid is a preferred stabilizing agent.
  • Compatible albumin moieties and serum proteins may also be incorporated in the stabilizing agents of the present invention.
  • Stabilizing agents comprising HSA in
  • radionuclide compositions for up to a day or more.
  • stabilizing agents such as ascorbic acid, alone or in combination with other stabilizing agents, would provide long-term stabilization of therapeutic radionuclide compositions.
  • Use of the stabilizing agents of the present invention precludes the necessity of adopting more stringent stabilization measures, such as performing the conjugation and/or purification under anaerobic conditions and using the preparation immediately after purification.
  • radionuclide compositions which may be attributed to the reoxidation of the radioactive constituent (e.g. 186 Re,
  • the stable therapeutic radionuclide compositions of the present invention comprise a variety of radio- labeled compositions.
  • Radio-immunoconjugates represent one preferred class of compositions wherein a therapeutic radioactive moiety is linked to a proteinaceous targeting moiety having specificity for certain target cells and/or tissue (s).
  • Therapeutic radionuclides contemplated for use in the compositions of the present invention include beta
  • radionuclides such as 186 Re, 188 Re,
  • Radioisotopes of the lanthanide series may exert therapeutic effects and may be suitable for use in the therapeutic radionuclide compositions of the present invention.
  • Reduced rhenium radionuclides including 186 Re and 188Re having an oxidation state of less than +7 are preferred for use in the therapeutic compositions of the present invention.
  • Suitable targeting moieties for incorporation in the therapeutic radionuclide compositions of the present invention include targeting moieties comprising proteins and polypeptides which may include carbohydrate moieties such as polysaccharides, glycoproteins, or other compounds having a carbohydrate moiety.
  • Preferred proteinaceous targeting agents include antibodies, receptors
  • cell surface receptors such as lectins
  • enzymes e.g., fibrinolytic enzymes
  • biologic response modifiers e.g., interleukins, interferons, lymphokines, erythropoietin, growth factors, colony stimulating
  • Microaggregated proteins such as
  • microaggregated albumin and the like, may also be used as targeting moieties.
  • Monoclonal antibodies or fragments thereof are especially preferred proteinaceous targeting moieties.
  • monoclonal antibodies that may be used are anti-TAC, or other antibodies that may be used.
  • interleukin-2 receptor antibodies 9.2.27 and NRML-05 to the 250 kilodalton human melanoma-associated proteoglycan; NRLU-10 to 37-40 kilodalton pancarcinoma glycoprotein;
  • NRCO-02 having colon specificity; and OVB-3 to an as yet unidentified tumor-associated antigen.
  • Antibodies derived from hybridomas or by means of genetic or protein engineering techniques may be employed.
  • the targeting moieties may be modified as desired, as long as the biological activity necessary for the intended therapeutic application is retained.
  • chimeric antibodies which are produced by
  • determining portions derived from non-human sources and other portions derived from human sources may be
  • Antibodies employed in the present invention may comprise intact molecules, fragments thereof, or functional
  • Exemplary antibody fragments include F(ab') 2 , Fab 1 , Fab, and Fv fragments, which may be
  • Radio-immunoconjugates comprising rhenium radionuclides are typically produced by stably binding the radionuclide in a chelating agent, and subsequently coupling a portion of the radionuclide metal chelate to a proteinaceous targeting moiety.
  • chelating agents are known in the art for chelating diagnostic agents such as 99m Tc, and many of these agents are
  • Metal chelating compounds having nitrogen and sulfur donor atoms such as
  • N 2 S 2 chelating agents dithiodiaminocarboxylic acids and dithiodiamidocarboxylic acids
  • N 3 S chelating agents thiotriaza chelating compounds
  • T is H or a sulfur protecting group
  • each X independently represents H 2 or O;
  • M is a radionuclide ion, to which 1 or 2 oxygen atoms may be bonded;
  • each R independently represents a substituent selected from the group consisting of hydrogen; alkyl; geminal dialkyl; a non-alkyl side chain of an amino acid other than cysteine (alkyl side chains being covered when R is an alkyl group); and -(CH 2 ) n -Z;
  • Z represents -COOH, a conjugation group, or a targeting compound
  • n is an integer of from 1 to about 4;
  • R' is H 2 ; -(CH 2 ) n -Z; or an alkyl group having one or more polar groups substituted thereon;
  • the compound comprises at least one -(CH 2 ) n -Z substituent wherein Z is a conjugation group or a
  • the conjugation group is a functional group which reacts with a group on the desired targeting moiety to bind the radionuclide metal chelate to the targeting agent.
