U.S. patent application number 10/027593 was filed with the patent office on 2002-10-17 for transcobalamin receptor binding conjugates useful for treating abnormal cellular proliferation.
Invention is credited to Collins, Douglas A., Hogenkamp, Henricus P.C..
Application Number | 20020151525 10/027593 |
Document ID | / |
Family ID | 26935575 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020151525 |
Kind Code |
A1 |
Collins, Douglas A. ; et
al. |
October 17, 2002 |
Transcobalamin receptor binding conjugates useful for treating
abnormal cellular proliferation
Abstract
An agent, composition and method for the treatment, prophylaxis
and/or diagnosis of proliferative disorders, which is highly and
efficiently absorbed at the site of abnormal cellular proliferation
is disclosed.
Inventors: |
Collins, Douglas A.;
(Rochester, MN) ; Hogenkamp, Henricus P.C.;
(Roseville, MN) |
Correspondence
Address: |
KING & SPALDING
191 PEACHTREE STREET, N.E.
ATLANTA
GA
30303-1763
US
|
Family ID: |
26935575 |
Appl. No.: |
10/027593 |
Filed: |
October 25, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60243082 |
Oct 25, 2000 |
|
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60243112 |
Oct 25, 2000 |
|
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Current U.S.
Class: |
514/80 ;
540/145 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 47/551 20170801; C07H 23/00 20130101 |
Class at
Publication: |
514/80 ;
540/145 |
International
Class: |
A61K 031/675; C07F
015/06 |
Claims
We claim:
1. A compound of the formula (I): 12or its enantiomer, diastereomer
or its pharmaceutically salt, wherein: (i) the wavy line in the
chemical structure indicates either a dative or covalent bond such
that there are three dative Co--N bonds and one covalent Co--N
bond, wherein, in the case of the dative bond, that valence of
nitrogen is completed either with a double bond with an adjacent
ring carbon or with a hydrogen; (ii) the dotted line in the
chemical structure indicates either a double or single bond such
that the double bond does not over-extend the valence of the
element (i.e. to give pentavalent carbons) and, in the case of a
single bond, the valence is completed with hydrogen; (iii) X is
hydrogen, cyano, halogen (Cl, F, Br or I), haloalkyl (including
CF.sub.3, CF.sub.2CF.sub.3, CH.sub.2CF.sub.3 and CF.sub.2Cl), NO,
NO.sub.2, NO.sub.3, phosphonate (including
alkyl-P(O).sub.2OR.sup.5), PR.sup.15R.sup.16R.sup.17, NH.sub.2,
NR.sup.15R.sup.16, OH, OR.sup.15, SR.sup.15, SCN, N.sub.3,
OC(O)R.sup.15, C(O).sub.2R.sup.15, C(O)R.sup.15,
OC(O)NR.sup.15R.sup.16 C(O).sub.2NR.sup.15R.sup.16,
C(O)NR.sup.15R.sup.16, P(O).sub.2OR.sup.15, S(O).sub.2OR.sup.16, a
purine or pyrimidine nucleoside or nucleoside analog, adenosyl,
5-FU, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkaryl, amino acid,
peptide, protein, carbohydrate, heteroalkyl, heterocycle,
heteroaryl or alkylheteroaryl; (iv) M is a monovalent heterocycle
or heteroaromatic, which is capable of +3 binding to the adjacent
sugar ring, and forming a dative bond with Co.sup.+3; (v) K is O,
S, NJ.sup.1, C(OH)H, CR.sup.100R.sup.101 or
C(R.sup.100)V.sup.8Z.sup.8; (vi) E is O or S; (vii) G.sup.1 is
hydrogen, alkyl, acyl, silyl, phosphate or L-T; (viii) Y.sup.1,
Y.sup.2, Y.sup.3, Y.sup.4, Y.sup.5, Y.sup.6 and
y.sup.7independently are O, S or NJ.sup.2; (ix) V.sup.1, V.sup.2,
V.sup.3, V.sup.4, V.sup.5, V.sup.6, V.sup.7 and V.sup.8
independently are O, S, NJ.sup.3, CR.sup.102R.sup.103 or a direct
bond; (x) Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.7 and
Z.sup.8 independently are R.sup.104 or L-T; (xi) each L is
independently a direct bond or linker, of a singular molecular
weight, to one or more T moieties, and that does not significantly
impair the ability of the TC- or IF-binding carrier to bind to a
transcobalamin receptor, optionally when bound to a transport
protein; (xii) each T independently comprises the residue of a
therapeutic and/or diagnostic agent effective for the treatment,
prophylaxis and/or diagnosis of a proliferative disorder,
optionally bound though a chelating moiety; (xiii) at least one of
Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4,Z.sup.5, Z.sup.7, Z.sup.8, K and
G.sup.1 is L-T; (xiv) J.sup.1, J.sup.2 and J.sup.3 independently
are hydrogen, alkyl, alkenyl, alkynyl, alkaryl, cycloalkyl, aryl,
cycloaryl, heteroalkyl, heterocycle, heteroaryl, hydroxyl, alkoxy
or amine; (xv) R.sub.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sub.11, R.sup.12,
R.sup.13 and R.sup.14 independently are hydrogen, lower alkyl,
lower alkenyl, lower alkynyl, lower cycloalkyl, heteroalkyl,
heterocyclic, lower alkoxy, azido, amino, lower alkylamino,
halogen, thiol, SO.sub.2, SO.sub.3, carboxylic acid, C.sub.1-6
carboxyl, hydroxyl, nitro, cyano, oxime or hydrazine; (xvi)
R.sup.13 and R.sup.14 optionally can form a double bond; (xvii)
R.sup.15, R.sup.16 and R.sup.17 are independently hydrogen, alkyl,
alkenyl, alkynyl, aryl, alkaryl or aralkyl group, heteroalkyl,
heterocycle or heteroaromatic; and (xviii) R.sup.100, R.sup.101,
R.sup.102, R.sup.103, and R.sup.104 are independently hydrogen,
alkyl, alkenyl, alkynyl, aryl, acyl, heteroaromatic, heteroaryl,
heteroalkyl, hydroxyl, alkoxy, cyano, azido, halogen, nitro,
SO.sub.2, SO.sub.3, thioalkyl or amino; (xix) wherein at least one
of Y, R, G, E, K, M and V is not as it is found in natural vitamin
B.sub.12.
2. The compound of claim 1, wherein at least one T is selected from
the group consisting of cisplatin, taxol, taxotere (docetaxel),
daunorubicin (daunomycin; rubidomycin), doxorubicin, rubidazone and
idarubicin (idamycin; 4-demethoxy-daunorubicin).
3. The compound of claim 1, wherein at least one T is a detectable
and/or therapeutic radionuclide.
4. The compound of any one of claims 1-3, wherein at least one L is
independently an amine, a polyamine, an amino acid, a poly(amino
acid) or peptide linker;
5. The compound of any one of claims 1-3, wherein at least one
--L-T is independently a poly(amino acid) residue bound to one or
more T.
6. The compound of claim 5, wherein at least one --L-T is
independently a poly-L-lysine
--NR'(CH((CH.sub.2).sub.4--NHR')CONR').sub.mR', wherein each R' is
independently hydrogen, lower alkyl or T; and m is 2-20.
7. The compound of any one of claims 1-3, wherein at least one
--L-T is independently a polyamine residue of the formula
--NR'(alkylene-NR--)nalk- yleneNR'R', wherein each R' is
independently hydrogen, lower alkyl or T and n is 1-20.
8. The compound of claim 7, wherein
--NR'(alkylene-NR').sub.nalkyleneNR' is selected from the group
consisting of --NR'(CH.sub.2).sub.3NR'(CH.sub.-
2).sub.4NR'(CH.sub.2).sub.3NR'R' (spermine);
--NR'(CH.sub.2).sub.3NR'(CH.s- ub.2).sub.4NR'R' (spermidine);
decamethylene tetraamine and pentamethylene hexamine.
9. The compound of any one of claims 1-3, wherein at least one
--L-T is independently a diamine residue of the formula
--NR'(alkylene).sub.xNR'R'- , wherein each R' is independently
hydrogen, lower alkyl or T and x is 2-20.
10. The compound of claim 9, wherein --NR'alkylene).sub.xNR'R' is
selected from the group consisting of 1,6-diaminohexane,
1,5-diaminopentane, 1,4-diaminobutane and 1,3-diaminopropane.
11. The compound of claim 1, wherein T is not a residue of a
therapeutic agent selected from the group consisting of hormone,
growth factor, interleukin, cytokines, lymphokines, GCSF, EPO,
interferon (.alpha., .beta., .gamma.), calcitonin, TRH,
vasopressin, desmopressin [Folia Endocrinologica Japonica 54, No.
5, p. 676-691 (1978)], oxytocin, insulin, Growth Hormone,
testosterone, somatotrophin, somatostatin (U.S. Pat. Nos. 4,087,390
and 4,100,117), SCGF, (stem cell growth factor), CGRP,
Erythropoietin, Colony Stimulating factors (GCSF, GM-CSF, CSF),
pregnant mare serum gonadotrophin (PMSG), human chorionic
gonadotrophin (HCG), Inhibin, PAI-2; neomycin, salbutamol,
pyrimethamine, penicillin G, methicillin, carbenicillin, pethidine,
xylazine, ketamine, mephenesin, GABA, iron dextran, nucleotide
analogues or ribozyme, prolactin, adrenocorticotropic hormone
(ACTH), melanocyte stimulating hormone (MSH), thyroid hormone
releasing hormone (TRH) (U.S. Pat. No. 4,100,152), thyroid
stimulating hormone (TSH), luteinizing hormone (LH), luteinizing
hormone releasing hormone (LHRH), follicle stimulating hormone
(FSH), oxytocin, calcitonin, parathyroid hormone, glucagon,
gastrin, secretin, pancreozymin, cholecystokinin angiotensin, human
placental lactogen, human chorionic gonadotropin (HCG), enkephalin
[U.S. Pat. No. 4,277,394, European patent application Publication
No. 31567], endorphin, kyotorphin, interleukins (I, II, and III),
tuftsin, thymopoietin, thymosin, thymostimulin, thymic humoral
factor (TFH), serum thymic factor (FTS) (U.S. Pat. No. 4,229,438),
thymic factors [Medicine in Progress 125, No. 10, p.835-843
(1983)], tumor necrosis factor (TNF), colony stimulating factor
(CSF), motilin, dinorphin, bombesin, neurotensin, cerulein,
bradykinin, urokinase, asparaginase, kallikrein, substance P
analogue and antagonist, nerve growth factor, blood coagulation
factors VIII and IX, lysozyme chloride, polymixin B, colistin,
gramicidin, bacitracin, protein synthesis stimulating peptides
(British patent No. 8232082), gastric inhibitory polypeptide (GIP),
vasoactive intestinal polypeptide (VIP), platelet-derived growth
factor (PDGF), growth hormone factor (GRF, somatocrinin), bone
morphogenetic protein (BMP), epidermal growth factor (EGF),
bleomycin, methotrexate, actinomycin D, mitomycin C, vinblastine
sulfate, vincristine sulfate, daunorubicin, adriamycin,
neocarzinostatin, cytosine arabinoside, fluorouracil,
tetrahydrofuryl-5-fluorouracil, krestin, picibanil, lentinan,
levamisole, bestatin, azimexon, glycyrrhizin, poly I:C, poly A:U
and poly ICLC, gentamicin, dibekacin, kanendomycin, lividomycin,
tobramycin, amikacin, fradiomycin, sisomicin, tetracycline
hydrochloride, oxytetracycline hydrochloride, rolitetracycline,
doxycycline hydrochloride, ampicillin, piperacillin, ticarcillin,
cephalothin, cephaloridine, cefotiam, cefsulodin, cefinenoxime,
cefinetazole, cefazolin, cefotaxime, cefoperazone, ceftizoxime,
moxolactam, latamoxef, thienamycin, sulfazecin, azthreonam, sodium
salicylate, sulpyrine, sodium flufenamate, sodium diclofenac,
sodium indomethacin, morphine hydrochloride, pethidine, levorphanol
tartrate, oxymorphone, ephedrine, methylephedrine, noscapine,
codeine phosphate, dihydrocodeine, phosphate, alloclamide,
chlophedianol, picoperidamine, cloperastine, protokylol,
isoproterenol, salbutamol, terbutaline sulfate, chlorpromazine,
prochlorperazine, trifluoperazine, atropine sulfate, scopolamine
methylbromide, pridinol methanesulfonate, tubocurarine chloride and
pancuronium bromide, sodium phenytoin, ethosuximide, sodium
acetazolamide, chlordiazepoxide hydrochloride, metoclopramide and
L-histidine monohydrochloride, imipramine, clomipramine,
noxiptiline, phenelzine sulfate, diphenhydramine, chlorpheniramine
maleate, tripelenamine, methdilazine, clemizole, diphenylpyraline,
methoxyphenamine, trans-p-oxocamphor, theophyllol, aminophylline,
etilefrine, propranolol, alprenolol, bufetolol, oxyprenolol,
oxyfedrine, diltiazem, tolazoline, hexobendine, bamethan sulfate,
hexamethonium bromide, pentolinium, mecamlamine, ecarazine,
clonidine, sodium glymidine, glypizide, phenformin, buformin,
metformin, sodium heparin, sodium citrate, thromboplastin,
thrombin, menadione sodium bisulfite, acetomenaphthone,
.epsilon.-amino-caproic acid, tranexamic acid, carbazochrome sodium
sulfonate, adrenochrome monoaminoguanidine methanesulfonate,
isoniazid, ethambutol, sodium p-aminosalicylate, prednisolone
succinate, prednisolone sodium phosphate, dexamethasone sodium
sulfate, betamethasone sodium phosphate, hexestrol phosphate,
hexestrol acetate, methimazole, levallorphan tartrate, nalorphine
hydrochloride and naloxone hydrochloride; a protein derived from or
immunogens against influenza, measles, Rubella, smallpox, yellow
fever, diphtheria, tetanus, cholera, plague, typhus, BCG,
tuberculosis causing agents, Haemophilus influenzae, Neisseria
catarrhalis, Klebsiella pneumoniae, pneumococci, streptococci; a
secretory product derived from diphtheria, tetanus, cholera,
plague, typhus, tuberculosis causing agents, Haemophilus
influenzae, Neisseria catarrhalis, Klebsiella pneumoniae,
pneumococci, streptococci, Streptococcus mutans, or is derived from
a malarial parasite or the causative agent of coccidiosis in
chickens.
12. A pharmaceutical composition for the treatment, prophylaxis
and/or diagnosis of a proliferative disorder in a host comprising a
compound of any one of claims 1-11, or the pharmaceutically
acceptable salt thereof, in combination with a pharmaceutically
acceptable carrier.
13. A pharmaceutical composition for the treatment, prophylaxis
and/or diagnosis of a proliferative disorder in a host comprising a
compound of any one of claims 1-11, or the pharmaceutically
acceptable salt thereof, optionally in a pharmaceutically
acceptable carrier, in combination with one or more other
therapeutic and/or diagnostic agent(s).
14. The pharmaceutical composition of claim 12 or 13, wherein the
host is a human.
15. A method for the treatment, prophylaxis and/or diagnosis of a
proliferative disorder in a host comprising administering an
effective amount of a compound of any one of claims 1-11, or the
pharmaceutically acceptable salt thereof, optionally in a
pharmaceutically acceptable carrier.
16. A method for the treatment, prophylaxis and/or diagnosis of a
proliferative disorder in a host comprising administering an
effective amount of a compound of any one of claims 1-11, or the
pharmaceutically acceptable salt thereof, optionally in a
pharmaceutically acceptable carrier, in combination or alternation
with one or more other effective therapeutic and/or diagnostic
agent.
17. The method of claim 15 or 16, wherein the host is a human.
18. A method for the treatment, prophylaxis and/or diagnosis of a
proliferative disorder other than neoplasms in a host comprising
administering an effective amount of a compound of the formula (I):
13or its enantiomer, diastereomer or its pharmaceutically
acceptable salt, wherein: (i) the wavy line in the chemical
structure indicates either a dative or covalent bond such that
there are three dative Co-N bonds and one covalent Co-N bond,
wherein, in the case of the dative bond, the valence of nitrogen is
completed either with a double bond with an adjacent ring carbon or
with a hydrogen; (ii) the dotted line in the chemical structure
indicates either a double or single bond such that the double bond
does not over-extend the valence of the element (i.e. to give
pentavalent carbons) and, in the case of a single bond, the valence
is completed with hydrogen; (iii) X is hydrogen, cyano, halogen
(Cl, F, Br or I), haloalkyl (including CF.sub.3, CF.sub.2CF.sub.3,
CH.sub.2CF.sub.3 and CF.sub.2Cl), NO, NO.sub.2, NO.sub.3,
phosphonate (including alkyl-P(O).sub.2OR.sup.15), PR.sup.15
R.sup.16R.sup.17, NH.sub.2, NR.sup.15 R.sup.6, OH, OR.sup.15,
SR.sup.15, SCN, N.sub.3, OC(O)R.sup.15, C(O).sub.2R.sup.15,
C(O)R.sup.15, OC(O)NR.sup.15R.sup.16, C(O).sub.2NR.sup.15R.sup.16,
C(O)NR.sup.15 R.sup.16, P(O).sub.2OR.sup.15, S(O).sub.2OR.sup.15, a
purine or pyrimidine nucleoside or nucleoside analog, adenosyl,
5-FU, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkaryl, amino acid,
peptide, protein, carbohydrate, heteroalkyl, heterocycle,
heteroaryl or alkylheteroaryl; (iv) M is a monovalent heterocycle
or heteroaromatic, which is capable of binding to the adjacent
sugar ring, and forming a dative bond with Co.sup.+3; (v) K is O,
S, NJ.sup.1, C(OH)H, CR.sup.100R.sup.101 or
C(R.sup.100)V.sup.8Z.sup.8; (vi) E is O or S; (vii) G.sup.1 is
hydrogen, alkyl, acyl, silyl, phosphate or L-T; (viii) Y.sup.1,
Y.sup.2, Y.sup.3, Y.sup.4, Y.sup.5 Y.sup.6 and Y.sup.7
independently are O, S or NJ.sup.2; (xi) V.sup.1, V.sup.2, V.sup.3,
V.sup.4, V.sup.5, V.sup.6, V.sup.7 and Z.sup.8 independently are O,
S, NJ.sup.3, CR.sup.102R.sup.103 or a direct bond; (X) Z.sup.1,
Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.7 and Z.sup.8
independently are R.sup.104 or L-T; (xi) each L is independently a
direct bond or linker, to one or more T moieties, and that does not
significantly impair the ability of the TC- or IF-binding carrier
to bind to a transcobalamin receptor, optionally when bound to a
transport protein; (xii) each T independently comprises the residue
of a therapeutic and/or diagnostic agent effective for the
treatment, prophylaxis and/or diagnosis of a proliferative
disorder, optionally bound though a chelating moiety; (xiii) at
least one of Z.sub.1, Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.7,
Z.sup.8, K and G.sup.1 is L-T; (xiv) J.sup.1, J.sup.2 and J.sup.3
independently are hydrogen, alkyl, alkenyl, alkynyl, alkaryl,
cycloalkyl, aryl, cycloaryl, heteroalkyl, heterocycle, heteroaryl,
hydroxyl, alkoxy or amine; (xv) R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11,
R.sup.12, R.sup.13 and R.sup.14independently are hydrogen, lower
alkyl, lower alkenyl, lower alkynyl, lower cycloalkyl, heteroalkyl,
heterocyclic, lower alkoxy, azido, amino, lower alkylamino,
halogen, thiol, SO.sub.2, SO.sub.3, carboxylic acid, C.sub.1-6
carboxyl, hydroxyl, nitro, cyano, oxime or hydrazine; (xvi)
R.sup.13 and R.sup.14 optionally can form a double bond; (xvii)
R.sup.15, R.sup.16 and R.sup.17 are independently hydrogen, alkyl,
alkenyl, alkynyl, aryl, alkaryl or aralkyl group, heteroalkyl,
heterocycle or heteroaromatic; and (xviii) R.sup.100, R.sup.101,
R.sup.102, R.sup.103, and R.sup.104 are independently hydrogen,
alkyl, alkenyl, alkynyl, aryl, acyl, heteroaromatic, heteroaryl,
heteroalkyl, hydroxyl, alkoxy, cyano, azido, halogen, nitro,
SO.sub.2, SO.sub.3, thioalkyl or amino; optionally in a
pharmaceutically acceptable carrier.
19. A method for the treatment, prophylaxis and/or diagnosis of a
proliferative disorder other than neoplasms in a host comprising
administering an effective amount of a compound of claim 18,
optionally in a pharmaceutically acceptable carrier, in combination
or alternation with one or more other effective therapeutic and/or
diagnostic agent(s).
20. The method of claim 18 or 19, wherein at least one L is a
linker of singular molecular weight.
21. The method of claim 18 or 19, wherein at least one L is
independently an amine, a polyamine, an amino acid, a poly(amino
acid) or peptide linker.
22. The method of claim 18 or 19, wherein at least one --L-T is
independently a poly(amino acid) residue bound to one or more
T.
23. The method of claim 22, wherein at least one --L-T is
independently a poly-L-lysine
--NR'(CH((CH.sub.2).sub.4--NHR')CONR').sub.mR', wherein each R' is
independently hydrogen, lower alkyl or T; and m is 2-20.
24. The method of claim 18 or 19, wherein at least one --L-T is
independently a polyamine residue of the formula
--NR'(alkylene-NR').sub.- nalkyleneNR'R', wherein each R' is
independently hydrogen, lower alkyl or T and n is 1-20.