  • Proteinaceous targeting moieties contain a variety of functional groups such as carboxylic acid (COOH) or free amine (-NH 2 ) groups, which are available for reaction with a suitable conjugation group on a chelating agent.
  • COOH carboxylic acid
  • -NH 2 free amine
  • an active ester on the chelating agent reacts with free amine groups on lysine residues of proteins to form amide bonds.
  • the protein and/or chelating agent may be derivatized to expose or attach additional reactive functional groups.
  • the derivatization may involve attachment of any of a number of linker molecules.
  • the derivatization may involve chemical treatment of the protein to generate, for
  • esters which may be utilized as conjugation groups represented by "Z" are those esters which provide a covalent, amide linkage with a polypeptide in an aqueous medium.
  • One or another of the reactive esters may be preferred, depending upon the particular radionuclide, the protein, and the conditions for conjugation, as is
  • derivatization may involve chemical treatment of the carbohydrate, such as glycol cleavage of the sugar moiety of a glycoprotein antibody with periodate to generate free aldehyde groups.
  • the free aldehyde groups on the antibody may be reacted with free amine or hydrazine conjugation groups on the chelating agent to bind the radionuclide metal chelate thereto.
  • Suitable metal chelating agents are disclosed in European Patent Office Publication Numbers 0 188 256 and 0 284 071, as well as U.S. Patent Application No.
  • compositions of the present invention are compositions of the present invention.
  • the chelating compound is radiolabeled to form the corresponding radionuclide metal chelate, and the chelate subsequently is attached to the targeting moiety in the "preformed chelate approach".
  • An alternative approach to preparing radiolabeled targeting moieties is the "postformed chelate approach", wherein the chelating compound is first attached to the protein or carbohydrate targeting molecule. The resulting conjugate is then reacted with a radionuclide metal to form a radionuclide metal chelate bound to the targeting molecule.
  • immunoconjugates is described below. Depending upon the therapeutic radionuclide employed, various techni-ques may be utilized to prepare the radionuclide metal chelate.
  • Perrhenate (ReO 4 -) i.s typi.cally reduced to ReO 3+ , ReO 2 +, or the like, prior to reaction with the chelating agent.
  • perrhenate may be reduced by reaction with stannous compounds or the like, and reacted with a transfer agent, such as citric acid, to form a weak rhenium citrate complex.
  • the reduced rhenium radionuclide citrate complex is then reacted with a chelating agent, and the radionuclide is transferred to the ligand (chelating agent). This reaction produces a radionuclide chelate intermediate having an active ester moiety, or another conjugation group.
  • a buffer pH 9.5 to 10
  • a buffered solution of the targeting moiety is then admixed with the chelated radionuclide.
  • the active ester moiety of the radionuclide metal chelate intermediate is typically linked to a lysine residue of a proteinaceous targeting agent by means of an amide bond, but other types of linkages may also be used.
  • the radionuclide compositions are purified to remove unreacted components, undesired reaction by-products, and
  • techniques may be employed, including techniques such as gel permeation columns, which provide separation based upon molecular weight, and ion exchange chromatography, such as QAE anion exchange, which provides separation based upon charge characteristics.
  • radio-immunoconjugates comprising rhenium radionuclide chelates linked to antibodies
  • other types of targeting moieties may be employed in the stable therapeutic radionuclide compositions of the present invention.
  • Therapeutic radionuclides may be linked to other proteinaceous targeting moieties using technology which is known in the art. Chelating agents and/or known linker molecules may be used to link radionuclides to other targeting agents, or direct labeling techniques may be employed.
  • Radio-immunoconjugates comprising radio-halogen labeled targeting agents are typically produced by radio-labeling small molecules and subsequently binding the radio-halogenated small molecules to proteinaceous targeting agents.
  • the therapeutically active agents are typically produced by radio-labeling small molecules and subsequently binding the radio-halogenated small molecules to proteinaceous targeting agents.
  • radionuclide 131 I is preferably incorporated into a small molecule having the formula X-Ar-R, wherein *X is a radiohalogen ( 131 I); Ar is an aromatic or heteroaromatic ring; and R is a short-chain substituent that does not highly activate ring Ar onto which the radiohalogen is substituted, and that bears a functional group suitable for conjugation to proteinaceous targeting moieties under mild, e.g., acylation, conditions that preserve the biological activity of the proteinaceous targeting agent.
  • Preferred Ar rings include benzene, pyridine, furan and thiophene.
  • the radiohalogen X is preferably para- or meta-positioned on the Ar ring relative to substituent R to render the radio-halogen less susceptible to catabolism by dehalogenase enzymes. Attachment of the radiohalogen to a carbon atom in the Ar ring is preferred over
  • Ar ring is not critical, and it may be mono-, bi-, tri-cyclic or contain a higher number of rings, but a monocyclic ring is
  • Ring Ar may consist of all carbon atoms, or it may contain heteroatoms such as nitrogen, oxygen or sulfur. Further substitution on the Ar ring, exclusive of
  • sulfonic acid carboxylic acid, or dialkyl amino group may be preferred to enhance solubility in a-gueous solutions.
  • R must not highly activate ring Ar toward electrophilic substitution. In other words, R cannot be a substituent that, when bound to Ar, increases the electron density of Ar on the order of the increase produced by a free hydroxy or primary amino substitution.
  • R should be a short-chain substituent so that unconjugated or cleaved radiohalogenated molecules can be rapidly removed by the kidneys.
  • R may contain an alkyl or other spacer chain between the aryl linkage and the functional group for protein conjugation, but such a spacer chain should preferably contain no more than 5, and most preferably no more than 3, straight-chain carbon atoms.
  • the R substituent should bear a functional group (termed "Q" herein) that is available for covalent attachment to corresponding functional groups (or
  • R substituents include imide ester, alkyl imide esters, amido alkyl imide esters, imidate ester, alkyl imidate esters, and amido alkyl imidate esters.
  • Suitable functional groups Q for conjugation to proteinaceous moieties include phenolic esters (e.g., para-nitrophenol), imide esters (e.g., succinimide ester), imidate esters, anhydrides, acylsuccinimides, aldehydes, isothiocyanates, thiol, diazo, amines, hydrazines, alkyl halides, Michael acceptor a,ß-unsaturated carbonyl
  • radiohalogenated small molecules of formula I wherein the R substituent bears a precursor of functional group Q include: carboxylie acid where Q is phenolic ester, imide ester, anhydride, acylsuccinimide, or maleimide; nitrile where Q is imidate ester; alcohol where Q is aldehyde; halide where Q is isothiocyanate, thiol, hydrazine, or amine; and amine where Q is diazo or maleimide.
  • radiohalogenated small molecules of this invention include the compounds having the following formulae:
  • X is a radiohalogen, such as 131 I;
  • n is an integer
  • the radiohalogen may be positioned anywhere on the aromatic ring Ar, but para- or meta-substitution is preferred in order to make the radiohalogen less
  • the spacer component (CH 2 ) n can be a straight- or branched-chain alkyl or heteroalkyl group containing up to 12 but preferably no more than 5
  • the alkyl spacer component should be shortened so that nonconjugated and chemically or
  • enzymatically cleaved radiohalogenated compounds can be rapidly cleared through the kidneys, rather than via fatty acid degradation pathways in the heart or liver.
  • a short alkyl or heteroalkyl spacer between the radiolabeled aryl ring and the protein may be desirable.
  • lodohippurate methyl 4-[ 131 I] iodobenzamidoacetimidate; and 4-[ 131 I]oodobenzamidoacetonitrile.
  • Methods for synthesizing compounds having the formula X-Ar-R are also provided. Briefly stated, the methodology described below may be used to metalate any positional isomer of a haloaromatic derivative bearing a functional group Q or a precursor to a functional group Q.
  • the roeta] aticn will employ a trialkyltin reagent such as Sn(n-Bu) 3 or SnMe 3 .
  • the resulting aryltin compound can be transmetalated in a site-specific reaction with one of the following organomercury or organoboron reagents: HgX 2 , Hg(OAc) 2 , BX 3 , or BZ 3 , wherein X is Br,I, or preferably Cl, and Z is alkyl or alkoxy.
  • organomercury or organoboron reagents HgX 2 , Hg(OAc) 2 , BX 3 , or BZ 3 , wherein X is Br,I, or preferably Cl, and Z is alkyl or alkoxy.
  • otherwise metalated compound is radiohalogenated via a demetalation reaction, preferably after functional group Q is formed.
  • Radiohalogenated small molecules for binding to proteinaceous agents are described in European Patent Application Publication Nos. 0 203 764 and 0 289 187, which are incorporated by reference herein in their entireties.