25. The method of claim 24, wherein
--NR'(alkylene-NR').sub.n-alkyleneNR' is selected from the group
consisting of --NR'(CH.sub.2).sub.3NR'(CH.sub.-
2).sub.4NR'--(CH.sub.2).sub.3NR'R'(spermine);
--NR'(CH.sub.2).sub.3NR'(CH.- sub.2).sub.4--NR'R' (spermidine);
deca-methylene tetraamine and pentamethylene hexamine.
26. The method of claim 18 or 19, wherein at least one --L-T is
independently a diamine residue of the formula --NR'
(alkylene).sub.xNR'R', wherein each R' is independently hydrogen,
lower alkyl or T and x is 2-20.
27. The method of claim 26, wherein --NR'(alkylene).sub.xNR'R' is
selected from the group consisting of 1,6-diaminohexane,
1,5-diaminopentane, 1,4-diaminobutane and 1,3-diaminopropane.
28. The method of any one of claims 18-27, wherein the host is a
human.
Description
[0001] This application claims priority to U.S. provisional
application no. 60/243,082 and U.S. provisional application no.
60/243,112, both filed on Oct. 25, 2000.
FIELD OF THE INVENTION
[0002] The present invention includes compounds, compositions and
methods for the treatment, prophylaxis and/or diagnosis of abnormal
cellular proliferation.
BACKGROUND OF THE INVENTION
[0003] Cellular differentiation, growth, function and death are
regulated by a complex network of mechanisms at the molecular level
in a multicellular organism. In the healthy animal or human, these
mechanisms allow the cell to carry out its designed function and
then die at a programmed rate.
[0004] Abnormal cellular proliferation, notably hyperproliferation,
can occur as a result of a wide variety of factors, including
genetic mutation, infection, exposure to toxins, autoimmune
disorders, and benign or malignant tumor induction.
[0005] There are a number of skin disorders associated with
cellular hyperproliferation. Psoriasis, for example, is a benign
disease of human skin generally characterized by plaques covered by
thickened scales. The disease is caused by increased proliferation
of epidermal cells of unknown cause. In normal skin the time
required for a cell to move from the basal layer to the upper
granular layer is about five weeks. In psoriasis, this time is only
6 to 9 days, partially due to an increase in the number of
proliferating cells and an increase in the proportion of cells
which are dividing (G. Grove, Int. J. Dermatol. 18:111, 1979).
Approximately 2% of the population the United States have
psoriasis, occurring in about 3% of Caucasian Americans, in about
1% of African Americans, and rarely in native Americans. Chronic
eczema is also associated with significant hyperproliferation of
the epidermis. Other diseases caused by hyperproliferation of skin
cells include atopic dermatitis, lichen planus, warts, pemphigus
vulgaris, actinic keratosis, basal cell carcinoma and squamous cell
carcinoma.
[0006] Other hyperproliferative cell disorders include blood vessel
proliferation disorders, fibrotic disorders, autoimmune disorders,
graft-versus-host rejection, tumors and cancers.
[0007] Blood vessel proliferative disorders include angiogenic and
vasculogenic disorders. Proliferation of smooth muscle cells in the
course of development of plaques in vascular tissue cause, for
example, restenosis, retinopathies and atherosclerosis. The
advanced lesions of atherosclerosis result from an excessive
inflammatory-proliferative response to an insult to the endothelium
and smooth muscle of the artery wall (Ross R., Nature 362:801-809
(1993)). Both cell migration and cell proliferation play a role in
the formation of atherosclerotic lesions.
[0008] Fibrotic disorders are often due to the abnormal formation
of an extracellular matrix. Examples of fibrotic disorders include
hepatic cirrhosis and mesangial proliferative cell disorders.
Hepatic cirrhosis is characterized by the increase in extracellular
matrix constituents resulting in the formation of a hepatic scar.
Hepatic cirrhosis can cause diseases such as cirrhosis of the
liver. An increased extracellular matrix resulting in a hepatic
scar can also be caused by viral infection such as hepatitis.
Lipocytes appear to play a major role in hepatic cirrhosis.
[0009] Mesangial disorders are brought about by abnormal
proliferation of mesangial cells. Mesangial hyperproliferative cell
disorders include various human renal diseases, such as
glomerulonephritis, diabetic nephropathy, malignant
nephrosclerosis, thrombotic microangiopathy syndromes, transplant
rejection, and glomerulopathies.
[0010] Another disease with a proliferative component is rheumatoid
arthritis. Rheumatoid arthritis is generally considered an
autoimmune disease that is thought to be associated with activity
of autoreactive T cells (See, e.g., Harris, E. D., Jr., The New
England Journal of Medicine, 322: 1277-1289 (1990)), and to be
caused by autoantibodies produced against collagen and IgE.
[0011] Other disorders that can include an abnormal cellular
proliferative component include Behcet's syndrome, acute
respiratory distress syndrome (ARDS), ischemic heart disease,
post-dialysis syndrome, leukemia, acquired immune deficiency
syndrome, vasculitis, lipid histiocytosis, septic shock and
inflammation in general.
[0012] A tumor, also called a neoplasm, is a new growth of tissue
in which the multiplication of cells is uncontrolled and
progressive. A benign tumor is one that lacks the properties of
invasion and metastasis and is usually surrounded by a fibrous
capsule. A malignant tumor (i.e., cancer) is one that is capable of
both invasion and metastasis. Malignant tumors also show a greater
degree of anaplasia (i.e., loss of differentiation of cells and of
their orientation to one another and to their axial framework) than
benign tumors.
[0013] Approximately 1.2 million Americans are diagnosed with
cancer each year, 8,000 of which are children. In addition, 500,000
Americans die from cancer each year in the United States alone.
Prostate and lung cancers are the leading causes of death in men
while breast and lung cancer are the leading causes of death in
women. It is estimated that cancer-related costs account for about
10 percent of the total amount spent on disease treatment in the
United States. (CNN.Cancer.Factshttp://-
www.cnn.com/HEALTH/9511/conquer cancer/facts/index.html, page 2 of
2, Jul. 18, 1999).
[0014] Proliferative disorders are currently treated by a variety
of classes of compounds including alkylating agents,
antimetabolites, natural products, enzymes, biological response
modifiers, miscellaneous agents, hormones and antagonists, such as
those listed below.
[0015] Alkylating Agents
[0016] Nitrogen Mustards: Mechlorethamine (Hodgkin's disease,
non-Hodgkin's lymphomas), Cyclophosphamide Ifosfamide (acute and
chronic lymphocytic leukemias, Hodgkin's disease, non-Hodgkin's
lymphomas, multiple myeloma, neuroblastoma, breast, ovary, lung,
Wilms' tumor, cervix, testis, soft-tissue sarcomas), Melphalan
(L-sarcolysin) (multiple myeloma, breast, ovary), Chlorambucil
(chronic lymphoctic leukemia, primary macroglobulinemia, Hodgkin's
disease, non-Hodgkin's lymphomas).
[0017] Ethylenimines and Methylmelamines: Hexamethylmelamine
(ovary), Thiotepa (bladder, breast, ovary).
[0018] Alkyl Sulfonates: Busulfan (chronic granuloytic
leukemia).
[0019] Nitrosoureas: Carmustine (BCNU) (Hodgkin's disease,
non-Hodgkin's lymphomas, primary brain tumors, multiple myeloma,
malignant melanoma), Lomustine (CCNU) (Hodgkin's disease,
non-Hodgkin's lymphomas, primary brain tumors, small-cell lung),
Semustine (methyl-CCNU) (primary brain tumors, stomach, colon),
Streptozocin (streptozocin) (malignant pancreatic insulinoma,
malignant carcinoin).
[0020] Triazenes: Dacarbazine (DTIC;
dimethyltriazenoimid-azolecarboxamide- ) (malignant melanoma,
Hodgkin's disease, soft-tissue sarcomas).
[0021] Antimetabolites
[0022] Folic Acid Analogs: Methotrexate (amethopterin) (acute
lymphocytic leukemia, choriocarcinoma, mycosis fungoides, breast,
head and neck, lung, osteogenic sarcoma).
[0023] Pyrimidine Analogs: Fluorouracil (5-fluorouracil; 5-FU)
Floxuridine (fluorodeoxyuridine; FUDR) (breast, colon, stomach,
pancreas, ovary, head and neck, urinary bladder, premalignant skin
lesions) (topical), Cytarabine (cytosine arabinoside) (acute
granulocytic and acute lymphocytic leukemias).
[0024] Purine Analogs and Related Inhibitors: Mercaptopurine
(6-mercaptopurine; 6-MP) (acute lymphocytic, acute granulocytic and
chronic granulocytic leukemia), Thioguanine (6-thioguanine: TG)
(acute granulocytic, acute lymphocytic and chronic granulocytic
leukemia), Pentostatin (2'-deoxycyoformycin) (hairy cell leukemia,
mycosis fungoides, chronic lymphocytic leukemia).
[0025] Vinca Alkaloids: Vinblastine (VLB) (Hodgkin's disease,
non-Hodgkin's lymphomas, breast, testis), Vincristine (acute
lymphocytic leukemia, neuroblastoma, Wilms' tumor,
rhabdomyosarcoma, Hodgkin's disease, non-Hodgkin's lymphomas,
small-cell lung).
[0026] Epipodophyl-lotoxins: Etoposide (testis, small-cell lung and
other lung, breast, 25Hodgkin's disease, non-Hodgkin's lymphomas,
acute granulocytic leukemia, Kaposi's sarcoma), Teniposide (testis,
small-cell lung and other lung, breast, Hodgkin's disease,
non-Hodgkin's lymphomas, acute granulocytic leukemia, Kaposi's
sarcoma).
[0027] Natural Products
[0028] Antibiotics: Dactinomycin (actinonmycin D) (choriocarcinoma,
Wilms' tumor rhabdomyosarcoma, testis, Kaposi's sarcoma),
Daunorubicin (daunomycin; rubidomycin) (acute granulocytic and
acute lymphocytic leukemias), Doxorubicin (soft tissue, osteogenic,
and other sarcomas; Hodgkin's disease, non-Hodgkin's lymphomas,
acute leukemias, breast, genitourinary thyroid, lung, stomach,
neuroblastoma), Bleomycin (testis, head and neck, skin and
esophagus lung, and genitourinary tract, Hodgkin's disease,
non-Hodgkin's lymphomas), Plicamycin (mithramycin) (testis,
malignant hypercalcema), Mitomycin (mitomycin C) (stomach, cervix,
colon, breast, pancreas, bladder, head and neck).
[0029] Enzymes: L-Asparaginase (acute lymphocytic leukemia).
[0030] Biological Response Modifiers: Interferon-alfa (hairy cell
leukemia, Kaposi's sarcoma, melanoma, carcinoid, renal cell, ovary,
bladder, non Hodgkin's lymphomas, mycosis fungoides, multiple
myeloma, chronic granulocytic leukemia).
[0031] Hormones and Antagonists
[0032] Estrogens: Diethylstibestrol Ethinyl estradiol (breast,
prostate)
[0033] Antiestrogen: Tamoxifen (breast).
[0034] Androgens: Testosterone propionate Fluxomyesterone
(breast).
[0035] Antiandrogen: Flutamide (prostate).
[0036] Gonadotropin-Releasing Hormone Analog: Leuprolide
(prostate).
[0037] Miscellaneous Agents
[0038] Platinum Coordination Complexes: Cisplatin (cis-DDP)
Carboplatin (testis, ovary, bladder, head and neck, lung, thyroid,
cervix, endometrium, neuroblastoma, osteogenic sarcoma).
[0039] Anthracenedione: Mixtozantrone (acute granulocytic leukemia,
breast).
[0040] Substituted Urea: Hydroxyurea (chronic granulocytic
leukemia, polycythemia vera, essential thrombocytosis, malignant
melanoma).
[0041] Methylhydrazine Derivative: Procarbazine (N-methylhydrazine,
MIH) (Hodgkin's disease).
[0042] Adrenocortical Suppressant: Miotane (o,p'-DDD) (adrenal
cortex), Aminoglutethimide (breast).
[0043] Adrenorticosteriods: Prednisone (acute and chronic
lymphocytic leukemias, non-Hodgkin's lymphomas, Hodgkin's disease,
breast).
[0044] Progestins: Hydroxprogesterone caproate,
Medroxyprogersterone acetate, Megestrol acetate (endometrium,
breast).
[0045] Toxicity associated with therapy for abnormally
proliferating cells, including cancer, is due in part to a lack of
selectivity of the drug for diseased versus normal cells. To
overcome this limitation, therapeutic strategies that increase the
specificity and thus reduce the toxicity of drugs for the treatment
of proliferative disorders are being explored. One such strategy
that is being aggressively pursued is drug targeting.
[0046] An objective of treatment and/or image targeting is to
deliver a therapeutic and/or diagnostic agent to a specific site of
action through a carrier system. Targeting achieves at least two
major aims of drug delivery. The first is to deliver the maximum
dose of therapeutic agent to diseased cells. The second is the
avoidance of uptake by normal, healthy cells. Thus, targeted drug
delivery systems result in enhancing drug accumulation in the
proliferative cells while decreasing exposure to healthy tissues.
As such, the efficacy is increased while the toxicity is decreased,
giving a better therapeutic index.
[0047] Two classes of compounds that are known to localize in
malignant tumors are the porphyrins and the related
phthalocyanines. The biochemical basis by which these compounds
achieve elevated concentration in malignant tumors is unknown, but
this observation has served as the rationale for the use of
hematoporphyrin derivatives in the photodyamic therapy of cancer
(Dougherty, T. J. et al., Porphyrin Photosensitization, 3-13, New
York: Plenum Publishing Corp. (1981)).
[0048] Cells undergoing rapid proliferation have been shown to
increase uptake of thymidine and methionine. (See, for example, M.
E. van Eijkeren et al., Acta Oncologica, 31, 539 (1992); K. Kobota
et al., J. Nucl. Med., 32, 2118 (1991) and K. Higashi et al., J.
Nucl. Med., 34, 773 (1993)). Since methylcobalamin is directly
involved with methionine synthesis and indirectly involved in the
synthesis of thymidylate and DNA, methyl-cobalamin as well as
Cobalt-57-cyanocobalamin have also been shown to have increased
uptake in rapidly dividing tissue (for example, see, B. A. Cooper
et al., Nature, 191, 393 (1961); H. Flodh, Acta Radiol. Suppl.,
284, 55 (1968); L. Bloomquist et al., Experientia, 25, 294 (1969)).
Additionally, upregulation in the number of transcobalamin II
receptors has been demonstrated in several malignant cell lines
during their accelerated thymidine incorporation and DNA synthesis.
See, J. Lindemans et al., Exp. Cell. Res., 184, 449 (1989); T.
Amagasaki et al., Blood, 26, 138 (1990) and J. A. Begly et al., J.
Cell Physiol., 156, 43 (1993). Bacteria naturally insert Cobalt-59
into the corrin ring of vitamin B.sub.12. Commercially this has
been exploited by the fermentative production of Co-56, Co-57,
Co-58, and Co-60 radiolabeled vitamin B.sub.12. For example, see
Chaiet et al., Science, 111, 601 (1950). Unfortunately Cobalt-57,
with a half life of 270.9 days, makes Co-57-cyanocobalamin
unsuitable for clinical tumor imaging. Other metal ions (cobalt,
copper and zinc) have been chemically inserted into naturally
occurring descobaltocorrinoids produced by Chromatium and
Streptomyces olivaceous. Attempts to chemically insert other metal
ions in these cobalt free corrinoid rings have been unsuccessful.
The placement of metals (cobalt, nickel, palladium, platinum,
rhodium, zinc, and lithium) into a synthetic corrin ring has not
presented any major difficulties. However, their instability and
cost to produce makes them impractical for biological assays.
Although Co-59 has a weakly paramagnetic quadrapolar nucleus in the
2.sup.+oxidation state, Co-59 exists in the 3.sup.+oxidation state
within the corrin ring of vitamin B.sub.12 and is diamagnetic.
Therefore, insertion of either a radioactive or paramagnetic metal
ion other than cobalt into the corrin ring does not seem feasible
at this time.
[0049] The structure of various forms of vitamin B.sub.12 is shown
in FIG. 1, wherein X is CN, OH, CH.sub.3 or 5'-deoxyadenosyl,
respectively. The term cobalamin is sometimes used to refer to the
entire molecule except the X group. The fundamental ring system
without cobalt (Co) or side chains is called corrin and the
octadehydrocorrin is called corrole. FIG. 1 is adapted from The
Merck Index, Merck & Co. (11th ed. 1989), wherein X is above
the plane defined by the corrin ring and the nucleotide is below
the plane of the ring. The corrin ring has attached seven
amidoalkyl (H.sub.2NC(O)Alk) substituents, at the 2, 3, 7, 8, 13,
18 and 23 positions, which can be designated a-g respectively. See
D. L. Anton et al, J. Amer. Chem. Soc., 102, 2215 (1980). The 2, 3,
7, 8 and 13 positions are shown in FIG. 1 as positions a-e,
respectively.
[0050] For several years after the isolation of vitamin B.sub.12 as
cyanocobalamin in 1948, it was assumed that cyanocobalamin and
possibly hydroxocobalamin, its photolytic breakdown product,
occurred in man. Since then it has been recognized that
cyanocobalamin is an artifact of the isolation of vitamin B.sub.12
and that hydroxocobalamin and the two coenzyme forms,
methylcobalamin and adenosylcobalamin, are the naturally occurring
forms of the vitamin.
[0051] Vitamin B.sub.12 (adenosyl-, cyano-, hydroxo- or
methylcobalamin) must be bound by the transport protein
Transcobalamin I, II, or III ("TC") to be biologically active, and
by Intrinsic Factor ("IF") if administered orally. Gastrointestinal
absorption of vitamin B.sub.12 occurs when the intrinsic
factor-vitamin B.sub.12 complex is bound to the intrinsic factor
receptor in the terminal ileum. Likewise, intravascular transport
and subsequent cellular uptake of vitamin B.sub.12 throughout the
body typically occurs through the transcobalamin transport protein
(I, II or III) and the cell membrane transcobalamin receptors,
respectively. After the transcobalamin vitamin B.sub.12 complex has
been internalized in the cell, the transport protein undergoes
lysozymal degradation, which releases vitamin B12 into the
cytoplasm. All forms of vitamin B.sub.12 can then be interconverted
into adenosyl-, hydroxo- or methylcobalamin depending upon cellular
demand. See, for example, A. E. Finkler et al., Arch. Biochem.
Biophys., 120, 79 (1967); C. Hall et al., J. Cell Physiol., 133,
187 (1987); M. E. Rappazzo et al., J. Clin. Invest., 51, 1915
(1972) and R. Soda et al., Blood, 65, 795 (1985).
[0052] A process for preparing .sup.125I-vitamin B.sub.12
derivatives is described in Niswender et al. (U.S. Pat. No.
3,981,863). In this process, vitamin B.sub.12 is first subjected to
mild hydrolysis to form a mixture of monocarboxylic acids, which
Houts, infra, disclosed to contain mostly the (e)-isomer. The
mixture is then reacted with a p-(aminoalkyl)phenol to introduce a
phenol group into the B.sub.12 acids (via reaction with one of the
free carboxylic acid groups). The mixed substituent B.sub.12
derivatives are then iodinated in the phenol-group substituent.
This U.S. patent teaches that the mixed .sup.125I-B.sub.12
derivatives so made are useful in the radioimmunoassay of B.sub.12,
using antibodies raised against the mixture.
[0053] T. M. Houts (U.S. Pat. No. 4,465,775) reported that the
components of the radiolabeled mixture of Niswender et al did not
bind with equal affinity to IF. Houts disclosed that radioiodinated
derivatives of the pure monocarboxylic (d)-isomer are useful in
assays of B.sub.12 in which IF is used.
[0054] U.S. Pat. Nos. 5,739,313; 6,004,533; 6,096,290 and PCT
Publication WO 97/18231 listing Collins and Hogenkamp as inventors
disclose radionuclide labeling of vitamin B.sub.12 through the
propionamide moieties on naturally occurring vitamin B.sub.12. The
inventors converted the propionamide moieties at the b-, d-, and
e-positions of the corrole ring to monocarboxylic acids, through a
mild hydrolysis, and separated the carboxylic acids by column
chromatography. The inventors then attached a bifunctional linking
moiety to the carboxylate function through an amide linkage, and a
chelating agent to the linking moiety again through an amide
linkage. The chelating moiety was then used to attach a
radionuclide to the vitamin that can be used for therapeutic and/or
diagnostic purposes. See also PCT Publications WO 00/62808; WO
01/28595 and WO 01/28592.
[0055] PCT Publication WO 98/08859 listing Grissom et al as
inventors discloses conjugates containing a bioactive agent and an
organocobalt complex in which the bioactive agent is covalently
bound directly or indirectly, via a spacer, to the cobalt atom. The
organocobalt complex can be cobalamin and the bioactive agent can
be a chemotherapeutic agent. However, only one bioactive agent
(i.e., chemotherapeutic agent) is attached to the organocobalt
complex (i.e., cobalamin) and the attachment is solely through the
cobalt atom (i.e., the 6-position of cobalamin). The bioactive
agent is released from the bioconjugate by the cleavage of the weak
covalent bond between the bioactive agent and the cobalt atom as a
result of normal displacement by cellular nucleophiles or enzymatic
action, or by application of an external signal (e.g., light,
photoexcitation, ultrasound, or the presence of a magnetic
field).
[0056] U.S. Pat. Nos. 5,428,023; 5,589,463 and 5,807,823 to
Russell-Jones et al. discloses a vitamin B12 conjugate for
delivering oral hormone formulations. Russell-Jones teaches that
the vitamin B.sub.12 conjugate must be capable of binding in vivo
to intrinsic factor, enabling uptake and transport of the complex
from the intestinal lumen of a vertebrate host to the systemic
circulation of the host. The hormones are attached to the vitamin
B.sub.12 through a hydrolyzed propionamide linkage on the vitamin.
The patent states that the method is useful for orally
administering hormones, bioactive peptides, therapeutic agents,
antigens, and haptens, and lists as therapeutic agents neomycin,
salbutamol cloridine, pyrimethamine, penicillin G, methicillin,
carbenicillin, pethidine, xylazine, ketamine hydrochloride,
mephanesin and iron dextran. U.S. Pat. No. 5,548,064 to
Russell-Jones et al. discloses a vitamin B.sub.12 conjugate for
delivering erythropoietin and granulocyte-colony stimulating
factor, using the same approach as the '023 patent.
[0057] U.S. Pat. No. 5,449,720 to Russell-Jones et al discloses
vitamin B.sub.12 linked through a polymer to various active agents
wherein the conjugate is capable of binding to intrinsic factor for
systemic delivery. In particular, the document discloses the
attachment of various polymeric linkers to the propionamide
positions of the vitamin B12 molecule, and the attachment of
various bioactive agents to the polymeric linker. Exemplary
bioactive agents include hormones, bioactive peptides and
polypeptides, antitumor agents, antibiotics, antipyretics,
analgesics, antiinflammatories, and haemostatic agents. Exemplary
polymers include carbohydrates and branched chain amino acid
polymers. The linkers used in '720 are polymeric. Importantly, the
linkers are described as exhibiting a mixture of molecular weights,
due to the polymerization process by which they are made. See in
particular, page 11, lines 25-26 wherein it is stated that the
polymer used in that invention is of uncertain size and/or
structure.
[0058] PCT Publication WO 99/65930 to Russell-Jones et al.
discloses the attachment of various agents to the 5'--OH position
on the vitamin B.sub.12 ribose ring. The publication indicates that
the system can be used to attach polymers, nanoparticles,
therapeutic agents, proteins and peptides to the vitamin. See also,
U.S. Pat. Nos. 6,262,253 "Vitamin B12 conjugates with GCSF,
analogues thereof and pharmaceutical compositions;" 6,221,397
"Surface cross-linked particles suitable for controlled delivery;"
6,159,502 "Oral delivery systems for microparticles;" 6,150,341
"Vitamin B12 derivatives and methods for their preparation;"
5,869,466 "Vitamin B12 mediated oral delivery systems for GCSF;"
and 5,548,064 "Vitamin B.sub.12 conjugates with EPO, analogues
thereof and pharmaceutical compositions."
[0059] U.S. Pat. No. 5,574,018 to Habberfield et al. discloses
conjugates of vitamin B12 in which a therapeutically useful protein
is attached to the primary hydroxyl site of the ribose moiety. The
patent lists erythropoietin, granulocyte-colony stimulating factor
and human intrinsic factor as therapeutically useful proteins, and
indicates that the conjugates are particularly well adapted for
oral administration.
[0060] U.S. Pat. No. 5,840,880 to Morgan, Jr. et al. discloses
vitamin B.sub.12 conjugates to which are linked receptor modulating
agents, which affect receptor trafficking pathways that govern the
cellular uptake and metabolism of vitamin B.sub.12. The receptor
modulating agents are linked to the vitamin at the b-, d-, or
e-position.
[0061] Other patent filings which describe uses of Vitamin B.sub.12
include U.S. Pat. No. 3,936,440 to Nath (Method of Labeling Complex
Metal Chelates with Radioactive Metal Isotopes); U.S. Pat. No.
4,209,614 to Bernstein et al., (Vitamin B.sub.12 Derivatives
Suitable for Radiolabeling); U.S. Pat. No. 4,279,859 (Simultaneous
Radioassay of Folate and Vitamin B.sub.12); U.S. Pat. No. 4,283,342
to Yollees (Anticancer Agents and Methods of Manufacture); U.S.
Pat. No. 4,301,140 to Frank et al (Radiopharmaceutical Method for
Monitoring Kidneys); U.S. Pat. No. 4,465,775 to Houts (Vitamin
B.sub.12 and labeled Derivatives for Such Assay); U.S. Pat. No.
5,308,606 to Wilson et al (Method of Treating and/or Diagnosing
Soft Tissue Tumors); U.S. Pat. No. 5,405,839 (Vitamin B.sub.12
Derivative, Preparation Process Thereof, and Use Thereof); U.S.
Pat. No. 5,449,720 to Russell-Jones et al., (Amplification of the
Vitamin B.sub.12 Uptake System Using Polymers); U.S. Pat. No.
5,589,463 to Russell Jones (Oral Delivery of Biologically Active
Substances Bound to Vitamin B.sub.12); U.S. Pat. No. 5,608,060 to
Axworthy et al (Biotinidase-Resistant Biotin-DOTA Conjugates); U.S.
Pat. No. 5,807,832 to Russell-Jones et al (Oral Delivery of
Biologically Active Substances Bound to Vitamin B.sub.12); U.S.
Pat. No. 5,869,465 to Morgan et al (Method of Receptor Modulation
and Uses Therefor); U.S. Pat. No. 5,869,466 to Russell-Jones et al
(vitamin B.sub.12 Mediated Oral Delivery systems for GCSF).
[0062] See also Ruma Banerjee, Chemistry and Biochemistry of
B.sub.12 John Wiley & Sons, Inc. (1999), and in particular Part
II, Section 15 of that book, entitled "Diagnostics and Therapeutic
Analogues of Cobalamin," by H. P. C. Hogenkamp, Douglas A. Collins,
Charles B. Grissom, and Frederick G. West.
[0063] Despite the above findings, there remains a need for new
compounds, compositions and methods to administer therapeutic
and/or diagnostic agents that have improved specificity, i.e.,
which can localize the active agent efficiently in proliferating
cells in high concentration compared to normal cells.
[0064] It is therefore an object of the invention to provide new
compounds and compositions for the treatment, prophylaxis and/or
diagnosis of abnormal cellular proliferation.
[0065] It is another object of the present invention to provide new
methods, including surgical and medical methods, for the treatment,
prophylaxis and/or diagnosis of proliferative conditions.
SUMMARY OF THE INVENTION
[0066] It has been discovered that an agent for the treatment,
prophylaxis and/or diagnosis of a proliferative disorder is highly
and effectively absorbed into a site of unwanted proliferation by
direct or indirect attachment to a compound that binds to a
transport protein for vitamin B.sub.12, i.e. transcobalamin I, II
or III, or intrinsic factor, (the TC- or IF-binding carrier) in a
manner that ultimately allows binding to a transcobalamin receptor
(TR).
[0067] The TC-or IF-binding carrier and therapeutic and/or
diagnostic agent useful to treat and/or image sites of
proliferative disease in the body, such as cancerous tumors, can
optionally be joined by means of a di- or multi-valent linking
moiety. The linker used to join the TC- or IF-binding carrier and
the active agent preferably has a single molecular weight, and does
not exhibit a molecular weight distribution, for example as found
in most polymers. The linker can range in size from small to large
molecular weight, as long as there is not a distribution of weights
in the linker. It is important to strictly control the uniformity
of size of the conjugate for predictability of therapeutic
performance.
[0068] The linkers preferably have a molecular weight below about
2000, more preferably below about 1900 or 1800, and even more
preferably below about 1500 or 1000.
[0069] Thus, in one embodiment the invention provides a therapeutic
and/or diagnostic conjugate having a high specificity for
abnormally proliferative cells, comprising (1) a TC- or IF-binding
carrier, and (2) a therapeutic and/or diagnostic agent linked
directly or through a linker to the TC- or IF-binding carrier,
wherein the linker has either (i) a unimodal (i.e., single) and
defined molecular weight, or (ii) a molecular weight less than
about 2000, and preferably, below 1900, 1800, or 1500.
[0070] In one embodiment, the TC- or IF-binding carrier is any
moiety that will bind to a transcobalamin receptor, optionally when
complexed with the transport protein, and can be linked to a
therapeutic and/or diagnostic agent. Methods for the assessment of
whether a moiety binds the TC receptor are known, and include those
described by Pathare, et al., (1996) Bioconjugate Chem. 7, 217-232;
and Pathare, et al., Bioconjugate Chem. 8, 161-172. An assay that
assesses binding to a mixture of transcobalamin I and II receptors
is found in Chaiken, et al, Anal. Biochem. 201, 197 (1992). An
unsaturated vitamin B.sub.12 binding capacity (UBBC) assay to
assess the in vitro binding of the conjugate to the transcobalamin
transport proteins is described by D. A. Collins and H. P. C.
Hogenkamp in J. Nuclear Medicine, 1997, 38, 717-723. See also
Fairbanks, V. F. Mayo Clinical Proc. 83, Vol 58, 203-204.
[0071] In a particular embodiment of the present invention, the TC
binding carrier or IF binding carrier is represented by formula
(I). 1
[0072] or its enantiomer, diastereomer or its pharmaceutically
acceptable salt or prodrug, wherein:
[0073] (i) the wavy line in the chemical structure indicates either
a dative or covalent bond such that there are three dative Co-N
bonds and one covalent Co-N bond, wherein, in the case of the
dative bond, the valence of nitrogen is completed either with a
double bond with an adjacent ring carbon or with a hydrogen;
[0074] (ii) the dotted line in the chemical structure indicates
either a double or single bond such that the double bond does not
over-extend the valence of the element (i.e. to give pentavalent
carbons) and, in the case of a single bond, the valence is
completed with hydrogen; wherein, in a preferred embodiment, the
bonding and stereochemistry of the compound is the same as that of
vitamin B.sub.12 as it exists in nature;
[0075] (iii) X is hydrogen, cyano, halogen (Cl, F, Br or I),
haloalkyl (including CF.sub.3, CF.sub.2CF.sub.3, CH.sub.2CF.sub.3
and CF.sub.2Cl), NO, NO.sub.2, NO.sub.3, phosphonate (including
alkyl-P(O).sub.2OR.sup.15)- , PR.sup.15R.sup.16R.sup.17, NH.sub.2,
NR.sup.15R.sup.16, OH, OR.sup.15, SR.sup.15, SCN, N.sub.3,
OC(O)R.sup.15, C(O).sub.2R.sup.15, C(O)R.sup.15,
OC(O)N.sup.15R.sup.16, C(O).sub.2NR.sup.15R.sup.16,
C(O)NR.sup.15R.sup.16, P(O).sub.2OR.sup.15, S(O).sub.2OR.sup.15, a
purine or pyrimidine nucleoside or nucleoside analog, including
adenosyl (preferably linked through a 5'-deoxy linkage) and 5-FU,
alkyl, alkenyl, alkynyl, aryl, aralkyl, alkaryl, amino acid,
peptide, protein, carbohydrate, heteroalkyl, heterocycle,
heteroaryl or alkylheteroaryl (in one embodiment that is less
preferred, X is L-T);
[0076] (iv) M is a monovalent heterocycle or heteroaromatic, which
is capable of binding to the adjacent sugar ring, and forming a
dative bond with Co.sup.+3, and is preferably a benzimidazole, a 5-
and/or 6-substituted benzimidazole, such as
5,6-dimethylbenzimidazole, 5-methyl-benzimidazole,
5-hydroxy-benzimidazole, 5-methoxy-benzimidazole, naphth-imidazole,
5-hydroxy-6-methyl-benzimidazole or
5-methoxy-6-methyl-benz-imidazole; or a purine or pyrimidine
including but not limited to adenine, 2-methyladenine,
2-methylmercaptoadenine, e-methiylsulfinyladenine,
2-methyl-sulfonyladenine and guanine; or a phenol, such as phenol
or p-cresol;
[0077] (v) K is O, S, NJ.sup.1, C(OH)H, CR.sup.100R.sup.101 or
C(R.sup.100)V.sup.8Z.sup.8;
[0078] (vi) E is O or S;
[0079] (vii) G.sup.1 is hydrogen, alkyl, acyl, silyl, phosphate or
L-T;
[0080] (viii) Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4, Y.sup.5, y.sup.6
and y.sup.7 independently are O, S or NJ.sup.2;
[0081] (ix) V.sup.1, V.sup.2, V.sup.3, V.sup.4, V.sup.5, V.sup.6,
V.sup.7 and V.sup.8 independently are O, S, NJ.sup.3,
CR.sup.102R.sup.103 or a direct bond;
[0082] (x) Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.7 and
Z.sup.8 independently are R.sup.104 or L-T;
[0083] (xi) each L is independently a direct bond or a linker to
one or more T moieties, and that does not significantly impair the
ability of the TC- or IF-binding carrier to bind to a
transcobalamin receptor, optionally when bound to a transport
protein;
[0084] (xii) each T independently comprises the residue of a
therapeutic and/or diagnostic agent, optionally bound though a
chelating moiety if necessary or desired (in one embodiment, T is a
therapeutic and/or diagnostic agent for the treatment, prophylaxis
and/or diagnosis of a proliferative disorder other than
cancer);
[0085] (xiii) at least one of Z.sup.1, z.sup.2, z.sup.3, z.sup.4,
z.sup.5, z.sup.7, Z.sup.8, K and G.sup.1 is L-T (in a preferred
embodiment, z.sup.2 comprises the sole L-T in the TC- or IF-binding
carrier);
[0086] (xiv) J.sup.1, J.sup.2 and J.sup.3 independently are
hydrogen, alkyl, alkenyl, alkynyl, alkaryl, cycloalkyl, aryl,
cycloaryl, heteroalkyl, heterocycle, heteroaryl, hydroxyl, alkoxy
or amine;
[0087] (xv) R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13
and R.sup.14 independently are hydrogen, lower alkyl, lower
alkenyl, lower alkynyl, lower cycloalkyl, heteroalkyl,
heterocyclic, lower alkoxy, azido, amino, lower alkylamino,
halogen, thiol, SO.sub.2, SO.sub.3, carboxylic acid, C.sub.1-6
carboxyl, hydroxyl, nitro, cyano, oxime or hydrazine;
[0088] (xvi) R.sup.13 and R.sup.14 optionally can form a double
bond;
[0089] (xvii) R.sup.15, R.sup.16 and R.sup.17 are independently
hydrogen, alkyl, alkenyl, alkynyl, aryl, alkaryl or aralkyl group,
heteroalkyl, heterocycle or heteroaromatic; and
[0090] (xviii) R.sup.100, R.sup.101, R.sup.102, R.sup.103, and
R.sup.104 are independently hydrogen, alkyl, alkenyl, alkynyl,
aryl, acyl, heteroaromatic, heteroaryl, heteroalkyl, hydroxyl,
alkoxy, cyano, azido, halogen, nitro, SO.sub.2, SO.sub.3, thioalkyl
or amino.
[0091] In a preferred embodiment, L is a linker, and in particular
a polymer linker, of singular molecular weight.
[0092] In naturally occurring vitamin B.sub.12, there is an
.alpha.-D-5,6-dimethylbenzimidazolyl ribose 3'-phosphate that is
bound through the phosphate to the B.sub.12 moiety and coordinated
to the cobalt ion. In a modified vitamin B.sub.12 TC- or IF-binding
carrier, the M-sugar component is likewise in an a-D configuration,
although other configurations (i.e., .alpha.-L, .beta.-D and
.beta.-L) are possible.
[0093] One of the biologically active forms of vitamin B.sub.12 has
a 5'-deoxyadenosyl moiety in the X position. Vitamin B.sub.12
catalysis occurs via the detachment and reattachment of the
methylene radical at the 5'-deoxy position of the adenosyl moiety.
In one embodiment, the selected substituent in the X position is
capable of similar catalysis.
[0094] In one particular embodiment the linker used to attach the
TC- or IF-binding carrier and the therapeutic and/or diagnostic
agent is a polyamine such as spermine or spermidine.
[0095] In another embodiment X comprises the residue of
5'-deoxyadenosine.
[0096] In one embodiment, the TC- or IF-binding carrier comprises
one or more therapeutic and/or diagnostic agents at each of one or
more of the b-, d-, or e-cobalamin positions, linked directly or
through a linker, and preferably through the b-position.
[0097] In another embodiment the TC- or IF-binding carrier of the
present invention comprises one or more therapeutic and/or
diagnostic agents at M, K or G.sup.1.
[0098] The present invention also provides a method of imaging a
disorder characterized by abnormal cell proliferation in an animal,
preferably a human, comprising administering to the animal an
effective amount of a TC- or IF-binding carrier of the present
invention.
[0099] The invention also provides the use of a compound of the
present invention for the manufacture of a medicament for the
treatment, prophylaxis and/or diagnosis of a disorder characterized
by abnormal cellular proliferation in an animal (e.g., a
human).
[0100] In one embodiment, the compound of formula I can be
understood to exclude compounds (and therapeutic methods using such
compounds) in which:
[0101] (i) X is cyano, hydroxyl, methyl, adenosine or L-T;
[0102] (ii) M is the residue of 5,6-dimethylbenzimidazole;
[0103] (iii) E is O;
[0104] (iv) K is C(OH)H;
[0105] (v) G.sup.1 is hydrogen;
[0106] (vi) y.sup.1, y.sup.2, y.sup.3, y.sup.4, y.sup.5, y.sup.6
and y.sup.7 are O;
[0107] (vii) L is a direct bond or a multivalent linker derived
from a dicarboxylic acid (C(O)OH-alkylene-C(O)OH), a diamine
(NH.sub.2-alkylene-NH.sub.2), an amino-carboxylic acid
(C(O)OH-alkylene-NH.sub.2), an amino acid, a peptide or a polymer
of one or amino acids;
[0108] (viii) J.sup.1, J.sup.2 and J.sup.3 are all hydrogen;
[0109] (ix) all of R.sup.1, R.sup.2, R.sup.4, R.sup.5, R.sup.8,
R.sup.9, R.sup.11, R.sup.12 and R.sup.15 are methyl and all of
R.sup.3, R.sup.6, R.sup.7, R.sup.10, R.sup.13 and R.sup.14 are
hydrogen; and/or
[0110] (x) V.sup.1Z.sup.1, V.sup.3Z.sup.3, V.sup.6Z.sup.6 and
V.sup.7Z.sup.7 are amino.
[0111] The present invention includes at least the following:
[0112] (a) a TC- or IF-binding carrier of the present invention
linked directly or via a linker to one or more therapeutic and/or
diagnostic agent(s), or the pharmaceutically acceptable salt or
prodrug thereof, for the treatment, prophylaxis and/or diagnosis of
a proliferative disorder;
[0113] (b) a pharmaceutical composition for the treatment,
prophylaxis and/or diagnosis of a proliferative disorder comprising
an effective amount of a TC- or IF-binding carriers of the present
invention linked directly or via a linker to one or more
therapeutic and/or diagnostic agent(s), or the pharmaceutically
acceptable salt or prodrug thereof, in combination with a
pharmaceutically acceptable carrier of diluent;
[0114] (c) a pharmaceutical composition for the treatment,
prophylaxis and/or diagnosis of a proliferative disorder comprising
an effective amount of a TC- or IF-binding carrier of the present
invention linked directly or via a linker to one or more
therapeutic and/or diagnostic agent(s), or the pharmaceutically
acceptable salt or prodrug thereof, optionally with a
pharmaceutically acceptable carrier or diluent, in combination with
one or more other effective therapeutic and/or diagnostic agent(s),
or the pharmaceutically acceptable salt or prodrug thereof;
[0115] (d) a method for the treatment, prophylaxis and/or diagnosis
of a proliferative disorder in a host, and in particular a human,
comprising administering an effective amount of a TC- or IF-binding
carrier of the present invention linked directly or via a linker to
one or more therapeutic and/or diagnostic agent(s), or the
pharmaceutically acceptable salt or prodrug thereof, optionally
with a pharmaceutically acceptable carrier or diluent;
[0116] (e) a method for the treatment, prophylaxis and/or diagnosis
of a proliferative disorder in a host, and in particular a human,
comprising administering an effective amount of a TC- or IF-binding
carrier of the present invention linked directly or via a linker to
one or more therapeutic and/or diagnostic agent(s), or the
pharmaceutically acceptable salt or prodrug thereof, optionally
with a pharmaceutically acceptable carrier or diluent in
combination or alternation with one or more other effective
therapeutic and/or diagnostic agent(s), or its pharmaceutically
acceptable salt or prodrug thereof;
[0117] (f) use of an effective amount of a TC- or IF-binding
carrier of the present invention linked directly or via a linker to
one or more therapeutic and/or diagnostic agent(s), or the
pharmaceutically acceptable salt or prodrug thereof, optionally
with a pharmaceutically acceptable carrier or diluent, for the
treatment, prophylaxis and/or diagnosis of a proliferative disorder
in a host, and in particular a human;
[0118] (g) use of an effective amount of a TC- or IF-binding
carrier of the present invention linked directly or via a linker to
one or more therapeutic and/or diagnostic agent(s), or the
pharmaceutically acceptable salt or prodrug thereof, optionally
with a pharmaceutically acceptable carrier or diluent, in
combination or alternation with one or more other effective
therapeutic and/or diagnostic agent(s), or the pharmaceutically
acceptable salt or prodrug thereof, for the treatment, prophylaxis
and/or diagnosis of a proliferative disorder;
[0119] (h) use of an effective amount of a TC- or IF-binding
carrier of the present invention linked directly or via a linker to
one or more therapeutic and/or diagnostic agent(s), or the
pharmaceutically acceptable salt or prodrug thereof, optionally
with a pharmaceutically acceptable carrier or diluent, in the
manufacture of a medicament for the treatment, prophylaxis and/or
diagnosis of a proliferative disorder in a host, and in particular
a human; and
[0120] (i) use of an effective amount of a TC- or IF-binding
carrier of the present invention linked directly or via a linker to
one or more therapeutic and/or diagnostic agent(s), or the
pharmaceutically acceptable salt or prodrug thereof, optionally
with a pharmaceutically acceptable carrier or diluent, in
combination or alternation with one or more other effective
therapeutic and/or diagnostic agent(s), or the pharmaceutically
acceptable salt or prodrug thereof, in the manufacture of a
medicament for the treatment, prophylaxis and/or diagnosis of a
proliferative disorder.