  • Vinyl radiohalogenated small molecules having the following formulae may also be linked to proteinaceous targeting agents:
  • X is a radiohalogen, such as I
  • R 1 and R 2 represent a hydrogen atom, alkyl or substituted alkyl group; an aryl or substituted aryl group; a heteroalkyl group; a heteroaryl group; or a mixed alkylaryl group; and Y represents any of the groups as for R 1 and R 2 , except that Y cannot be hydrogen, and bears a functional group suitable for binding to protein under conditions that preserve the biological activity of the protein.
  • These compounds can be coupled to proteins such as monoclonal antibodies as described above to provide reagents for diagnostic and therapeutic applications.
  • Stabilizing agents are introduced into the therapeutic radionuclide compositions of the present invention.
  • Suitable stabilizing agents include,
  • antioxidants and challenging agents which are suitable for in vivo human administration.
  • Stabilizing agents comprising antioxidants such as ascorbic acid, gentisic acid, reductic acid, derivatives thereof, such as nicotinamide complexes thereof, and functionally similar compounds; challenging agents such as DTPA, EDTA, and the like; and pharmaceutically acceptable salts, esters, amides, and mixtures thereof, are preferred stabilizing agents.
  • Ascorbic acid is an especially preferred
  • the stable compositions of the present invention comprise an "effective amount" of a stabilizing agent, which represents an amount of stabilizing agent sufficient to maintain the therapeutic radionuclide in a reduced state and stably bound to the proteinaceous targeting moiety.
  • a stabilizing agent represents an amount of stabilizing agent sufficient to maintain the therapeutic radionuclide in a reduced state and stably bound to the proteinaceous targeting moiety.
  • therapeutic radionuclide compositions comprising radio-immunoconjugates are injected into human patients in a volume of about 20-30 ml.
  • Such patient preparations preferably comprise from about 1 mg/ml to about 15 mg/ml stabilizing agent, e.g., antioxidant or challenging agent, and most preferably about 7-10 mg/ml stabilizing agent.
  • the total amount of stabilizing agent in each radionuclide patient preparation is therefore from about 20 mg to about 500 mg, and preferably from about 120 mg to about 300 mg.
  • the amount of stabilizing agent required to provide the desired stability increases as the mass and
  • Stabilizing agents of the present invention may be added to the therapeutic radionuclide composition after purification.
  • the purification column is conditioned with an aqueous solution comprising the stabilizing agent prior to elution of the radionuclide composition, and the purified product is eluted with a solution comprising the
  • aqueous ascorbic acid solutions at concentrations of about 1 mg/ml or more, and preferably about 20 mg/ml, are preferred for conditioning and eluting the purification column.
  • Aqueous solutions of DTPA and EDTA are likewise preferably provided at
  • HSA human serum albumin
  • Compatible albumin moieties and serum proteins are those that do not induce a substantial deleterious immune response upon administration to a patient.
  • Suitable albumin moieties may include, for example, whole (native) human serum albumin (HSA) and non-human albumin moieties derived from natural as well as engineered sources; albumin moieties having an altered primary, secondary, tertiary or quaternary structure that exhibit properties similar or superior to native (whole or fragmented) albumin moieties; albumin derivatives
  • albumin moieties that may have a secondary and/or tertiary and/or quaternary structure different from that of native albumin, as disclosed in U.S. patent application No. 07/459,068.
  • Numerous serum proteins may also exhibit stabilizing effects and may be employed as stabilizing agents of the present invention.
  • HSA like ascorbic acid, is readily available at sufficiently high purity levels; it does not react with the therapeutic radionuclide compounds; and it may be administered in vivo to human patients over a wide range of dosage levels.
  • HSA is preferably admixed with the
  • radionuclide composition after purification after purification.
  • the therapeutic radionuclide compositions of the present invention are intended for injection into humans or other mammalian hosts. Accordingly, appropriate manufacturing and in vitro storage practices must be observed to provide suitable sterile, pyrogen-free
  • compositions are used. Although not necessary, it is preferable to use a pharmaceutically acceptable extender or filler to dilute the stabilizer and the (optional) carrier to simplify metering the requisite small quantities of such compounds.
  • a pharmaceutically acceptable extender or filler to dilute the stabilizer and the (optional) carrier to simplify metering the requisite small quantities of such compounds.
  • Sodium chloride and glucose are preferred carriers; sodium chloride is especially preferred because it facilitates provision of an isotonic solution.