[0121] In a preferred embodiment, a therapeutic and/or diagnostic
agent and the TC- or IF-binding carrier, or a pharmaceutically
acceptable salt or prodrug thereof, is delivered to the site of
unwanted proliferation in a manner that bypasses, or at least does
not rely on, the gastrointestinal route of absorption via the
vitamin B.sub.12 intrinsic factor binding protein. Preferred modes
of administration are parenteral, intraperitoneal, intravenous,
intradermal, epidural, intraspinal, intrasternal, intra-articular,
intra-synovial, intrathecal, intra-arterial, intracardiac,
intramuscular, intranasal, subcutaneous, intraorbital,
intracapsular, topical, transdermal patch, via rectal, vaginal or
urethral administration including via suppository, percutaneous,
nasal spray, surgical implant, internal surgical paint, infusion
pump, or via catheter. In one embodiment, the agent and carrier are
administered in a slow release formulation such as a direct tissue
injection or bolus, implant, microparticle, microsphere,
nanoparticle or nanosphere.
[0122] It is preferred that the TC- or IF-binding carrier and the
therapeutic and/or diagnostic agent be administered parenterally
and not orally to increase the effectiveness of the agent, for
example, in the case of imaging, to decrease the exposure of normal
cells to the diagnostic agent. It is known that the ileal receptor
for intrinsic factor-cobalamin complex is present in the
gastrointestinal tract in only very small quantities, and on oral
delivery of vitamin B.sub.12 into the alimentary system the ileal
receptor can only absorb approximately two micrograms per day of
vitamin B.sub.12 for systemic delivery. Even assuming a small
amount of systemic absorption via passive transport of a large oral
dose, this level of administration is insufficient for the
treatment, prophylaxis and/or diagnosis of a proliferative
disorder.
[0123] In an alternative embodiment, it has been discovered that an
agent for the treatment, prophylaxis and/or diagnosis of a
proliferative disorder can be highly and effectively absorbed into
a site of unwanted proliferation by direct or indirect attachment
to a compound that binds to the intrinsic factor (IF-binding
carrier), wherein the IF-binding carrier and therapeutic and/or
diagnostic agent are administered using any of the methods listed
above, for example, parenterally.
[0124] The TC- or IF-binding carrier and the therapeutic and/or
diagnostic agent, or a pharmaceutically acceptable salt or prodrug
thereof, can be administered in the course of surgical or medical
treatment, prophylaxis and/or diagnosis of the afflicted site. For
example, the TC- or IF-binding carrier and active agent can be
positioned directly at the site of proliferation during the course
of surgery either by painting the formulation (with or without a
controlled release matrix) onto the surface of the afflicted area
or by depositing a bolus of material in a suitable matrix that is
released into the afflicted area over time. In another embodiment,
the TC- or IF-binding carrier and the active agent are administered
directly into the proliferative mass via injection or catheter.
[0125] In another embodiment, the TC- or IF-binding carrier and the
therapeutic and/or diagnostic agent is combined with either
intrinsic factor or a transcobalamin carrier protein, or both, and
administered parenterally, for example, via intravenous,
intramuscular, direct injection or catheter, to the afflicted
location.
BRIEF DESCRIPTION OF THE FIGURES
[0126] FIG. 1 illustrates a compound of formula I, wherein X is CN,
OH, CH.sub.3, adenosyl or a residue of a peptide or amino acid. The
compound of formula I can be cyanocobalamin (X is CN),
hydroxocobalamin (X is OH), methylcobalamin (X is CH.sub.3),
adenosylcobalamin (X is adenosyl), or a cobalamin conjugate (X is a
residue of a peptide or amino acid).
[0127] FIG. 2 illustrates a synthesis of a compound wherein a
residue of a compound of formula I is linked to poly-L-lysine, 8
units to 11 units, linked to DTPA.
[0128] FIG. 3 illustrates a synthesis of representative compounds
of the invention (8, 9) wherein a residue of a compound of formula
I is linked to a non-metallic radionuclide (e.g., Fluorine-18).
[0129] FIG. 4 illustrates a synthesis of a compound of the present
invention (7) wherein a residue of a compound of formula I is
linked to a non-metallic radionuclide (e.g., Fluorine-18) through a
linker.
[0130] FIG. 5 illustrates a synthesis of a compound of the present
invention (10) wherein a residue of a compound of formula I is
linked to a non-metallic radionuclide (e.g., Fluorine-18) through a
linker.
[0131] FIG. 6 illustrates a synthesis of a compound of the present
invention (11) wherein a residue of a compound of formula I is
linked to a peptide residue that comprises a non-metallic
radionuclide (e.g., Fluorine-18).
[0132] FIG. 7 illustrates a synthesis of a compound wherein a
residue of a vitamin B.sub.12 analog is attached to a linker, which
is bound to a residue of a chemotherapeutic agent.
DETAILED DESCRIPTION OF THE INVENTION
[0133] It has been discovered that an agent for the treatment,
prophylaxis and/or diagnosis of a proliferative disorder is highly
and effectively absorbed into a site of unwanted proliferation by
direct or indirect attachment to a compound that binds to a
transport protein for vitamin B.sub.12, i.e. transcobalamin I, II
or III, or intrinsic factor, (the TC- or IF-binding carrier) in a
manner that ultimately allows binding to a transcobalamin receptor
(TR).
[0134] Therefore, the present invention includes at least the
following:
[0135] (a) a TC- or IF-binding carrier of the present invention
linked directly or via a linker to one or more therapeutic and/or
diagnostic agent(s), or the pharmaceutically acceptable salt or
prodrug thereof, for the treatment, prophylaxis and/or diagnosis of
a proliferative disorder;
[0136] (b) a pharmaceutical composition for the treatment,
prophylaxis and/or diagnosis of a proliferative disorder comprising
an effective amount of a TC- or IF-binding carriers of the present
invention linked directly or via a linker to one or more
therapeutic and/or diagnostic agent(s), or the pharmaceutically
acceptable salt or prodrug thereof, in combination with a
pharmaceutically acceptable carrier of diluent;
[0137] (c) a pharmaceutical composition for the treatment,
prophylaxis and/or diagnosis of a proliferative disorder comprising
an effective amount of a TC- or IF-binding carrier of the present
invention linked directly or via a linker to one or more
therapeutic and/or diagnostic agent(s), or the pharmaceutically
acceptable salt or prodrug thereof, optionally with a
pharmaceutically acceptable carrier or diluent, in combination with
one or more other effective therapeutic and/or diagnostic agent(s),
or the pharmaceutically acceptable salt or prodrug thereof;
[0138] (d) a method for the treatment, prophylaxis and/or diagnosis
of a proliferative disorder in a host, and in particular a human,
comprising administering an effective amount of a TC- or IF-binding
carrier of the present invention linked directly or via a linker to
one or more therapeutic and/or diagnostic agent(s), or the
pharmaceutically acceptable salt or prodrug thereof, optionally
with a pharmaceutically acceptable carrier or diluent;
[0139] (e) a method for the treatment, prophylaxis and/or diagnosis
of a proliferative disorder in a host, and in particular a human,
comprising administering an effective amount of a TC- or IF-binding
carrier of the present invention linked directly or via a linker to
one or more therapeutic and/or diagnostic agent(s), or the
pharmaceutically acceptable salt or prodrug thereof, optionally
with a pharmaceutically acceptable carrier or diluent in
combination or alternation with one or more other effective
therapeutic and/or diagnostic agent(s), or its pharmaceutically
acceptable salt or prodrug thereof;
[0140] (f) use of an effective amount of a TC- or IF-binding
carrier of the present invention linked directly or via a linker to
one or more therapeutic and/or diagnostic agent(s), or the
pharmaceutically acceptable salt or prodrug thereof, optionally
with a pharmaceutically acceptable carrier or diluent, for the
treatment, prophylaxis and/or diagnosis of a proliferative disorder
in a host, and in particular a human;
[0141] (g) use of an effective amount of a TC- or IF-binding
carrier of the present invention linked directly or via a linker to
one or more therapeutic and/or diagnostic agent(s), or the
pharmaceutically acceptable salt or prodrug thereof, optionally
with a pharmaceutically acceptable carrier or diluent, in
combination or alternation with one or more other effective
therapeutic and/or diagnostic agent(s), or the pharmaceutically
acceptable salt or prodrug thereof, for the treatment, prophylaxis
and/or diagnosis of a proliferative disorder;
[0142] (h) use of an effective amount of a TC- or IF-binding
carrier of the present invention linked directly or via a linker to
one or more therapeutic and/or diagnostic agent(s), or the
pharmaceutically acceptable salt or prodrug thereof, optionally
with a pharmaceutically acceptable carrier or diluent, in the
manufacture of a medicament for the treatment, prophylaxis and/or
diagnosis of a proliferative disorder in a host, and in particular
a human; and
[0143] (i) use of an effective amount of a TC- or IF-binding
carrier of the present invention linked directly or via a linker to
one or more therapeutic and/or diagnostic agent(s), or the
pharmaceutically acceptable salt or prodrug thereof, optionally
with a pharmaceutically acceptable carrier or diluent, in
combination or alternation with one or more other effective
therapeutic and/or diagnostic agent(s), or the pharmaceutically
acceptable salt or prodrug thereof, in the manufacture of a
medicament for the treatment, prophylaxis and/or diagnosis of a
proliferative disorder.
[0144] The TC-or IF-binding carrier and therapeutic and/or
diagnostic agent useful to treat and/or image sites of
proliferative disease in the body, such as cancerous tumors, can
optionally be joined by means of a di- or multi-valent linking
moiety. The linker used to join the TC- or IF-binding carrier and
the active agent preferably has a single molecular weight, and does
not exhibit a molecular weight distribution, for example as found
in most polymers. The linker can range in size from small to large
molecular weight, as long as there is not a distribution of weights
in the linker. It is important to strictly control the uniformity
of size of the conjugate for predictability of therapeutic
performance.
[0145] The linkers preferably have a molecular weight below about
2000, more preferably below about 1900 or 1800, and even more
preferably below about 1500 or 1000.
[0146] Thus, in one embodiment the invention provides a non-oral or
oral pharmaceutical formulation comprising a therapeutic and/or
diagnostic conjugate having a high specificity for abnormally
proliferative cells, comprising (1) a transcobalamin (TC) or
intrinsic factor (IF) binding carrier, and (2) a therapeutic and/or
diagnostic agent linked directly or through a linker to the TC- or
IF-binding carrier.
[0147] In a particular embodiment the invention provides a
therapeutic and/or diagnostic conjugate having a high specificity
for abnormally proliferative cells, comprising (1) a transcobalamin
(TC) or intrinsic factor (IF) binding carrier, and (2) a
therapeutic and/or diagnostic agent linked directly or through a
linker to the TC- or IF-binding carrier, wherein the linker has
either (i) a unimodal (i.e., single) and defined molecular weight,
or (ii) a molecular weight less than about 2000, and preferably,
below 1900, 1800 or 1500.
[0148] I. Definitions
[0149] The following definitions and term construction are
intended, unless otherwise indicated:
[0150] Specific and preferred values listed below for radicals,
substituents and ranges, are for illustration only; they do not
exclude other defined values or other values within defined ranges
for the radicals and substituents.
[0151] Halo is fluoro, chloro, bromo or iodo.
[0152] Alkyl, alkoxy, alkenyl, alkynyl, etc. denote both straight
and branched groups; but reference to an individual radical such as
"propyl" embraces only the straight chain radical, a branched chain
isomer such as "isopropyl" being specifically referred to.
[0153] The term heterocycle or heterocyclic, as used herein except
where noted represents a stable 5- to 7-membered monocyclic or
stable 8- to 11-membered bicyclic heterocyclic ring which is either
saturated or unsaturated, and which consists of carbon atoms and
from one to three heteroatoms selected from the group consisting of
N, O and S; and wherein the nitrogen and sulfur heteroatoms may
optionally be oxidized, and the nitrogen heteroatom may optionally
be quatemized, and including any bicyclic group in which any of the
above-defined heterocyclic rings is fused to a benzene ring. The
heterocyclic ring may be attached at any heteroatom or carbon atom
that results in the creation of a stable structure.
[0154] The term alkyl, as used herein, unless otherwise specified,
refers to a saturated straight, branched, or cyclic, primary,
secondary, or tertiary hydrocarbon of C.sub.1 to C.sub.10, and
specifically includes methyl, ethyl, propyl, isopropyl,
cyclopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl,
isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl,
cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, and
2,3-dimethylbutyl. The term includes both substituted and
unsubstituted alkyl groups. Moieties with which the alkyl group can
be substituted are selected from the group consisting of hydroxyl,
amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano,
sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate,
either unprotected, or protected as necessary, as known to those
skilled in the art, for example, as taught in Greene, et al.,
Protective Groups in Organic Synthesis, John Wiley and Sons, Second
Edition, 1991, hereby incorporated by reference.
[0155] The term lower alkyl, as used herein, and unless otherwise
specified, refers to a C.sub.1 to C.sub.4 saturated straight,
branched, or if appropriate, a cyclic (for example, cyclopropyl)
alkyl group, including both substituted and unsubstituted forms.
Unless otherwise specifically stated in this application, when
alkyl is a suitable moiety, lower alkyl is preferred. Similarly,
when alkyl or lower alkyl is a suitable moiety, unsubstituted alkyl
or lower alkyl is preferred.
[0156] The terms "alkenyl" and "alkynyl" refer to alkyl moieties
wherein at least one saturated C--C bond is replaced by a double or
triple bond. Thus, (C.sub.2-C.sub.6)alkenyl can be vinyl, allyl,
1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl,
1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1- hexenyl,
2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl. Similarly,
(C.sub.2-C.sub.6)alkynyl can be ethynyl, 1-propynyl, 2-propynyl,
1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl,
3-pentynyl, 4-pentynyl, 1- hexynyl, 2-hexynyl, 3-hexynyl,
4-hexynyl, or 5-hexynyl.
[0157] The term "alkylene" refers to a saturated, straight chain,
divalent alkyl radical of the formula --(CH.sub.2).sub.n--, wherein
n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[0158] As used herein, with exceptions as noted, "aryl" is intended
to mean any stable monocyclic, bicyclic or tricyclic carbon ring of
up to 8 members in each ring, wherein at least one ring is aromatic
as defined by the Huckel 4n+2 rule. Examples of aryl ring systems
include phenyl, naphthyl, tetrahydronaphthyl and biphenyl. The aryl
group can be substituted with one or more moieties selected from
the group consisting of hydroxyl, amino, alkylamino, arylamino,
alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic
acid, phosphate, or phosphonate, either unprotected, or protected
as necessary, as known to those skilled in the art, for example, as
taught in Greene, et al., Protective Groups in Organic Synthesis,
John Wiley and Sons, Second Edition, 1991.
[0159] The term purine or pyrimidine base includes, but is not
limited to, adenine, N.sup.6-alkylpurines, N.sup.6-acylpurines
(wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl),
N.sup.6-benzylpurine, N.sup.6-halopurine, N.sup.6-vinylpurine,
N.sup.6-acetylenic purine, N.sup.6-acyl purine, N 6-hydroxyalkyl
purine, N.sup.6-thioalkyl purine, N.sup.2-alkylpurines,
N.sup.2-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine,
5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2-
and/or 4-mercapto-pyrimidine, uracil, 5-halouracil, including
5-fluorouracil, C.sup.5-alkylpyrimidines,
C.sup.5-benzylpyrimidines, C.sup.5-halopyrimidines,
C.sup.5-vinylpyrimidine, C.sup.5-acetylenic pyrimidine,
C.sup.5-acyl pyrimidine, C.sup.5-hydroxyalkyl purine,
C.sup.5-amidopyrimidine, C.sup.5-cyanopyrimidine,
C.sup.5-nitropyrimidine, C.sup.5-aminopyrimidine- ,
N.sup.2-alkylpurines, N.sup.2-alkyl-6-thiopurines, 5-azacytidinyl,
5-azauracilyl, triazolopyridinyl, imidazolopyridinyl,
pyrrolopyrimidinyl, and pyrazolopyrimidinyl. Purine bases include,
but are not limited to, guanine, adenine, hypoxanthine,
2,6-diamino-purine and 6-chloropurine. Functional oxygen and
nitrogen groups on the base can be protected as necessary or
desired. Suitable protecting groups are well known to those skilled
in the art, and include trimethylsilyl, dimethylhexylsilyl,
t-butyldimethylsilyl and t-butyldiphenylsilyl, trityl, alkyl
groups, and acyl groups such as acetyl and propionyl,
methanesulfonyl, and p-toluenesulfonyl.
[0160] The term heteroalkyl refers to an alkyl group that contains
a heteroatom in the alkyl chain, including O, S, N, or P, and
wherein the heteroatom can be attached to other substituents
(including R.sup.15) to complete the valence. Nonlimiting examples
of heteroalkyl moieties include polyoxyalkylene, and when divalent,
--(CH.sub.2O).sub.n-- wherein n is an integer of from 0 to 20.
[0161] The term acyl refers to a carboxylic acid ester in which the
non-carbonyl moiety of the ester group is selected from straight,
branched, or cyclic alkyl or lower alkyl, alkoxyalkyl including
methoxymethyl, aralkyl including benzyl, aryloxyalkyl such as
phenoxymethyl, aryl including phenyl optionally substituted with
halogen, C.sub.1 to C.sub.4 alkyl or C.sub.1 to C.sub.4 alkoxy,
sulfonate esters such as alkyl or aralkyl sulphonyl including
methanesulfonyl, the mono, di or triphosphate ester, trityl or
monomethoxytrityl, substituted benzyl, trialkylsilyl (e.g.
dimethyl-t-butylsilyl) or diphenylmethylsilyl. Aryl groups in the
esters optimally comprise a phenyl group. The term "lower acyl"
refers to an acyl group in which the non-carbonyl moiety is lower
alkyl.
[0162] The term heteroaryl or heteroaromatic, as used herein,
refers to an aromatic moiety that includes at least one sulfur,
oxygen, nitrogen or phosphorus in the aromatic ring. The term
heterocyclic refers to a nonaromatic cyclic group wherein there is
at least one heteroatom, such as oxygen, sulfur, nitrogen or
phosphorus in the ring. Nonlimiting examples of heteroaryl and
heterocyclic groups include furyl, firanyl, pyridyl, pyrimidyl,
thienyl, isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl,
benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, benzothienyl,
isobenzofuryl, pyrazolyl, indolyl, isoindolyl, benzimidazolyl,
purinyl, carbazolyl, oxazolyl, thiazolyl, isothiazolyl,
1,2,4-thiadiazolyl, isooxazolyl, pyrrolyl, quinazolinyl,
cinnolinyl, phthalazinyl, xanthinyl, hypoxanthinyl, thiophene,
furan, pyrrole, isopyrrole, pyrazole, imidazole, 1,2,3-triazole,
1,2,4-triazole, oxazole, isoxazole, thiazole, isothiazole,
pyrimidine or pyridazine, and pteridinyl, aziridines, thiazole,
isothiazole, 1,2,3-oxadiazole, thiazine, pyridine, pyrazine,
piperazine, pyrrolidine, oxaziranes, phenazine, phenothiazine,
morpholinyl, pyrazolyl, pyridazinyl, pyrazinyl, quinoxalinyl,
xanthinyl, hypoxanthinyl, pteridinyl, 5-azacytidinyl,
5-aza-uracilyl, triazolopyridinyl, imidazolopyridinyl,
pyrrolopyrimidinyl, pyrazolopyrimidinyl, adenine, N6-alkylpurines,
N6-benzylpurine, N6-halopurine, N6-vinypurine, N6-acetylenic
purine, N6-acyl purine, N6-hydroxyalkyl purine, N6-thioalkyl
purine, thymine, cytosine, 6-azapyrimidine, 2-mercaptopyrmidine,
uracil, N5-alkyl-pyrimidines, N5-benzyl-pyrimidines,
N5-halopyrimidines, N5-vinyl-pyrimidine, N5-acetylenic pyrimidine,
N5-acyl pyrimidine, N5-hydroxyalkyl purine, and N6-thioalkyl
purine, and isoxazolyl. The heteroaromatic and heterocyclic
moieties can be optionally substituted as described above for aryl,
including substituted with one or more substituent selected from
halogen, haloalkyl, alkyl, alkoxy, hydroxy, carboxyl derivatives,
amido, amino, alkylamino, dialkylamino. The heteroaromatic can be
partially or totally hydrogenated as desired. As a nonlimiting
example, dihydropyridine can be used in place of pyridine.
Functional oxygen and nitrogen groups on the heteroaryl group can
be protected as necessary or desired. Suitable protecting groups
are well known to those skilled in the art, and include
trimethylsilyl, dimethylhexylsilyl, t-butyldi-methylsilyl, and
t-butyldiphenylsilyl, trityl or substituted trityl, alkyl groups,
acyl groups such as acetyl and propionyl, methanesulfonyl, and
p-toluenesulfonyl.
[0163] The term aralkyl, as used herein, and unless otherwise
specified, refers to an aryl group as defined above linked to the
molecule through an alkyl group as defined above. The term alkaryl,
as used herein, and unless otherwise specified, refers to an alkyl
group as defined above linked to the molecule through an aryl group
as defined above.
[0164] The term alkoxy, as used herein, and unless otherwise
specified, refers to a moiety of the structure --O-alkyl, wherein
alkyl is as defined above.
[0165] The term amino, as used herein, refers to a moiety
represented by the structure --NR.sub.2, and includes primary
amines, and secondary, and tertiary amines substituted by alkyl
(i.e. alkylamino). Thus, R.sub.2 may represent two hydrogens, two
alkyl moieties, or one hydrogen and one alkyl moiety.
[0166] The term amido, as used herein, refers to a moiety
represented by the structure --C(O)NR.sub.2, wherein R.sub.2 is as
defined for amino.
[0167] As used herein, an "amino acid" is a natural amino acid
residue (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl,
Hyp, Ile, Leu Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D
or L form, or an unnatural amino acid (e.g. phosphoserine;
pbosphothreonine; phosphotyrosine; gamma-carboxyglutamate; hippuric
acid; octahydroindole-2-carboxylic acid; statine;
1,2,3,4,-tetrahydroisoquinoli- ne-3-carboxylic acid; penicillamine;
omithine; citrulline; a-methyl-alanine; para-benzoylphenylalanine;
pbenylglycine; propargyl-glycine; sarcosine; and tert-butylglycine)
residue having one or more open valences. Other unnatural amino
acids include those represented by the formula NH.sub.2
(CH.sub.2).sub.y COOH, wherein y=2-20, and preferably 2-12, and
include the aminoalkanoic acids such as .epsilon.-amino caproic
acid (H.sub.2N--(CH.sub.2).sub.5--COOH).
[0168] The term also comprises natural and unnatural amino acids
bearing amino protecting groups such as acetyl, acyl,
trifluoroacetyl, and benzyloxycarbonyl), as well as natural and
unnatural amino acids protected at carboxy with protecting groups
such as a C.sub.1-C.sub.6 alkyl, phenyl or benzyl ester and amide.
Other suitable amino and carboxy protecting groups are known to
those skilled in the art. See for example, T. W. Greene, Protecting
Groups in Organic Synthesis; Wiley: New York, 1981; D. Voet,
Biochemistry, Wiley: New York, 1990; L. Stryer, Biochemistry,
(3.sup.rd Ed), W. H. Freeman and Co.: New York, 1975; J. March,
Advanced Organic Chemistry, Reactions, Mechanisms and Structure,
(2nd Ed.), McGraw Hill: New York, 1977; F. Carey and R. Sundberg,
Advanced Organic Chemistry, Part B: Reactions and Synthesis,
(.sub.2nd Ed.), Plenum: New York, 1977; and references cited
therein.
[0169] As used herein, a "peptide" is a sequence of 2 to 25 amino
acids (e.g. as defined hereinabove) or peptidic residues having one
or more open valences. The sequence may be linear or cyclic. For
example, a cyclic peptide can be prepared or may result from the
formation of disulfide bridges between two cysteine residues in a
sequence.
[0170] The term "residue" is used throughout the specification to
describe any pharmaceutically acceptable form of a therapeutic
and/or diagnostic agent, which, upon administration to a patient,
does not inhibit the action of the active agent. As a non-limiting
example, a pharmaceutically acceptable residue of an active agent
is one that is modified to facilitate binding to the TC- or
IF-binding agent, covalently, ionically or through a chelating
agent, such that the modification does not inhibit the biological
action of the active agent, in that it does not inhibit the drugs
ability to modulate abnormal cellular proliferation. In a preferred
embodiment, the residue refers to the active agent with an open
valence state such that covalent bonding to the compound is
possible. This open valence state can be achieved by any means
known in the art, including the methodology described herein. In a
preferred embodiment, the open valence state is achieved through
the removal of an atom, such as hydrogen, to activate a functional
group.
[0171] As used herein, the term "substantially free of" or
"substantially in the absence of" refers to a composition that
includes at least 85 or 90% by weight, preferably 95% to 98% by
weight, and even more preferably 99% to 100% by weight, of the
designated enantiomer of that TC- or IF-binding agent. In a
preferred embodiment, in the methods and compounds of this
invention, the compounds are substantially free their
enantiomers.
[0172] Similarly, the term "isolated" refers to a composition that
includes at least 85 or 90% by weight, preferably 95% to 98% by
weight, and even more preferably 99% to 100% by weight, of the TC-
or IF-binding agent, the remainder comprising other chemical
species, including diastereomers or enantiomers.
[0173] The term "independently" is used herein to indicate that the
variable that is independently applied varies independently from
application to application. Thus, in a compound such as R"YR",
wherein R" is "independently carbon or nitrogen," both R" can be
carbon, both R" can be nitrogen, or one R" can be carbon and the
other R" nitrogen.
[0174] The term "host," as used herein, refers to a multicellular
organism in which proliferative disorders can occur, including
animals, and preferably a human. Alternatively, the host is any
abnormally proliferating cell, whose replication or function can be
altered by the compounds of the present invention. The term host
specifically refers to any cell line that abnormally proliferates,
either from natural or unnatural causes (for example, from genetic
mutation or genetic engineering, respectively), and animals, in
particular, primates (including chimpanzees) and humans. In most
animal applications of the present invention, the host is a human
patient. Veterinary applications, in certain indications, however,
are clearly anticipated by the present invention (such as bovine
viral diarrhea virus in cattle, hog cholera virus in pigs, and
border disease virus in sheep).
[0175] II. TC- or IF-Binding Carrier
[0176] In one embodiment, the TC- or IF-binding carrier is any
ligand that will bind effectively to a vitamin B.sub.12 transport
protein (i.e. transcobalamin I, II or III or intrinsic factor) and
which when appropriately linked to a therapeutic and/or diagnostic
agent and bound to a transport protein, will fit into a
transcobalamin receptor. Suitable carriers may be ascertained using
any one of several means known in the art, including competitive
binding assays with the receptor modulating agent competing with
native vitamin B.sub.12. Methods for the assessment of whether a
moiety binds the TC receptor are known, and include those described
by Pathare et al., Bioconjugate Chem. 1996, 7, 217-232; and
Pathare, et al., Bioconjugate Chem. 8, 161-172. An assay that
assesses binding to a mixture of transcobalamin I and II receptors
is found in Chaiken, et al, Anal. Biochem. 1992, 201, 197. An
unsaturated Vitamin B.sub.12 binding capacity (UBBC) assay to
assess the in vitro binding of the conjugate to the transcobalamin
proteins is described by D. A. Collins and H. P. C. Hogenkamp in J.
Nuclear Medicine, 1997, 38, 717-723. See also Fairbanks, V. F. Mayo
Clinical Proc. 83, Vol 58, 203-204. See also Fairbanks, V. F. Mayo
Clinical Proc. 83, Vol 58, 203-204. The TC- or IF-binding carrier
preferably displays a binding affinity of at least 50% of the
binding affinity displayed by vitamin B.sub.12, more preferably at
least 75% and even more preferably at least 90%.
[0177] In one embodiment, the therapeutic and/or diagnostic agent
is bound directly or indirectly through an amide residue at the
b-position, as illustrated in FIG. 1.
[0178] In another embodiment, the TC- or IF-binding carrier of the
present invention is represented by formula (I), 2
[0179] or its enantiomer, diastereomer or its pharmaceutically
acceptable salt or prodrug, wherein:
[0180] (i) the wavy line in the chemical structure indicates either
a dative or covalent bond such that there are three dative Co-N
bonds and one covalent Co-N bond, wherein, in the case of the
dative bond, the valence of nitrogen is completed either with a
double bond with an adjacent ring carbon or with a hydrogen;
[0181] (ii) the dotted line in the chemical structure indicates
either a double or single bond such that the double bond does not
over-extend the valence of the element (i.e. to give pentavalent
carbons) and, in the case of a single bond, the valence is
completed with hydrogen; wherein, in a preferred embodiment, the
bonding and stereochemistry of the compound is the same as that of
vitamin B.sub.12 as it exists in nature;
[0182] (iii) X is hydrogen, cyano, halogen (Cl, F, Br or I),
haloalkyl (including CF.sub.3, CF.sub.2CF.sub.3, CH.sub.2CF.sub.3
and CF.sub.2Cl), NO, NO.sub.2, NO.sub.3, phosphonate (including
alkyl-P(O).sub.2OR), PR.sup.13R.sup.16R.sup.17, NH.sub.2,
NR.sup.15R.sup.16, OH, OR.sup.15, SR.sup.15, SCN, N.sub.3,
OC(O)R.sup.15, C(O).sub.2R.sup.15, C(O)R.sup.15,
OC(O)NR.sup.15R.sup.16, C(O).sub.2NR.sup.15R.sup.16,
C(O)NR.sup.15R.sup.16, P(O).sub.2OR.sup.15, S(O).sub.2OR.sup.15, a
purine or pyrimidine nucleoside or nucleoside analog, including
adenosyl (preferably linked through a 5'-deoxy linkage) and 5-FU,
alkyl, alkenyl, alkynyl, aryl, aralkyl, alkaryl, amino acid,
peptide, protein, carbohydrate, heteroalkyl, heterocycle,
heteroaryl or alkylheteroaryl (in one embodiment that is less
preferred, X is L-T);
[0183] (iv) M is a monovalent heterocycle or heteroaromatic, which
is capable of binding to the adjacent sugar ring, and forming a
dative bond with Co.sup.+3, and is preferably a benzimidazole, a 5-
and/or 6- substituted benzimidazole, such as
5,6-dimethylbenzimidazole, 5-methyl-benzimidazole,
5-hydroxy-benzimidazole, 5-methoxy-benzimidazole, naphth-imidazole,
5-hydroxy-6-methyl-benzimidazole or
5-methoxy-6-methyl-benz-imidazole; or a purine or pyrimidine
including but not limited to adenine, 2-methyladenine,
2-methylmercaptoadenine, e-methylsulfinyladenine,
2-methyl-sulfonyladenine and guanine; or a phenol, such as phenol
or p-cresol;
[0184] (v) K is O, S, NJ.sup.1, C(OH)H, CR.sup.100R.sup.101 or
C(R.sup.100)V.sup.8Z.sup.8;
[0185] (vi) E is O or S;
[0186] (vii) G.sup.1 is hydrogen, alkyl, acyl, silyl, phosphate or
L-T;
[0187] (viii) Y.sup.1, y.sup.2, y.sup.3, y.sup.4, y.sup.5, y.sup.6
and Y.sup.7 independently are O, S or NJ.sup.2;
[0188] (ix) V.sup.1, V.sup.2, V.sup.3, V.sup.4, V.sup.5, V.sup.6,
V.sup.7 and V.sup.8 independently are O, S, NJ.sup.3,
CR.sup.102R.sup.103 or a direct bond;
[0189] (x) Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.7 and
Z.sup.8 independently are R.sup.104 or L-T;
[0190] (xi) each L is independently a direct bond or a linker to
one or more T moieties, and that does not significantly impair the
ability of the TC- or IF-binding carrier to bind to a
transcobalamin receptor, optionally when bound to a transport
protein;
[0191] (xii) each T independently comprises the residue of a
therapeutic and/or diagnostic agent, optionally bound though a
chelating moiety if necessary or desired (in one embodiment, T is a
therapeutic and/or diagnostic agent for the treatment, prophylaxis
and/or diagnosis of a proliferative disorder other than
cancer);
[0192] (xiii) at least one of Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4,
Z.sup.5, Z.sup.7, Z.sup.8, K and G.sup.1 is L-T (in a preferred
embodiment, Z.sup.2 comprises the sole L-T in the TC- or IF-binding
carrier); (xiv) J.sup.1, J.sup.2 and J.sup.3 independently are
hydrogen, alkyl, alkenyl, alkynyl, alkaryl, cycloalkyl, aryl,
cycloaryl, heteroalkyl, heterocycle, heteroaryl, hydroxyl, alkoxy
or amine;
[0193] (xv) R.sup.1, R.sup.2, R R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13
and R.sup.14 independently are hydrogen, lower alkyl, lower
alkenyl, lower alkynyl, lower cycloalkyl, heteroalkyl,
heterocyclic, lower alkoxy, azido, amino, lower alkylamino,
halogen, thiol, SO.sub.2, SO.sub.3, carboxylic acid, C.sub.1-6
carboxyl, hydroxyl, nitro, cyano, oxime or hydrazine;
[0194] (xvi) R.sup.13 and R.sup.14 optionally can form a double
bond;
[0195] (xvii) R.sup.15, R.sup.16 and R.sup.17 are independently
hydrogen, alkyl, alkenyl, alkynyl, aryl, alkaryl or aralkyl group,
heteroalkyl, heterocycle or heteroaromatic; and (xviii) R.sup.100,
R.sup.101, R.sup.102, R.sup.103, and R.sup.104 are independently
hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, heteroaromatic,
heteroaryl, heteroalkyl, hydroxyl, alkoxy, cyano, azido, halogen,
nitro, SO.sub.2, SO.sub.3, thioalkyl or amino.
[0196] In a preferred embodiment, L is a linker, and in particular
a polymer linker, of singular molecular weight.
[0197] In naturally occurring vitamin B12, there is an
.alpha.-D-5,6-dimethylbenzimidazolyl ribose 3'-phosphate that is
bound through the phosphate to the B12 moiety and coordinated to
the cobalt ion. In a modified vitamin B.sub.2 TC- or IF-binding
carrier, the M-sugar component is likewise in an .alpha.-D
configuration, although other configurations (i.e., .alpha.-L,
.beta.-D and .beta.-L) are possible.
[0198] One of the biologically active forms of vitamin B.sub.12 has
a 5'-deoxyadenosyl moiety in the X position. Coenzyme B.sub.12
catalysis occurs via the detachment and reattachment of the
methylene radical at the 5'-deoxy position of the vitamin.
[0199] In one particular embodiment the linker used to conjugate
the TC- or IF-binding carrier and the therapeutic and/or diagnostic
agent is a polyamine such as spermine or spermidine.
[0200] Because vitamin B.sub.12 is preferentially taken up by
abnormally proliferating cells, the TC- or IF-binding
carrier/active agent of the present invention provides a delivery
system capable of targeting abnormally proliferative cells, and
selectively treating and/or imaging a greater proportion of such
cells in relation to healthy cells. A wide range of analogs and
derivatives are capable of attaining these properties, as reflected
by the above referenced chemical structure and variables.
[0201] The TC- or IF-binding carrier can be modified in any manner
that does not interfere with its fundamental ability to bind a
transcobalamin transport protein, and thereafter bind the TC
receptor. In one embodiment, however, each variable on the vitamin
B.sub.12 structure independently either (i) retains its natural
vitamin B.sub.12 structure, (ii) imparts imaging and/or
anti-proliferative properties to the cobalamin conjugate, (iii)
renders the cobalamin conjugate more water soluble, or more stable,
(iv) increases the bioavailability of the carrier; (v) increases or
at least does not decrease the binding affinity of the carrier for
the TC-binding or IF-binding protein over vitamin B.sub.12; or (vi)
imparts another characteristic that is desired for pharmaceutical
or diagnostic performance.
[0202] The therapeutic and/or diagnostic agent can be linked to the
TC-binding or IF-binding moiety through a number of positions,
including any of the V-Z moieties, the X moiety, the M moiety, the
K moiety and/or the G.sup.1 moiety, though as mentioned above at
least one of Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.7,
Z.sup.8, M, K and G.sup.1 moieties comprises a therapeutic and/or
diagnostic agent. In one embodiment a therapeutic and/or diagnostic
agent is linked to the TC- or IF-binding carrier through Z.sup.2,
Z.sup.4, and/or Z.sup.5 (i.e. one or more of Z.sup.2, Z.sup.4, and
Z.sup.5 is L-T, and T is a therapeutic and/or diagnostic agent). In
a more particular embodiment a therapeutic and/or diagnostic agent
is linked to the TC- or IF-binding carrier through the Z.sup.2
moiety (i.e. Z.sup.2 is L-T, and T is a therapeutic and/or
diagnostic agent). In each of the foregoing embodiments, the Z
moiety or moieties not containing a therapeutic and/or diagnostic
agent preferably retain its natural vitamin B.sub.12 configuration,
in which VZ is NH.sup.2. Alternatively, the Z moieties not
containing a therapeutic and/or diagnostic agent may comprise a
secondary or tertiary amino analog of NH.sup.2 substituted by one
or two of J.sup.1.
[0203] In any Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.6,
Z.sup.7, Z.sup.8, X, M, K, or G.sup.1 moieties through which a
therapeutic and/or diagnostic agent is linked, it will be
understood that such moiety may comprise more than one therapeutic
and/or diagnostic agent, or a combination of therapeutic and/or
diagnostic agents, i.e., each T can independently comprise the
residue of one or more therapeutic and/or diagnostic agents bound
to L through one or more chelating moieties. More specifically, in
a series of embodiments, each T can comprise 1, 2, 3, 4, 5, 6, 7,
8, 9 or 10 therapeutic and/or diagnostic agents bound through one
or more chelating moieties.
[0204] R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12, and
R.sup.13 independently represent moieties that do not interfere
with binding between the compound and the transcobalamin transport
protein or receptor. Vitamin B.sub.12 can be modified through these
moieties to modulate physical properties of the molecule, such as
water solubility, stability or .lambda..sub.max. Preferred groups
for enhancing water solubility include heteroalkyl, amino,
C.sub.1-6 alkylamino, C.sub.1-6 alcohol, C.sub.1-6 carboxylic acid
and SO.sub.3.sup.-.
[0205] In another embodiment, one, some or all of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.10, R.sup.11, R.sup.12, and R.sup.13 independently assume
their natural roles in vitamin B12. Thus, one, some, or all of
R.sup.1, R.sup.2, R.sup.4, R.sup.5, R.sup.8, R.sup.9, R.sup.11,
R.sup.12 and R.sup.15 are independently methyl in one embodiment,
and one, some, or all of R.sup.3, R.sup.6, R.sup.7, R.sup.10,
R.sup.13 and R.sup.14 are independently hydrogen.
[0206] In another embodiment, one, some, or all of Y.sup.1,
y.sup.2, Y.sup.3, Y.sup.4, Y.sup.3, Y.sup.5, Y.sup.6 and Y.sup.7
assume their natural roles in vitamin B.sub.12, and are O.
Similarly, in another embodiment V.sup.6 assumes its natural role
in vitamin B.sub.12, and is NH, or a primary amine analog thereof
substituted by J.sup.1.
[0207] In still another embodiment, position X assumes its natural
role in vitamin B.sub.12, i.e. as cyano, hydroxyl, methyl or
5'-deoxyadenosyl, most preferably 5'-deoxyadenosyl.
[0208] In another embodiment M is the radical of a purine or
pyrimidine base. In another embodiment M is the radical of
adenosine, guanine, cytosine, uridine or thymine. In still another
embodiment M is the radical of 5,6-dimethylbenzimidazole.
[0209] In still another embodiment K is CH(OH).
[0210] In yet another embodiment E is O.
[0211] In another embodiment G.sup.1 is OH.
[0212] In still another embodiment, all constituents of the
conjugate assume their natural roles in vitamin B.sub.12, except
for the moieties through which any therapeutic and/or diagnostic
agents are linked. The therapeutic and/or diagnostic agent(s) are
preferably linked to the vitamin B.sub.12 structure through
Z.sup.2, Z.sup.4 and/or Z.sup.5, and even more preferably through
the Z.sup.2 moieties.
[0213] In still another embodiment, T is not a residue of a
therapeutic agent selected from the group consisting of hormone,
growth factor, interleukin, cytokines, lymphokines, GCSF, EPO,
interferon (.alpha., .beta., .gamma.), calcitonin, TRH,
vasopressin, desmopressin [Folia Endocrinologica Japonica 54, No.