  • Stable therapeutic radionuclide compositions according to the present invention may be diluted as necessary and administered to mammalian hosts by
  • Therapeutic 186Re compositions suitable for in vivo administration preferably have radioactivity levels of about 25 to about 600 mCi activity, and
  • compositions prepared for m vivo patient treatment preferably have relatively high specific
  • antibodies or fragments, of about 4 ⁇ Ci/ ⁇ g Ab to about 8 ⁇ Ci/ ⁇ g Ab.
  • the level of radioactivity and the specific activity of preparations desirably
  • administered to a patient will depend upon numerous factors, including the targeting moiety, the physical characteristics and health of the patient, the condition being treated, and the like.
  • the purity of the eluted therapeutic radionuclide composition must be determined prior to administration to a patient to confirm that the purity level is
  • Purity measurements may be determined by a variety of techniques, including instant thin layer chromatography (ITLC) and high pressure liquid chromatography (HPLC). ITLC using a 12% TCA solvent is preferred for rapid determinations, and it provides an accurate measurement of the percentage of rhenium
  • HPLC with Zorbax diol using 0.2M phosphate buffer at pH 7 as the mobile phase may be used for identifying impurities and decomposition products and verifying purity levels.
  • Preparation of 186Re radionuclide compositions for clinical therapeutic applications involves several process steps: (1) formation of the 186 Re ligand ester; (2) conjugation of the 186 Re ligand ester to the targeting moiety; (3) purification of the 186Re reaction mixture; and (4) final dilution and testing prior to patient administration. All procedures involving manipulation of radionuclides should be carried out in a suitably shielded environment, such as a laminar flow hood shielded with clear lead glass bricks, lead bricks, and a lead sheet to reduce radiation exposure during the radiolabeling
  • soluble perrhenate ( 186 ReO 4 -) having the desired activity, generally from about 50 mCi to about 800 mCi, and having a mass of from about 0.01 to about 0.3 mg, is transferred to a reaction vial.
  • Soluble perrhenate ( 186 ReO 4 -) may be obtained from the University of Missouri Research Reactor.
  • the soluble perrhenate is reacted with a transfer agent composition comprising about 1 mg stannous chloride; 25 mg citric acid; 1 mg gentisic acid; and 75 mg lactose.
  • the ratio of stannous chloride reducing agent: rhenium is from about 1:1 to about 10:1 on a molar basis to provide effective reduction, and the ratio of citric acid:rhenium is from about 25:1 to about 500:1 to efficiently generate the citrate/rhenium
  • the chelating agent is dissolved in an organic solvent such as isopropanol, and is introduced in quantities of from about 250 ⁇ g to about 800 ⁇ g to achieve
  • 186 Re:chelating agent offering ratios of about 1:1 to about 1:1.5, preferably about 1:1.3.
  • the chelate reaction mixture is heated to 85-95°C, and the reaction proceeds for about 30 minutes.
  • the desired product of the chelating reaction is a chelated radionuclide ( 186 Re) having an active ester moiety
  • MAGG-GABA One preferred chelating agent for rhenium radionuclide composition is referred to as MAGG-GABA and has the following structure in the chelated, active ester form:
  • Ester purity of the reaction mixture may be quantified by chromatographic techniques.
  • radionuclide is then conjugated to a lysine residue of the proteinaceous targeting moiety.
  • Additional carbonate buffer may be introduced to elevate the pH of the conjugation
  • the reaction mixture is then purified by size exclusion using, for example, Sephadex G- 25 columns, or by ion exchange chromatography.
  • the purification column is preconditioned with aqueous solution comprising a stabilizing agent, and stabilizing solution is
  • the conjugation reaction mixture is diluted as necessary, for patient administration, or it may be divided into several portions for testing purposes.
  • immunoconjugate preparation was eluted using a PBS solution, and the purified eluted conjugate was collected in a vial containing a 5% solution of HSA (about 220 mg HSA) as a stabilizing agent.
  • HSA about 220 mg HSA
  • Preparation I was about 25 mCi, and the total activity in Preparation II was about 100 mCi. Preparation I was divided into two groups, with one sample purified, collected and stored at 4°C, and the other sample
  • Stability of the radionuclide preparation was measured at various time intervals to monitor the purity level of the preparation. Stability was measured by ITLC (12% TCA solvent), as the percentage of radioactivity remaining bound in radio-immunoconjugate complexes.
  • NR2AD is a non-target specific (irrelevant) monoclonal antibody.