5, p. 676-691 (1978)], oxytocin, insulin, Growth Hormone,
testosterone, somatotrophin, somatostatin (U.S. Pat. Nos. 4,087,390
and 4,100,117), SCGF, (stem cell growth factor), CGRP,
Erythropoietin, Colony Stimulating factors (GCSF, GM-CSF, CSF),
pregnant mare serum gonadotrophin (PMSG), human chorionic
gonadotrophin (HCG), Inhibin, PAI-2; neomycin, salbutamol,
pyrimethamine, penicillin G, methicillin, carbenicillin, pethidine,
xylazine, ketamine, mephenesin, GABA, iron dextran, nucleotide
analogues or ribozyme, prolactin, adrenocorticotropic hormone
(ACTH), melanocyte stimulating hormone (MSH), thyroid hormone
releasing hormone (TRH) (U.S. Pat. No. 4,100,152), thyroid
stimulating hormone (TSH), luteinizing hormone (LH), luteinizing
hormone releasing hormone (LHRH), follicle stimulating hormone
(FSH), oxytocin, calcitonin, parathyroid hormone, glucagon,
gastrin, secretin, pancreozymin, cholecystokinin angiotensin, human
placental lactogen, human chorionic gonadotropin (HCG), enkephalin
[U.S. Pat. No. 4,277,394, European patent application Publication
No. 31567], endorphin, kyotorphin, interleukins (I, II, and III),
tuftsin, thymopoietin, thymosin, thymostimulin, thymic humoral
factor (TFH), serum thymic factor (FTS) (U.S. Pat. No. 4,229,438),
thymic factors [Medicine in Progress 125, No. 10, p.835-843
(1983)], tumor necrosis factor (TNF), colony stimulating factor
(CSF), motilin, dinorphin, bombesin, neurotensin, cerulein,
bradykinin, urokinase, asparaginase, kallikrein, substance P
analogue and antagonist, nerve growth factor, blood coagulation
factors VIII and IX, lysozyme chloride, polymixin B, colistin,
gramicidin, bacitracin, protein synthesis stimulating peptides
(British patent No. 8232082), gastric inhibitory polypeptide (GIP),
vasoactive intestinal polypeptide (VIP), platelet-derived growth
factor (PDGF), growth hormone factor (GRF, somatocrinin), bone
morphogenetic protein (BMP), epidermal growth factor (EGF),
bleomycin, methotrexate, actinomycin D, mitomycin C, vinblastine
sulfate, vincristine sulfate, daunorubicin, adriamycin,
neocarzinostatin, cytosine arabinoside, fluorouracil,
tetrahydrofuryl-5-fluorouracil, krestin, picibanil, lentinan,
levamisole, bestatin, azimexon, glycyrrhizin, poly I:C, poly A:U
and poly ICLC, gentamicin, dibekacin, kanendomycin, lividomycin,
tobramycin, amikacin, fradiomycin, sisomicin, tetracycline
hydrochloride, oxytetracycline hydrochloride, rolitetracycline,
doxycycline hydrochloride, ampicillin, piperacillin, ticarcillin,
cephalothin, cephaloridine, cefotiam, cefsulodin, cefinenoxime,
cefinetazole, cefazolin, cefotaxime, cefoperazone, ceftizoxime,
moxolactam, latamoxef, thienamycin, sulfazecin, azthreonam, sodium
salicylate, sulpyrine, sodium flufenamate, sodium diclofenac,
sodium indomethacin, morphine hydrochloride, pethidine, levorphanol
tartrate, oxymorphone, ephedrine, methylephedrine, noscapine,
codeine phosphate, dihydrocodeine, phosphate, alloclamide,
chlophedianol, picoperidamine, cloperastine, protokylol,
isoproterenol, salbutamol, terbutaline sulfate, chlorpromazine,
prochlorperazine, trifluoperazine, atropine sulfate, scopolamine
methylbromide, pridinol methanesulfonate, tubocurarine chloride and
pancuronium bromide, sodium phenytoin, ethosuximide, sodium
acetazolamide, chlordiazepoxide hydrochloride, metoclopramide and
L-histidine monohydrochloride, imipramine, clomipramine,
noxiptiline, phenelzine sulfate, diphenhydramine, chlorpheniramine
maleate, tripelenamine, methdilazine, clemizole, diphenylpyraline,
methoxyphenamine, trans-p-oxocamphor, theophyllol, aminophylline,
etilefrine, propranolol, alprenolol, bufetolol, oxyprenolol,
oxyfedrine, diltiazem, tolazoline, hexobendine, bamethan sulfate,
hexamethonium bromide, pentolinium, mecamlamine, ecarazine,
clonidine, sodium glymidine, glypizide, phenformin, buformin,
metformin, sodium heparin, sodium citrate, thromboplastin,
thrombin, menadione sodium bisulfite, acetomenaphthone,
epsilon.-amino-caproic acid, tranexamic acid, carbazochrome sodium
sulfonate, adrenochrome monoaminoguanidine methanesulfonate,
isoniazid, ethambutol, sodium p-aminosalicylate, prednisolone
succinate, prednisolone sodium phosphate, dexamethasone sodium
sulfate, betamethasone sodium phosphate, hexestrol phosphate,
hexestrol acetate, methimazole, levallorphan tartrate, nalorphine
hydrochloride and naloxone hydrochloride; a protein derived from or
immunogens against influenza, measles, Rubella, smallpox, yellow
fever, diphtheria, tetanus, cholera, plague, typhus, BCG,
tuberculosis causing agents, Haemophilus influenzae, Neisseria
catarrhalis, Klebsiella pneumoniae, pneumococci, streptococci; a
secretory product derived from diphtheria, tetanus, cholera,
plague, typhus, tuberculosis causing agents, Haemophilus
influenzae, Neisseria catarrhalis, Klebsiella pneumoniae,
pneumococci, streptococci, Streptococcus mutans, or is derived from
a malarial parasite or the causative agent of coccidiosis in
chickens.
[0214] The above discussion has demonstrated how the various
variables associated with the cobalamin conjugates of the present
invention can be independently varied to more particularly define
specific classes of cobalamin conjugates encompassed by this
invention. It is to be understood that the modification of one
variable can be made independently of the modification of any other
variable. Moreover, any number of embodiments can be defined by
modifying two or more of the variables in such embodiments. A few
of such embodiments are described below for purposes of
exemplification.
[0215] Subembodiment 1
[0216] X is 5'-deoxyadenosyl; M is a monovalent heterocycle that is
capable of binding to the adjacent sugar ring, and forming a dative
bond with Co.sup.+3, optionally substituted by L-T; K is O, S,
NJ.sup.1, CR.sup.100R.sup.101, or C(R.sup.100)V.sup.8Z.sup.8; E is
O or S; G.sup.1 is hydrogen, alkyl, acyl, silyl, phosphate, or L-T;
Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4, Y.sup.5, Y.sup.6 and Y.sup.7
independently are O, S or NJ.sup.2; V.sup.1, V.sup.2, V.sup.3,
V.sup.4, V.sup.5, V.sup.6, V.sup.7 and V.sup.8 independently are O,
S or NJ.sup.3; CR.sup.102R.sup.103, or a direct bond; Z, Z.sup.2,
Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.7 and Z.sup.8 independently are
R.sup.104 or L-T; each L is independently a direct bond or the
residue of a multivalent moiety that does not significantly impair
the ability of the compound to bind transcobalamin or intrinsic
factor proteins; each T independently comprises the residue of one
or more radionuclides; at least one of Z.sup.1, Z.sup.2, Z.sup.3,
Z.sup.4, Z.sup.5, Z.sup.7 and Z.sup.8, M, K, or G.sup.1 comprises a
radionuclide; J.sup.1, J.sup.2 and J.sup.3 independently are
hydrogen, alkyl, alkenyl, alkynyl, alkaryl, cycloalkyl, aryl,
cycloaryl, heterocycle, heteroaryl, hydroxyl, alkoxy, or amine;
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14
and R.sup.15 retain their natural vitamin B.sub.12 configuration;
and
[0217] R.sup.100, R.sup.101, R.sup.102, R.sup.103, and R.sup.104
are independently hydrogen, alkyl, alkenyl, alkynyl, hydroxyl,
alkoxy, cyano, azido, halogen, nitro, SO.sub.2, SO.sub.3,
thioalkyl, or amino.
[0218] Subembodiment 2
[0219] X is 5'-deoxyadenosyl; M, K, E and G.sup.1 retain their
natural vitamin B12 configuration; Y.sup.1, Y.sup.2, Y.sup.3,
Y.sup.4, Y.sup.5, Y.sup.6 and Y.sup.7 independently are O, S or
NJ.sup.2; V.sup.1, V.sup.2, V.sup.3, V.sup.4, V.sup.5, V.sup.6,
V.sup.7 and V.sup.8 independently are O, S or NJ.sup.3;
CR.sup.102R.sup.103, or a direct bond; Z.sup.1, Z.sup.2, Z.sup.3,
Z.sup.4, Z.sup.5, Z.sup.7 and Z.sup.8 independently are R.sup.104
or L-T; each L is independently a direct bond or the residue of a
multivalent moiety that does not significantly impair the ability
of the compound to bind transcobalamin or intrinsic factor
proteins; each T independently comprises the residue of one or more
radionuclides; at least one of Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4,
Z.sup.5, Z.sup.7 and Z.sup.8, M, K, or G.sup.1 comprises a
radionuclide; J.sup.1, J.sup.2 and J.sup.3 independently are
hydrogen, alkyl, alkenyl, alkynyl, alkaryl, cycloalkyl, aryl,
cycloaryl, heterocycle, heteroaryl, hydroxyl, alkoxy, or amine;
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14
and R.sup.15 independently are hydrogen, lower alkyl, lower
alkenyl, lower alkynyl, lower cycloalkyl, heterocyclic, lower
alkoxy, azido, amino, lower alkylamino, halogen, thiol, SO.sub.2,
SO.sub.3, carboxylic acid, C.sup.1-6 carboxyl, hydroxyl, nitro,
cyano, oxime or hydrazine; R.sup.13 and R.sup.14 optionally can
come together to form a double bond; and R.sup.100, R.sup.101,
R.sup.102, R.sup.103, and R.sup.104 are independently hydrogen,
alkyl, alkenyl, alkynyl, hydroxyl, alkoxy, cyano, azido, halogen,
nitro, SO.sub.2, SO.sub.3, thioalkyl, or amino.
[0220] Subembodiment 3
[0221] X is 5'-deoxyadenosyl; M is a monovalent heterocycle that is
capable of binding to the adjacent sugar ring, and forming a dative
bond with Co.sup.+3, optionally substituted by L-T; K is O, S,
NJ.sup.1, CR.sup.100R.sup.101, or C(R.sup.100)V.sup.8Z.sup.8; E is
O or S; G.sup.1 is hydrogen, alkyl, acyl, silyl, phosphate, or L-T;
Y.sup.1 , Y.sup.2, Y.sup.3, Y.sup.4, Y.sup.5, Y.sup.6 and Y.sup.7
independently are O, S or NJ.sup.2; V.sup.1, V.sup.2, V.sup.3,
V.sup.4, V.sup.5, V.sup.6, V.sup.7 and V.sup.8 independently are O,
S or NJ.sup.3; CR.sup.102R.sup.103, or a direct bond; Z.sup.1,
Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.7 and Z.sup.8
independently are R.sup.104 or L-T; each L is independently a
direct bond or the residue of a multivalent moiety that does not
significantly impair the ability of the compound to bind
transcobalamin or intrinsic factor proteins; each T independently
comprises the residue of one or more radionuclides; at least one of
Z.sup.2, Z.sup.4, or Z.sup.5 comprises a radionuclide, the
remaining Z moieties retaining their natural vitamin B.sub.12
configuration; J.sup.1, J.sup.2 and J.sup.3 independently are
hydrogen, alkyl, alkenyl, alkynyl, alkaryl, cycloalkyl, aryl,
cycloaryl, heterocycle, heteroaryl, hydroxyl, alkoxy, or amine;
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14
and R.sup.15 independently are hydrogen, lower alkyl, lower
alkenyl, lower alkynyl, lower cycloalkyl, heterocyclic, lower
alkoxy, azido, amino, lower alkylamino, halogen, thiol, SO.sub.2,
SO.sub.3, carboxylic acid, C.sub.1-6 carboxyl, hydroxyl, nitro,
cyano, oxime or hydrazine; R.sup.13 and R.sup.14 optionally can
come together to form a double bond; and R.sup.100, R.sub.101,
R.sub.102, R.sub.103, and R.sup.104 are independently hydrogen,
alkyl, alkenyl, alkynyl, hydroxyl, alkoxy, cyano, azido, halogen,
nitro, SO.sub.2, SO.sub.3, thioalkyl, or amino.
[0222] Subembodiment 4
[0223] X is hydrogen, cyano, amino, amido, hydroxyl,
5'-deoxyadenosyl, L-T, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,
aralkyl, heterocycle, or heteroaryl, or alkylheteroaryl; M, K, E
and G.sup.1 retain their natural vitamin B12 configuration;
Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4, Y.sup.5, Y.sup.6 and Y.sup.7
independently are O, S or NJ.sup.2; V.sup.1, V.sup.2, V.sup.3,
V.sup.4, V.sup.5, V.sup.6, V.sup.7 and V.sup.8 independently are O,
S or NJ.sup.3;CR.sup.102R.sup.103, or a direct bond;
Z.sup.1,Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.7 and Z.sup.8
independently are R.sup.104 or L-T; each L is independently a
direct bond or the residue of a multivalent moiety that does not
significantly impair the ability of the compound to bind
transcobalamin or intrinsic factor proteins; each L is
independently a direct bond or the residue of a multivalent moiety
that does not significantly impair the ability of the compound to
bind transcobalamin or intrinsic factor proteins; each T
independently comprises the residue of one or more radionuclides;
at least one of Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.5,
Z.sup.7, Z.sup.8, M, K, and G.sup.1 comprises a radionuclide;
J.sup.2 and J.sup.3 independently are hydrogen, pepperoni alkyl,
alkenyl, alkynyl, alkaryl, cycloalkyl, aryl, cycloaryl,
heterocycle, heteroaryl, hydroxyl, alkoxy, or amine; R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14 and
R.sup.15 retain their natural vitamin B12 configuration; and
R.sup.100, R.sup.101, R.sup.102, R.sup.103, and R.sup.104 are
independently hydrogen, alkyl, alkenyl, alkynyl, hydroxyl, alkoxy,
cyano, azido, halogen, nitro, SO.sub.2, SO.sub.3, thioalkyl, or
amino.
[0224] Subembodiment 5
[0225] X is hydrogen, cyano, amino, amido, hydroxyl,
5'-deoxyadenosyl, L-T, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,
aralkyl, heterocycle, or heteroaryl, or alkylheteroaryl; M, K, E
and G.sup.1 retain their natural vitamin B12 configuration;
Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4, Y.sup.5, Y.sup.6 and Y.sup.7
independently are O, S or NJ.sup.2; V.sup.1, V.sup.2, V.sup.3,
V.sup.4, V.sup.5, V.sup.6, V.sup.7 and V.sup.8 independently are O,
S or NJ.sup.3; CR.sup.102R.sup.103, or a direct bond; Z.sup.1,
Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.7 and Z.sup.8
independently are R.sup.104 or L-T; each L is independently a
direct bond or the residue of a multivalent moiety that does not
significantly impair the ability of the compound to bind
transcobalamin or intrinsic factor proteins; each L is
independently a direct bond or the residue of a multivalent moiety
that does not significantly impair the ability of the compound to
bind transcobalamin or intrinsic factor proteins; each T
independently comprises the residue of one or more radionuclides;
at least one of Z.sup.2, Z.sup.4, or Z.sup.5 comprises a
radionuclide, the remaining Z moieties retaining their natural
vitamin B.sub.12 configuration; J.sup.1, J.sup.2 and J.sup.3
independently are hydrogen, alkyl, alkenyl, alkynyl, alkaryl,
cycloalkyl, aryl, cycloaryl, heterocycle, heteroaryl, hydroxyl,
alkoxy, or amine; R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12 ,
R.sup.13 , R.sup.14 and R.sup.15 independently are hydrogen, lower
alkyl, lower alkenyl, lower alkynyl, lower cycloalkyl,
heterocyclic, lower alkoxy, azido, amino, lower alkylamino,
halogen, thiol, SO.sub.2, SO.sub.3, carboxylic acid, C.sub.1-6
carboxyl, hydroxyl, nitro, cyano, oxime or hydrazine; R.sup.13 and
R.sup.14 optionally can come together to form a double bond; and
R.sup.100, R.sup.101, R.sup.102, R.sup.103 and R.sup.104are
independently hydrogen, alkyl, alkenyl, alkynyl, hydroxyl, alkoxy,
cyano, azido, halogen, nitro, SO.sub.2, SO.sub.3, thioalkyl, or
amino.
[0226] Subembodiment 6
[0227] X is hydrogen, cyano, amino, amido, hydroxyl,
5'-deoxyadenosyl, L-T, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,
aralkyl, heterocycle, or heteroaryl, or alkylheteroaryl; M, K, E
and G.sup.1 retain their natural vitamin B.sub.12 configuration;
Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4, Y.sup.5, Y.sup.6 and Y.sup.7
independently are O, S or NJ.sup.2; V.sup.1, V.sup.2, V.sup.3,
V.sup.4, V.sup.5, V.sup.6, V.sup.7 and V.sup.8 independently are O,
S or NJ.sup.3; CR.sup.102R.sup.103,or a direct bond; Z.sup.1,
Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.7 and Z.sup.8
independently are R.sup.104 or L-T; each L is independently a
direct bond or the residue of a multivalent moiety that does not
significantly impair the ability of the compound to bind
transcobalamin or intrinsic factor proteins; each L is
independently a direct bond or the residue of a multivalent moiety
that does not significantly impair the ability of the compound to
bind transcobalamin or intrinsic factor proteins; each T
independently comprises the residue of one or more radionuclides;
at least one of Z.sup.2, Z.sup.4, or Z.sup.5 comprises a
radionuclide, the remaining Z moieties retaining their natural
vitamin B.sub.12 configuration; J.sup.1, J.sup.2 and J.sup.3
independently are hydrogen, alkyl, alkenyl, alkynyl, alkaryl,
cycloalkyl, aryl, cycloaryl, heterocycle, heteroaryl, hydroxyl,
alkoxy, or amine; R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12,
R.sup.13, R.sup.14 and R.sup.15independently are hydrogen, lower
alkyl, lower alkenyl, lower alkynyl, lower cycloalkyl,
heterocyclic, lower alkoxy, azido, amino, lower alkylamino,
halogen, thiol, SO.sub.2, SO.sub.3, carboxylic acid, C.sub.1-6
carboxyl, hydroxyl, nitro, cyano, oxime or hydrazine; R.sup.13 and
R.sup.14 optionally can come together to form a double bond; and
R.sup.100, R.sup.101, R.sup.102 R.sup.103and R.sup.104 are
independently hydrogen, alkyl, alkenyl, alkynyl, hydroxyl, alkoxy,
cyano, azido, halogen, nitro, SO.sub.2, SO.sub.3, thioalkyl, or
amino.
[0228] Subembodiment 7
[0229] X is 5'-deoxyadenosyl; M, K, E and G.sup.1 retain their
natural vitamin B.sub.12 configuration; Y.sup.1, Y.sup.2, Y.sup.3,
Y.sup.4, Y.sup.5, Y.sup.6 and Y.sup.7 independently are O, S or
NJ.sup.2; V.sup.1, V.sup.2, V.sup.3, V.sup.4, V.sup.5, V.sup.6,
V.sup.7 and V.sup.8 independently are O, S or NJ.sup.3;
CR.sup.102R.sup.103, or a direct bond; Z.sup.1, Z.sup.2, Z.sup.3,
Z.sup.4, Z.sup.5, Z.sup.7 and Z.sup.8 independently are R.sup.104
or L-T; each L is independently a direct bond or the residue of a
multivalent moiety that does not significantly impair the ability
of the compound to bind transcobalamin or intrinsic factor
proteins; each L is independently a direct bond or the residue of a
multivalent moiety that does not significantly impair the ability
of the compound to bind transcobalamin or intrinsic factor
proteins; each T independently comprises the residue of one or more
radionuclides; at least one of Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4,
Z.sup.5, Z.sup.7, Z.sup.8, M, K, and G.sup.1 comprises a
radionuclide; J.sup.2 and J.sup.3 independently are hydrogen,
alkyl, alkenyl, alkynyl, alkaryl, cycloalkyl, aryl, cycloaryl,
heterocycle, heteroaryl, hydroxyl, alkoxy, or amine; R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8,
R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14 and
R.sup.15 retain their natural vitamin B.sub.12 configuration; and
R.sup.100, R.sup.101, R.sup.102, R.sup.103, and R.sup.104 are
independently hydrogen, alkyl, alkenyl, alkynyl, hydroxyl, alkoxy,
cyano, azido, halogen, nitro, SO.sub.2, SO.sub.3, thioalkyl, or
amino.
[0230] Subembodiment 8
[0231] X is 5'-deoxyadenosyl; M, K, E, and G.sup.1 retain their
natural vitamin B.sub.12 configuration; Y.sup.1, Y.sup.2, Y.sup.3,
Y.sup.4, Y.sup.5, Y.sup.6 and Y.sup.7 independently are O, S or
NJ.sup.2; V.sup.1, V.sup.2, V.sup.3, V.sup.4, V.sup.5, V.sup.6,
V.sup.7 and V.sup.8 independently are O, S or NJ.sup.3;
CR.sup.102R.sup.103, or a direct bond; Z.sup.1, Z.sup.2, Z.sup.3,
Z.sup.4, Z.sup.5, Z.sup.7 and Z.sup.8 independently are R.sup.104
or L-T; each L is independently a direct bond or the residue of a
multivalent moiety that does not significantly impair the ability
of the compound to bind transcobalamin or intrinsic factor
proteins; each L is independently a direct bond or the residue of a
multivalent moiety that does not significantly impair the ability
of the compound to bind transcobalamin or intrinsic factor
proteins; each T independently comprises the residue of one or more
radionuclides; at least one of Z.sup.2, Z.sup.4, or Z.sup.5
comprises a radionuclide, the remaining Z moieties retaining their
natural vitamin B.sub.12 configuration; J.sup.1, J.sup.2 and
J.sup.3 independently are hydrogen, alkyl, alkenyl, alkynyl,
alkaryl, cycloalkyl, aryl, cycloaryl, heterocycle, heteroaryl,
hydroxyl, alkoxy, or amine; R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11,
R.sup.12 , R.sup.13, R.sup.14 and R.sup.15 independently are
hydrogen, lower alkyl, lower alkenyl, lower alkynyl, lower
cycloalkyl, heterocyclic, lower alkoxy, azido, amino, lower
alkylamino, halogen, thiol, SO.sub.2, SO.sub.3, carboxylic acid,
C.sub.1-6 carboxyl, hydroxyl, nitro, cyano, oxime or hydrazine;
R.sup.13 and R.sup.14 optionally can come together to form a double
bond; and R.sup.100, R.sup.101, R.sup.102, R.sup.103, and R.sup.104
are independently hydrogen, alkyl, alkenyl, alkynyl, hydroxyl,
alkoxy, cyano, azido, halogen, nitro, SO.sub.2, SO.sub.3,
thioalkyl, or amino.
[0232] Subembodiment 9
[0233] X is hydrogen, cyano, amino, amido, hydroxyl,
5'-deoxyadenosyl, L-T, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,
aralkyl, heterocycle, or heteroaryl, or alkylheteroaryl; M, K, E,
and G.sup.1 retain their natural vitamin B.sub.12 configuration;
Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4, Y.sup.5, Y.sup.6 and Y.sup.7
independently are O, S or NJ.sup.2; V.sup.1, V.sup.2, V.sup.3,
V.sup.4, V.sup.5, V.sup.6, V.sup.7 and V.sup.8 independently are O,
S or NJ.sup.3; CR.sup.102R.sup.103, or a direct bond; Z.sup.1,
Z.sup.2, Z.sup.3, Z.sup.4, Z.sup.5, Z.sup.7 and Z.sup.8
independently are R.sup.104 or L-T; each L is independently a
direct bond or the residue of a multivalent moiety that does not
significantly impair the ability of the compound to bind
transcobalamin or intrinsic factor proteins; each L is
independently a direct bond or the residue of a multivalent moiety
that does not significantly impair the ability of the compound to
bind transcobalamin or intrinsic factor proteins; each T
independently comprises the residue of one or more radionuclides;
at least one of Z.sup.2, Z.sup.4, or Z.sup.5 comprises a
radionuclide, the remaining Z moieties retaining their natural
vitamin B.sub.12 configuration; J.sup.1, J.sup.2 and J.sup.3
independently are hydrogen, alkyl, alkenyl, alkynyl, alkaryl,
cycloalkyl, aryl, cycloaryl, heterocycle, heteroaryl, hydroxyl,
alkoxy, or amine; R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12,
R.sup.13, R.sup.14 and R.sup.15 all retain their natural vitamin
B.sub.12 configuration; and R.sup.100, R.sup.101, R.sup.102,
R.sup.103, and R.sup.104 are independently hydrogen, alkyl,
alkenyl, alkynyl, hydroxyl, alkoxy, cyano, azido, halogen, nitro,
SO.sub.2, SO.sub.3, thioalkyl, or amino.
[0234] Subembodiment 10
[0235] X is 5'-deoxyadenosyl; M, K, E, and G.sup.1 retain their
natural vitamin B.sub.12 configuration; Y.sup.1, Y.sup.2, Y.sup.3,
Y.sup.4, Y.sup.5, Y.sup.6 and Y.sup.7 independently are O, S or
NJ.sup.2; V.sup.1, V.sup.2, V.sup.3, V.sup.4, V.sup.5, V.sup.6,
V.sup.7 and V.sup.8 independently are O, S or NJ.sup.3;
CR.sup.102R.sup.103, or a direct bond; Z.sup.1, Z.sup.2, Z.sup.3,
Z.sup.4, Z.sup.5, Z.sup.7 and Z.sup.8 independently are R.sup.104
or L-T; each L is independently a direct bond or the residue of a
multivalent moiety that does not significantly impair the ability
of the compound to bind transcobalamin or intrinsic factor
proteins; each L is independently a direct bond or the residue of a
multivalent moiety that does not significantly impair the ability
of the compound to bind transcobalamin or intrinsic factor
proteins; each T independently comprises the residue of one or more
radionuclides; at least one of Z.sup.2, Z.sup.4, or Z.sup.5
comprises a radionuclide, the remaining Z moieties retaining their
natural vitamin B.sub.12 configuration; J.sup.1, J.sup.2 and
J.sup.3 independently are hydrogen, alkyl, alkenyl, alkynyl,
alkaryl, cycloalkyl, aryl, cycloaryl, heterocycle, heteroaryl,
hydroxyl, alkoxy, or amine; R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11,
R.sup.12, R.sup.13, R.sup.14 and R.sup.15 all retain their natural
vitamin B.sub.12 configuration; and R.sup.100, R.sup.101,
R.sup.102, R.sup.103 and R.sup.104 are independently hydrogen,
alkyl, alkenyl, alkynyl, hydroxyl, alkoxy, cyano, azido, halogen,
nitro, SO.sub.2, SO.sub.3, thioalkyl, or amino.
[0236] III. Linkers
[0237] The TC- or IF-binding carrier and therapeutic and/or
diagnostic agent useful to treat and/or image sites of
proliferative disease in the body, such as cancerous tumors, can be
combined by means of a di- or multi-valent linking moiety. The
linker used to join the TC binding carrier and the therapeutic
and/or diagnostic agent preferably has a single molecular weight,
and does not exhibit a molecular weight distribution, for example
as found in most polymers. The linker can range in size from small
to large molecular weight, as long as there is not a distribution
of weights in the linker. It is important to strictly control the
uniformity of size of the conjugate for predictability of
therapeutic performance.
[0238] The linkers preferably have a molecular weight below about
2000, more preferably below about 1000, and even more preferably
below about 500.
[0239] As noted above, L is the residue of a linker molecule that
conjugates one or more therapeutic and/or diagnostic agents to the
TC or IF-binding carrier. The structure of the linker from which L
is derived (in any one of the Z.sup.1, Z.sup.2, Z.sup.3, Z.sup.4,
Z.sup.5, Z.sup.6, Z.sup.7, X, M, K or G.sup.1 moieties) is not
crucial, provided it does not significantly impair the ability of
the conjugate to bind to the transcobalamin or IF transport protein
or receptor. L is preferably any multivalent molecule (divalent or
greater) that does not significantly impair the ability of the TC
carrier to bind to the transcobalamin transport protein or
receptor. The ability of vitamin B.sub.12 or any other TC-binding
carrier to bind to the transcobalamin transport protein or receptor
is "significantly impaired" when attaching a linking moiety to the
B.sub.12 or TC-binding carrier lessens the affinity of the vitamin
B.sub.12 or the TC-binding carrier for the transcobalamin transport
protein to which the vitamin B.sub.12 or TC-binding carrier is most
readily bound by 50% or more. The unsaturated vitamin B.sub.12
binding capacity (UBBC) assay described by D. A. Collins and H. P.
C. Hogenkamp in J. Nuclear Medicine, 1997, 38, 717-723 can be used
to compare the relative affinities of ligands for this
receptor.
[0240] In one embodiment the linker is of precise molecular weight
and does not posses a molecular weight distribution. In one
embodiment, the linker has a molecular weight less than about
2,500, 2,000, 1900, 1800, 1,500, 1,000 or 500.
[0241] A particularly preferred linker is one having multiple sites
for conjugation to one or more therapeutic and/or diagnostic
agents, wherein the linker has a unimodal molecular weight.
Recombinant protein production techniques can be employed to obtain
poly(amino acid) linkers of substantially constant molecular
weight.
[0242] In one embodiment the linker is an amino acid, or a polymer
or peptide formed from a plurality of amino acids. The polymer or
peptide can be derived from one or more amino acids. The amino
acid, poly(amino acid) or peptide can link T to V through the
carboxy terminus or the amino terminus. The amino acid residue,
peptide residue, or poly(amino acid) residue can conveniently be
linked to V and T through an amide (e.g., --N(R)C(--O)-- or
--C(.dbd.O)N(R)--), ester (e.g., --OC(.dbd.O)-- or --C(.dbd.O)O--),
ether (e.g., --O--), ketone (e.g., --C(.ident.O)--), thioether
(e.g., --S--), sulfinyl (e.g., --S(O)--), sulfonyl (e.g.,
--S(O).sub.2--), or a direct (e.g., C--C bond) linkage, wherein
each R is independently H or (C.sub.1--C.sub.14) alkyl.
[0243] Peptide derivatives can be prepared as disclosed in U.S.
Pat. Nos. 4,612,302; 4,853,371; and 4,684,620. Peptide sequences
specifically recited herein are written with the amino terminus on
the left and the carboxy terminus on the right, but are meant to
also include the opposite flow. Particularly suitable peptides and
poly(amino acids) comprise from 2 to about 20 amino acids, from 2
to about 15 amino acids, or from 2 to about 12 amino acids.
[0244] One exemplary poly(amino acid) is poly-L-lysine
((--NHCH((CH.sub.2).sub.4--NH.sub.2)CO--).sub.m--Q, wherein Q is H,
(C.sub.1--C.sub.14)alkyl, or a suitable carboxy protecting group,
and m is from 2 to about 20, from about 5 to about 15, or from
about 8 to about 11. The polylysine offers multiple primary amine
sites to which anti-proliferative agents can be readily attached.
Alternatively, the linkers can be formed with multiple cysteines,
to provide free thiols, or multiple glutamates or aspartates, to
provide free carboxyls for conjugation using suitable
carbodiimides. Similarly the linker can contain multiple histidines
or tyrosines for conjugation. Other exemplary poly(amino acid)
linkers are poly-L-glutamic acid, poly-L-aspartic acid,
poly-L-histidine, poly-L-ornithine, poly-L-serine,
poly-L-threonine, poly-L-tyrosine, poly-L-lysine-L-phenylalanine or
poly-L-lysine-L-tyrosin- e. When the linker is derived from a
poly(amino acid) other than polylysine, the linker is, in a series
of embodiments, prepared from 2 to about 30 amino acids, 5 to about
20 amino acids, or 8 to about 15 amino acids.
[0245] In another particular embodiment L is a polyamine residue
(having at least three amino moieties) of the following chemical
structure: NR'(alkylene-NR').sub.nalkyleneNR', wherein n is from 1
to 20, the carbon length of alkylene can vary within the n units,
and each R' is independently hydrogen, lower alkyl, or T. N is
preferably from 1 to 10. Moreover, L preferably has a backbone
along its longest length of no more than 100, 75, 50, 40, 30, 20 or
15 atoms. Exemplary polyamines from which L can be derived include
spermine (H.sub.2N(CH.sub.2).sub.3NH(CH.sub.2).s-
ub.4NH(CH.sub.2).sub.3NH.sub.2), spermidine
(H.sub.2N(CH.sub.2).sub.3NH(CH- .sub.2).sub.4NH.sub.2),
decamethylene tetraamine, and pentamethylene hexamine. These
linkers are a definite size and thus provide consistent and
predictable targeting by the cobalamin conjugate, in addition to
multiple binding sites for the therapeutic and/or diagnostic
agent.
[0246] In another embodiment L is a diamine represented by the
formula NH.sub.2 (CH.sub.2).sub.x NH.sub.2, in which x is 2-20, and
preferably 2-12. Thus, the linker can be prepared from
1,6-diaminohexane, 1,5-diaminopentane, 1,4-diaminobutane and
1,3-diaminopropane.
[0247] Other suitable linkers are formed from the covalent linkage
of various water soluble molecules with amino acids, peptides,
poly(amino acids), polyamines, polyoxyalkylenes, polyanhydrides,
polyesters, polyamides, polyglycolides and diamines. Suitable water
soluble molecules include, for example, polyethylene glycol, and
dicarboxylic monosaccharides such as glucaric acid, galactaric acid
and xylaric acid.
[0248] Other suitable linkers include those represented by the
formula HO(O)C(CH.sub.2).sub.xC(O)OH, in which x is 2-20, and
preferably 2-12. Thus, the linker can be prepared from succinic
acid, glutaric acid, adipic acid, suberic acid, sebacic acid,
azelaic acid or maleic acid. Still other suitable linkers comprise
carboxylic acid derivatives that yield an amide upon reaction with
an amine. Such reactive groups include, by way of example,
carboxylic acid halides such as acid chlorides and bromides;
carboxylic acid anhydrides such as acetic anhydrides and
trifluoroacetic anhydrides; esters such as p-nitrophenyl esters and
N-hydroxysuccinimide esters; and imidazolides. Techniques for using
such linkers are described in detail in Bodanszky, Principles of
Peptide Synthesis, Springer Verlag, Berlin, 1984.
[0249] In one embodiment, the linker is modified to facilitate its
conjugation either to V and/or T. Suitable molecules for modifying
the linker include: disuccinimidyl suberate (DSS),
bis(sulfosuccinimidyl) suberate (BSS), ethylene
glycolbis(succinimidylsuccinate) (EGS), ethylene
glycolbis(sulfosuccinimidyl-succinate) (Sulfo-EGS),
p-aminophenyl-acetic acid, dithiobis(succinimidyl-propionate)
(DSP), 3,3'-dithiobis-(sulfosucc- inimidyl-propionate) (DTSSP),
disuccinimidyl tartarate (DST), disulfosuccinimidyl tartarate
(Sulfo-DST), bis(2-(succinimidooxycarbonylo- xy)-ethylene)sulfone
(BSOCOES), bis(2-(sulfo-succinimidooxycarbonyloxy)-et-
hylene)sulfone (Sulfo-BSOCOES), dimethyl adipimidate 2HCl (DMA),
dimethyl pimelimidate 2HCl (DMP), and dimethyl suberimidate 2HCl
(DMS).
[0250] In a particular embodiment, nonmetallic radioisotopes can
conveniently be linked to the vitamin B.sub.12 structure through a
residue of a peptide having the following formula: 3
[0251] wherein each M is independently a non-metallic radionuclide;
each R is independently (C.sub.1-C.sub.14)alkyl,
(C.sub.2-C.sub.14)alkenyl, (C.sub.2-C.sub.14)alkynyl,
(C.sub.1-C.sub.14)alkoxy, hydroxy, cyano, nitro, halo,
trifluoromethyl, N(R.sub.a) (R.sub.b), (C1 -C.sub.14)alkanoyl,
(C.sub.2-C.sub.14)alkanoyloxy, (C.sub.6-C.sub.10)aryl, or
(C.sub.3-C.sub.8)cycloalkyl wherein R.sub.a and R.sub.b are each
independently H or (C.sub.1-C.sub.14)alkyl; P; Q is H,
(C.sub.1-C.sub.14)alkyl, or a suitable carboxy protecting group; n
is 2 to about 20; I is 1-5, j is 0-4 and I+j is .ltoreq.5; or a
pharmaceutically acceptable salt thereof. Specifically, i can be 1,
j can be 0, M can be a positron emitter such as Fluorine-18,
Bromine-76 or Iodine-124, or a gamma emitter such as Iodine-123 or
Iodine-131, and n can be about 6 to about 12.
[0252] Biodegradable Linkers
[0253] Various degradable linkers can be used to link the
TC-binding or IF-binding moiety to the antiproliferative agent. The
desired linkers can degrade under biological conditions such as by
enzymatic cleavage or by systemic pH or temperature. Alternatively,
these linkers can be induced to degrade by external manipulation
such as changes in pH, temperature, ultrasound, magnetic field,
radiation (i.e. UV radiation) or light.
[0254] U.S. Pat. No. 5,639,885 entitled "Redox amino acids and
peptides containing them;" U.S. Pat. No. 5,637,601 entitled
"Anticholinergic compounds, compositions and methods of treatment;"
U.S. Pat. No. 5,624,894 entitled "Brain-enhanced delivery of
neuroactive peptides by sequential metabolism;" U.S. Pat. No.
5,618,826 entitled "Anticholinergic compounds, compositions and
methods of treatment;" U.S. Pat. No. 5,618,803 entitled "Targeted
drug delivery via phosphonate derivatives;" U.S. Pat. No. 5,610,188
entitled "Anticholinergic compounds, compositions and methods of
treatment;" U.S. Pat. No. 5,525,727 entitled "Brain-specific drug
delivery;" U.S. Pat. No. 5,418,244 entitled "Anticholinergic
compounds, compositions and methods of treatment;" U.S. Pat. No.
5,413,996 entitled "Targeted drug delivery via phosphonate
derivatives;" U.S. Pat. No. 5,389,623 entitled "Redox carriers for
brain-specific drug delivery;" U.S. Pat. No. 5,296,483 entitled
"Brain-specific analogues of centrally acting amines;" U.S. Pat.
No. 5,258,388 entitled "Anticholinergic compounds, compositions and
methods of treatment;" U.S. Pat. No. 5,231,089 entitled "Method of
improving oral bioavailability of carbamazepine;" U.S. Pat. No.
5,223,528 entitled "Anticholinergic compounds, compositions and
methods of treatment;" U.S. Pat. No. 5,187,158 Brain-specific drug
delivery;" U.S. Pat. No. 5,177,064 entitled "Targeted drug delivery
via phosphonate derivatives;" U.S. Pat. No. 5,155,227 entitled
"Compounds for site-enhanced delivery of radionuclides;" U.S. Pat.
No. 5,136,038 entitled "Radiopharmaceuticals and chelating agents
useful in their preparation;" U.S. Pat. No. 5,087,618 entitled
"Redox carriers for brain-specific drug delivery;" U.S. Pat. No.
5,079,366 entitled "Quaternary pyridinium salts;" U.S. Pat. No.
5,053,215 entitled "NMR-assayable ligand-labeled trifluorothymidine
containing composition and method for diagnosis of HSV infection;"
U.S. Pat. No. 5,024,998 entitled "Pharmaceutical formulations for
parenteral use;" U.S. Pat. No. 5,017,618 entitled "Labile
derivatives of ketone analogs of
3-substituted-1-alkylamino-2-propanols and their use as
beta-adrenergic blockers;" U.S. Pat. No. 5,017,566 entitled "Redox
systems for brain-targeted drug delivery;" U.S. Pat. No. 5,008,257
entitled "Brain-specific drug delivery;" U.S. Pat. No. 5,002,935
entitled "Improvements in redox systems for brain-targeted drug
delivery;" U.S. Pat. No. 4,983,586 entitled "Pharmaceutical
formulations for parenteral use;" U.S. Pat. No. 4,963,688 entitled
"Compounds for site-enhanced delivery of radionuclides and uses
thereof;" U.S. Pat. No. 4,963,682 entitled "Novel
radiopharmaceuticals and chelating agents useful in their
preparation;" U.S. Pat. No. 4,933,438 entitled "Brain-specific
analogues of centrally acting amines;" U.S. Pat. No. 4,900,837
entitled "Brain-specific drug delivery of steroid sex hormones
cleaved from pyridinium carboxylates and dihydropyridine
carboxylate precursors;" U.S. Pat. No. 4,892,737 entitled
"Composition and method for enhancing permeability of topical
drugs;" U.S. Pat. No. 4,888,427 entitled "Amino acids containing
dihydropyridine ring systems for site-specific delivery of peptides
to the brain;" 4,880,921 entitled "Brain-specific drug delivery;"
35. 4,863,911 entitled "Method for treating male sexual
dysfunction;" U.S. Pat. No. 4,829,070 entitled "Novel redox
carriers for brain-specific drug delivery;" U.S. Pat. No. 4,824,850
entitled "Brain-specific drug delivery;" U.S. Pat. No. 4,801,597
entitled "Certain inositol-nicotinate ester derivatives and
polyionic complexes therefore useful for treating diabetes meuitus,
hyperlipidemia and lactic acidosis;" U.S. Pat. No. 4,771,059
entitled "Brain-specific analogues of centrally acting amines;"
U.S. Pat. No. 4,727,079 entitled "Brain-specific dopaminergic
activity involving dihydropyridine carboxamides, dihydroquinoline
and isoquinoline carboxamides;" U.S. Pat. No. 4,540,564 entitled
"Brain-specific drug delivery;" and U.S. Pat. No. 4,479,932
entitled "Brain-specific drug delivery" to Nicholas S. Bodor, et
al., disclose several biodegradable linkers that target the brain.
For example, a lipoidal form of dihydropyridine pyridinium salt
redox carrier, DHC, linked to a centrally acting drug which can be
reduced and biooxidized to pass through the blood brain barrier.
The dihydropyridine nucleus readily and easily penetrates the blood
brain barrier in increased concentrations; furthermore, the in vivo
oxidation of the dihydropyridine moiety to the ionic pyridinium
salts thereby prevents its elimination from the brain, while
elimination from the general circulation is accelerated, resulting
in a prolongedly sustained brain-specific drug activity. This
dihydropyridine can be incorporated into the linkers set forth
above for biodegradation.
[0255] Additionally U.S. Pat. No. 4,622,218 entitled
"Testicular-specific drug delivery," discloses linkers that can
specifically deliver drugs to the testes in much the same manner,
and which can be used in the linkers of the present invention. The
lipoidal form [D--DHC] of a dihydropyridine pyridinium salt redox
carrier, e.g. 1,4-dihydrotrigonelline, penetrates the blood-testis
barrier. Oxidation of the dihydropyridine carrier moiety in vivo to
the ionic pyridinium salt type drug/carrier entity [D--QC].sup.+
prevents elimination thereof from the testes, while elimination
from the general circulation is accelerated, resulting in
significant and prolongedly sustained testicular-specific drug
activity.
[0256] Margerum, et al. in U.S. Pat. No. 5,976,493 discloses the
use of polychelant compounds which are degradable in vivo to
release excretable fragments for diagnostic imaging which also are
suitable in the linkers of the present invention. These compounds
contain a linker moiety which is metabolically cleavable to release
macrocyclic monochelant fragments, wherein the macrocyclic skeleton
preferably has 9 to 25 ring members, and a biotolerable polymer,
preferably a substantially monodisperse polymer. Other suitable
linkers are disclosed, for example, in Krejcarek et al.
(Biochemical and Biophysical Research Communications 77: 581
(1977)) (mixed anhydrides), Hnatowich et al. (Science 220: 613
(1983))(cyclic anhydrides), U.S. Pat. 5,637,684 to Cook, et al.
(Phosphoramidate and phosphorothioamidate oligomeric
compounds).
[0257] Other suitable biodegradable polymers from which the linker
can be formed are the polyanhydrides and polyorthoesters, which
take advantage of labile backbone linkages (see: Domb et al.
Macromolecules, 22, 3200, 1989; and Heller et al. Biodegradable
Polymers as Drug Delivery Systems, Dekker, NY: 1990). Other linker
materials include hydrogels, such as the
PEG-oligoglycolyl-acrylates disclosed in U.S. Pat. No. 5,626,863 to
Hubbell et al. Other biodegradable linkers are formed from
oligoglycolic acid is a poly(a-hydroxy acid), polylactic acid,
polycaprolactone, polyorthoesters, polyanhydrides and
polypeptides.