  • the total (initial) preparation activity was about 137 mCi, and a 186 Re:Ab offering of 2.67:1 produced conjugates having a specific activity of 6.0 ⁇ Ci/ ⁇ g Ab.
  • the preparation was made using the standard radiolabeling and conjugation techniques described above. The conjugation reaction mixture was divided into tnree samples and purified on PD-10 columns.
  • Preparation I was the control preparation, wherein the radio-immunoconjugate mixture was eluted with PBS, and no stabilizing agent was employed.
  • Preparation II was the control preparation, wherein the radio-immunoconjugate mixture was eluted with PBS, and no stabilizing agent was employed.
  • Re-MAGG-GABA-NRCO-02 F(ab') 2 radio-immunoconjugates having a relatively high specific activity were prepared according to the standard radio-labeling and conjugation techniques described above.
  • the total (initial) preparation activity was about 216 mCi, and the specific activity was 7.3 ⁇ Ci/ ⁇ g Ab.
  • Preparation I was a standard control preparation in which a stabilizer was not used and the conjugate reaction mixture was eluted with
  • Example II a stabilization technique employing 20 mg/ml ascorbic acid solution for conditioning the purification column and eluting the radio-immunoconjugate mixture in combination with HSA provided in the collection vial, provides significantly enhanced long term stability.
  • the control preparation which contained no stabilizing element, exhibited only marginally acceptable purity immediately after purification. Long-term stability (nearly 6 days) of radio-labeled immunoconjugates was achieved at a high
  • Example III The experiment reported in Example III was repeated to test the stability of 186 Re-MAGG-GABA-NRLU-10 radio-immunoconjugates.
  • the preparation had a specific activity of 8.2 ⁇ Ci/ ⁇ g Ab.
  • the preparation was divided into two samples and Preparations I and II purified as described in Example III. Experimental results are shown below:
  • radio-immunoconjugates 186 Re MAGG- GABA-NR2AD radio-immunoconjugates were prepared according to the standard radiolabeling and conjugation techniques described above. The preparation had an activity of about 5.1 ⁇ Ci/ ⁇ g Ab. Each purification column was treated with a conditioning solution containing the stabilizer, and the radio-immunoconjugate mixture was eluted using the
  • the stabilizers used were as

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WO1993002652A3 (en) * 1991-08-01 1993-03-18 Hybritech Inc Modified haptens useful as imaging and therapeutic agents
WO1998055154A1 (en) * 1997-06-03 1998-12-10 Coulter Pharmaceutical, Inc. Radioprotectant for peptides labeled with radioisotope
US6174513B1 (en) * 1994-03-16 2001-01-16 Mallinckrodt Inc. Stabilization of peptides and proteins for radiopharmaceutical use
EP3027228A4 (en) * 2013-08-01 2017-06-07 Rochester Institute of Technology Modular imaging agents containing amino acids and peptides

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WO2008056481A1 (fr) * 2006-11-09 2008-05-15 Nihon Medi-Physics Co., Ltd. Agent d'imagerie de diagnostic radioactif

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US4837003A (en) * 1984-09-13 1989-06-06 Mallinckrodt, Inc. Radiolabeled antibody fragments
US4897255A (en) * 1985-01-14 1990-01-30 Neorx Corporation Metal radionuclide labeled proteins for diagnosis and therapy
US4793987A (en) * 1985-04-26 1988-12-27 Amersham International Plc Stabilized radiolabelled compounds
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993002652A3 (en) * 1991-08-01 1993-03-18 Hybritech Inc Modified haptens useful as imaging and therapeutic agents
US6174513B1 (en) * 1994-03-16 2001-01-16 Mallinckrodt Inc. Stabilization of peptides and proteins for radiopharmaceutical use
WO1998055154A1 (en) * 1997-06-03 1998-12-10 Coulter Pharmaceutical, Inc. Radioprotectant for peptides labeled with radioisotope
US5961955A (en) * 1997-06-03 1999-10-05 Coulter Pharmaceutical, Inc. Radioprotectant for peptides labeled with radioisotope
US6338835B1 (en) 1997-06-03 2002-01-15 Coulter Pharmaceutical, Inc. Radioprotectant for peptides labeled with radioisotope
EP3027228A4 (en) * 2013-08-01 2017-06-07 Rochester Institute of Technology Modular imaging agents containing amino acids and peptides
US10610608B2 (en) 2013-08-01 2020-04-07 Rochester Institute Of Technology Modular imaging agents containing amino acids and peptides

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