[0258] Nonlimiting examples of U.S. Patents that describe
controlled release formulations suitable for use as linking agents
are: U.S. Pat. No. 5,356,630 to Laurencin et al. (Delivery System
for Controlled Release of Bioactive Factors); ; U.S. Pat. No.
5,797,898 to Santini, Jr. et al (Microchip Drug Delivery Devices);
U.S. Pat. No. 5,874,064 to Edwards et al. (Aerodynamically Light
Particles for Pulmonary Drug Delivery); U.S. Pat. No. 5,548,035 to
Kim et al. (Biodegradable Copolymer as Drug Delivery Matrix
Comprising Polyethyleneoxide and Aliphatic Polyester Blocks); U.S.
Pat. No. 5,532,287 to Savage et al. (Radiation Cured Drug Release
Controlling Membrane); U.S. Pat. No. 5,284,831 to Kahl et al. (Drug
Delivery Porphyrin Composition and Methods); U.S. Pat. No.
5,741,329 to Agrawal et al. (Methods of Controlling the pH in the
Vicinity of Biodegradable Implants); U.S. Pat. No. 5,820,883 to
Tice et al. (Methods for Delivering Bioactive Agents into and
Through the Mucosally-Associated Lymphoid Tissues and Controlling
Their Release);U.S. Pat. No. 5,955,068 to Gouin et al.
(Biodegradable Polyanhydrides Derived from Dimers of Bile Acids,
and Use Thereof as Controlled Drug Release Systems); U.S. Pat. No.
6,001,395 to Coombes et al. (Polymeric Lamellar Substrate Particles
for Drug Delivery); U.S. Pat. No. 6,013,853 to Athanasiou et al.
(Continuous Release Polymeric Implant Carriers); U.S.
[0259] U.S Pat. No. 6,060,582 to Hubbell et al. (Photopolymerizable
Biodegradable Hydrogels as Tissue Contacting Materials and
Controlled Release Carriers); U.S. Pat. No. 6,113,943 to Okada et
al. (Sustained-Release Preparation Capable of Releasing a
Physiologically Active Substance); and PCT Publication No. WO
99/59548 to Oh et al. (Controlled Drug Delivery System Using the
Conjugation of Drug to Biodegradable Polyester); U.S. Pat. No.
6,123,861 (Fabrication of Microchip Drug Delivery Devices); U.S.
Pat. No. 6,060,082 (Polymerized Liposomes Targeted to M cells and
Useful for Oral or Mucosal Drug Delivery); U.S. Pat. No. 6,041,253
(Effect of Electric Field and Ultrasound for Transdermal Drug
Delivery); U.S. Pat. No. 6,018,678 (Transdermal protein delivery or
measurement using low-frequency sonophoresis); U.S. Pat. No.
6,007,845 Nanoparticles And Microparticles Of Non-Linear
Hydrophilic-Hydrophobic Multiblock Copolymers; U.S. Pat. No.
6,004,534 Targeted Polymerized Liposomes For Improved Drug
Delivery; U.S. Pat. No. 6,002,961 Transdermal Protein Delivery
Using Low-Frequency Sonophoresis; U.S. Pat. No. 5,985,309
Preparation Of Particles For Inhalation; U.S. Pat. No. 5,947,921
Chemical And Physical Enhancers And Ultrasound For Transdermal Drug
Delivery; U.S. Pat. No. 5,912,017 Multiwall Polymeric Microspheres;
U.S. Pat. No. 5,911,223 Introduction Of Modifying Agents Into Skin
By Electroporation; U.S. Pat. No. 5,874,064 Aerodynamically Light
Particles For Pulmonary Drug Delivery; U.S. Pat. No. 5,855,913
Particles Incorporating Surfactants For Pulmonary Drug Delivery;
U.S. Pat. No. 5,846,565 Controlled Local Delivery Of
Chemotherapeutic Agents For Treating Solid Tumors; U.S. Pat. No.
5,837,752 Semi-Interpenetrating Polymer Networks; U.S. Pat. No.
5,814,599 Transdermal Delivery Of Encapsulated Drugs; U.S. Pat. No.
5,804,178 Implantation Of Cell-Matrix Structure Adjacent Mesentery,
Omentum Or Peritoneum Tissue; U.S. Pat. No. 5,797,898 Microchip
Drug Delivery Devices; U.S. Pat. No. 5,770,417 Three-Dimensional
Fibrous Scaffold Containing Attached Cells For Producing
Vascularized Tissue In Vivo; U.S. U.S. Pat. No. 5,770,193
Preparation Of Three-Dimensional Fibrous Scaffold For Attaching
Cells To Produce Vascularized Tissue In Vivo; U.S. Pat. No.
5,762,904 Oral Delivery Of Vaccines Using Polymerized Liposomes;
U.S. Pat. No. 5,759,830 Three-Dimensional Fibrous Scaffold
Containing Attached Cells For Producing Vascularized Tissue In
Vivo; U.S. Pat. No. 5,749,847 Delivery Of Nucleotides Into
Organisms By Electroporation; U.S. Pat. No. 5,736,372 Biodegradable
Synthetic Polymeric Fibrous Matrix Containing Chondrocyte For In
Vivo Production Of A Cartilaginous Structure; U.S. Pat. No.
5,718,921 Microspheres Comprising Polymer And Drug Dispersed There
Within; U.S. Pat. No. 5,696,175 Preparation Of Bonded Fiber
Structures For Cell Implantation; U.S. Pat. No. 5,667,491 Method
For Rapid Temporal Control Of Molecular Transport Across Tissue;
U.S. Pat. No. 5,654,381 Functionalized Polyester Graft Copolymers;
U.S. Pat. No. 5,651,986 Controlled Local Delivery Of
Chemotherapeutic Agents For Treating Solid Tumors; U.S. Pat. No.
5,629,009 Delivery System For Controlled Release Of Bioactive
Factors; U.S. Pat. No. 5,626,862 Controlled Local Delivery Of
Chemotherapeutic Agents For Treating Solid Tumors; U.S. Pat. No.
5,593,974 Localized Oligonucleotide Therapy; U.S. Pat. No.
5,578,325 Nanoparticles And Microparticles Of Non-Linear
Hydrophilic-Hydrophobic Multiblock Copolymers; U.S. Pat. No.
5,562,099 Polymeric Microparticles Containing Agents For Imaging;
U.S. Pat. No. 5,545,409 Delivery System For Controlled Release Of
Bioactive Factors; U.S. Pat. No. 5,543,158 Biodegradable Injectable
Nanoparticles; U.S. Pat. No. 5,514,378 Biocompatible Polymer
Membranes And Methods Of Preparation Of Three Dimensional Membrane
Structures; U.S. Pat. No. 5,512,600 Preparation Of Bonded Fiber
Structures For Cell Implantation; U.S. Pat. No. 5,500,161 Method
For Making Hydrophobic Polymeric Microparticles; U.S. Pat. No.
5,487,390 Gas-filled polymeric microbubbles for ultrasound imaging;
U.S. Pat. No. 5,399,665 Biodegradable polymers for cell
transplantation; U.S. Pat. No. 5,356,630 Delivery system for
controlled release of bioactive factors; U.S. Pat. No. 5,330,768
Controlled drug delivery using polymer/pluronic blends; U.S. Pat.
No. 5,286,763 Bioerodible polymers for drug delivery in bone; U.S.
Pat. No. 5,149,543 lonically cross-linked polymeric microcapsules;
U.S. Pat. No. 5,128,420 Method of making hydroxamic acid polymers
from primary amide polymers; U.S. Pat. No. 5,122,367 Polyanhydride
bioerodible controlled release implants for administration of
stabilized growth hormone; U.S. Pat. No. 5,100,668 Controlled
release systems containing heparin and growth factors; U.S. Pat.
No. 5,019,379 Unsaturated polyanhydrides; U.S. Pat. No. 5,010,167
Poly(amide-and imide-co-anhydride) for biological application; U.S.
Pat. No. 4,948,587 Ultrasound enhancement of transbuccal drug
delivery; U.S. Pat. No. 4,946,929 Bioerodible articles useful as
implants and prostheses having predictable degradation rates; U.S.
Pat. No. 4,933,431 One step preparation of poly(amide-anhydride);
U.S. Pat. No. 4,933,185 System for controlled release of
biologically active compounds; U.S. Pat. No. 4,921,757 System for
delayed and pulsed release of biologically active substances; U.S.
Pat. No. 4,916,204 Pure polyanhydride from dicarboxylic acid and
coupling agent; U.S. Pat. No. 4,906,474 Bioerodible polyanhydrides
for controlled drug delivery; U.S. Pat. No. 4,900,556 System for
delayed and pulsed release of biologically active substances; U.S.
Pat. No. 4,898,734 Polymer composite for controlled release or
membrane formation; U.S. Pat. No. 4,891,225 Bioerodible
polyanhydrides for controlled drug delivery; U.S. Pat. No.
4,888,176 Controlled drug delivery high molecular weight
polyanhydrides; U.S. Pat. No. 4,886,870 Bioerodible articles useful
as implants and prostheses having predictable degradation rates;
U.S. Pat. No. 4,863,735 Biodegradable polymeric drug delivery
system with adjuvant activity; U.S. Pat. No. 4,863,611
Extracorporeal reactors containing immobilized species; U.S. Pat.
No. 4,861,627 Preparation of multiwall polymeric microcapsules;
U.S. Pat. No. 4,857,311 Polyanhydrides with improved hydrolytic
degradation properties; U.S. Pat. No. 4,846,786 Bioreactor
containing suspended, immobilized species; U.S. Pat. No. 4,806,621
Biocompatible, bioerodible, hydrophobic, implantable polyimino
carbonate article; U.S. Pat. No. 4,789,724 Preparation of anhydride
copolymers; U.S. Pat. No. 4,780,212 Ultrasound enhancement of
membrane permeability; U.S. Pat. No. 4,779,806 Ultrasonically
modulated polymeric devices for delivering compositions; U.S. Pat.
No. 4,767,402 Ultrasound enhancement of transdermal drug delivery;
U.S. Pat. No. 4,757,128High molecular weight polyanhydride and
preparation thereof; U.S. Pat. No. 4,657,543 Ultrasonically
modulated polymeric devices for delivering compositions; U.S. Pat.
No. 4,638,045 Non-peptide polyamino acid bioerodible polymers; U.S.
Pat. No. 4,591,496 Process for making systems for the controlled
release of macromolecules.
[0260] The above discussion has demonstrated how the various
variables associated with the cobalamin conjugates of the present
invention can be independently varied to more particularly define
specific classes of cobalamin conjugates encompassed by this
invention. It is to be understood that the modification of one
variable can be made independently of the modification of any other
variable. Moreover, any number of embodiments can be defined by
modifying two or more of the variables in such embodiments. A few
of such embodiments are described below for purposes of
exemplification.
[0261] Subembodiments 11-20
[0262] Any one of subembodiments 1-10, wherein the linker has a
substantially constant molecular weight.
[0263] Subembodiments 21-30
[0264] Any one of subembodiments 1-10, wherein the linker is a
polyamine of the following chemical structure:
NR'(alkylene-NR').sub.nalkyleneNR', wherein n is from 1 to 20, the
carbon length of alkylene can vary within the n units, and each R'
is independently hydrogen, lower alkyl, or T.
[0265] Subembodiments 31-40
[0266] Any one of subembodiments 1-10, wherein the linker is
spermine, spermidine, decamethylene tetraamine or pentamethylene
hexamine.
[0267] IV. Chelating Group
[0268] Chelating groups can be used to link radionuclides to the
TC- or IF-binding carrier of the present invention, either directly
or via a linker. Any suitable chelating group can be employed.
Suitable chelating groups include those disclosed in U.S. Pat. No.
5,739,313. Other suitable chelating groups are the thiazoline
derivatives disclosed in U.S. Pat. No. 6,083,966, the pyridinones
disclosed in U.S. Pat. No. 5,892,029, and the catecholaurates
disclosed in U.S. Pat. No. 5,514,695.
[0269] In one embodiment, the chelating group can be NTA, HEDTA,
DCTA, RP414, MDP, DOTATOC, CDTA, HYNIC, EDTA, DTPA, TETA, DOTA,
DOTMP, DCTA, 15N4, 9N3, 12N3, or MAG3 (or another suitable
polyamino acid chelator), which are described herein below, or a
phosphonate chelator (e.g. EDMT). In a preferred embodiment, the
chelating group is DTPA.
[0270] DTPA is diethylenetriaminepentaacetic acid; TETA is
1,4,8,11-tetraaza-cyclo-tetradecane-N,N',N",N'"-tetraacetic acid;
DOTA is 1,4,7,10-tetraaza-cyclododecane-N,N',N",N'"-tetraacetic
acid; 15N4 is
1,4,8,12-tetraazacyclo-pentadecane-N,N',N",N'"-tetra-acetic acid;
9N3 is 1,4,7-triazacyclononane-N,N',N"-triacetic acid; 12N3 is
1,5,9-triazacyclo-dodecane-N,N',N"-triacetic acid; polyaminoacid
chelators, such as MAG3 is
(N-(N-(N-((benzoylthio)acetyl)glycyl)glycyl)gl- ycine); and DCTA is
a cyclohexane-based metal chelator of the formula 4
[0271] wherein R.sup.3 may by (C.sub.1-C.sub.4)alkyl or
CH.sub.2CO.sub.2--, which may be attached through positions 4 or 5,
or through the group R.sup.3 and which carries from 1 to 4
detectable metal or nonmetal cations (M*), monovalent cations, or
the alkaline earth metals. Thus, with metals of oxidation state +1,
each individual cyclohexane-based molecule may carry up to 4 metal
cations (where both R.sup.3 groups are CH.sub.2COOM*). As is more
likely, with higher oxidation states, the number of metals will
decrease to 2 or even 1 per cyclohexane skeleton. This formula is
not intended to limit the molecule to any specific
stereochemistry.
[0272] NTA, HEDTA and DCTA are disclosed in Poster Sessions,
Proceedings of the 46th Annual Meeting, J. Nuc. Med., p. 316, No.
1386. RP414 is disclosed in Scientific Papers, Proceedings of the
46th Annual Meeting, J. Nuc. Med., p. 123, No. 499. MDP is
disclosed in Scientific Papers, Proceedings of the 46th Annual
Meeting, J. Nuc. Med., p. 102, No. 413. DOTATOC is disclosed in
Scientific Papers, Proceedings of the 46th Annual Meeting, J. Nuc.
Med., p. 102, No. 414 and Scientific Papers, Proceedings of the
46th Annual Meeting, J. Nuc. Med., p. 103, No. 415. CDTA is
disclosed in Poster Sessions, Proceedings of the 46th Annual
Meeting, J. Nuc. Med., p. 318, No. 1396. 20HYNIC is disclosed in
Poster Sessions, Proceedings of the 46th Annual Meeting, J. Nuc.
Med., p. 319, No. 1398.
[0273] Bifunctional chelators (i.e., chelating groups) based on
macrocyclic ligands in which conjugation is via an activated arm
attached to the carbon backbone of the ligand can also be employed
as a chelating group, as described by M. Moi et al., J. Amer.
Chem., Soc., 49, 2639 (1989)
(2-p-nitrobenzyl-1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraa-
cetic acid); S. V. Deshpande et al., J. Nucl. Med., 31, 473 (1990);
G. Kuser et al., Bioconj. Chem., 1,345 (1990); C. J. Broan et al.,
J. C. S. Chem. Comm., 23, 1739 (1990); and C. J. Anderson et al.,
J. Nucl. Med. 36, 850 (1995)
(6-bromoacetamido-benzyl-1,4,8,11-tetraazacyclotetadecane--
N,N',N",N'"-tetraacetic acid (BAT)).
[0274] In addition, the chelator or chelating group can be any of
the chelating groups disclosed in Scientific Papers, Proceedings of
the 46th Annual Meeting, J. Nuc. Med., Wednesday, Jun. 9, 1999, p.
124, No. 500.
[0275] Specifically, the chelating group can be any one of the
carbonyl complexes disclosed in Waibel et al., Nature
Biotechnology, 897-901, Vol. 17, September 1999; or Sattelberger et
al., Nature Biotechnology, 849-850, Vol. 17, September 1999.
[0276] Specifically, the detectable chelating group can be any one
of the carbonyl complexes disclosed in Waibel et al., Nature
Biotechnology, 897-901, Vol. 17, September 1999; or Sattelberger et
al., Nature Biotechnology, 849-850, Vol. 17, September 1999,
further comprising a metallic radionuclide. More specifically, the
detectable chelating group can be any one of the carbonyl complexes
disclosed in Waibel et al., Nature Biotechnology, 897-901, Vol. 17,
September 1999; or Sattelberger et al., Nature Biotechnology,
849-850, Vol. 17, September 1999, further comprising
Technetium-99m, Rhenium-186, or Rhenium-188.
[0277] V. Detectable and/or Therapeutic Radionuclides
[0278] As used herein, a "detectable radionuclide" is any suitable
radionuclide (i.e., radioisotope) capable of being detected in a
diagnostic procedure in vivo or in vitro. Suitable detectable
radionuclides include metallic radionuclides (i.e., metallic
radioisotopes) and non-metallic radionuclides (i.e., non-metallic
radioisotopes).
[0279] Suitable metallic radionuclides (i.e., metallic
radioisotopes or metallic paramagnetic ions) include Antimony-124,
Antimony-125, Arsenic-74, Barium-103, Barium-140, Beryllium-7,
Bismuth-206, Bismuth-207, Cadmium-109, Cadmium-115m, Calcium-45,
Cerium-139, Cerium-141, Cerium-144, Cesium-137, Chromium-51,
Cobalt-55, Cobalt-56, Cobalt-57, Cobalt-58, Cobalt-60, Cobalt-64,
Copper-67, Erbium-169, Europium-152, Gallium-64, Gallium-68,
Gadolinium-153, Gadolinium-157 Gold-195, Gold-199, Hafnium-175,
Hafnium-175-181, Hohnium-166, Indium-110, Indium-111, Iridium-192,
Iron-55, Iron-59, Krypton-85, Lead-210, Lutetium-177, Manganese-54,
Mercury-197, Mercury-203, Molybdenum-99, Neodymium-147,
Neptunium-237, Nickel-63, Niobium-95, Osmium-185+191,
Palladium-103, Platinum-195m, Praseodymium-143, Promethium-147,
Protactinium-233, Radium-226, Rhenium-186, Rhenium-188,
Rubidium-86, Ruthenium-103, Ruthenium-106, Scandium-44,
Scandium-46, Selenium-75, Silver-110, Silver-111, Sodium-22,
Strontium-85, Strontium-89, Strontium-90, Sulfur-35, Tantalum-182,
Technetium-99m, Tellurium-125, Tellurium-132, Thallium-204,
Thorium-228, Thorium-232, Thallium-170, Tin-113, Tin-114, Tin-117m,
Titanium-44, Tungsten-185, Vanadium-48, Vanadium-49, Ytterbium-169,
Yttrium-86, Yttrium-88, Yttrium-90, Yttrium-91, Zinc-65, and
Zirconium-95.
[0280] The compounds of the invention can also comprise one or more
(e.g., 1, 2, 3, or 4) non-metallic radionuclide which can be
directly linked to a residue of the compound of formula I at any
synthetically feasible site, or can be linked to a residue of the
compound of formula I, by a linker, at any synthetically feasible
site. Suitable linkers are described herein. In addition, suitable
points of attachment of the compound of formula I for the
non-metallic radionuclide, either directly or by a linker, are also
described herein. The invention also provides compounds having more
than one non-metallic radionuclide attached to a compound of
formula I, either directly, or by a linker.
[0281] Specifically, the non-metallic radionuclide can be a
non-metallic paramagnetic atom (e.g., Fluorine-i 9); or
non-metallic positron emitting radionuclide (e.g., Carbon-11,
Fluorine-18, Iodine-12 or Bromine-76), or a nonmetallic gamma
emitting radionuclide such as Iodine-123 or Iodine-131. Fluorine-19
is a suitable non-metallic paramagnetic for use the compounds of
the present invention in part because there is typically little or
no background noise associated with the diagnostic use of fluorine
in the body of a mammal (e.g., human).
[0282] As used herein, a "therapeutic chelating group" is a
chelating group comprising a metallic radionuclide (e.g., a
metallic radioisotope) that possesses therapeutic efficacy against
cancer or other neoplastic cells in vivo or in vitro. Any suitable
chelating group can be employed.
[0283] Specifically, the therapeutic chelating group can be any of
the carbonyl complexes disclosed in Waibel et al., Nature
Biotechnology, 897-901, Vol. 17, September 1999; or Sattelberger et
al., Nature Biotechnology, 849-850, Vol. 17, September 1999,
further comprising a metallic radionuclide. More specifically, the
therapeutic chelating group can be any of the carbonyl complexes
disclosed in Waibel et al., Nature Biotechnology, 897-901, Vol. 17,
September 1999; or Sattelberger et al., Nature Biotechnology,
849-850, Vol. 17, September 1999, further comprising Rhenium-186 or
Rhenium-188.
[0284] VI. Antiproliferative Agents
[0285] As used herein, the therapeutic agent is an
antiproliferative agent that decreases the hyperproliferation of
cells. Proliferative disorders are currently treated by a variety
of classes of compounds includes alkylating agents,
antimetabolites, natural products, enzymes, biological response
modifiers, miscellaneous agents, hormones and antagonists, such as
those listed in the Background of the Invention. Any of the
antiproliferative agents listed in the Background, any listed
below, or any other such agent known or discovered to exhibit an
antiproliferative effect that can be more effectively delivered by
conjugation to a TC- or IF-binding carrier can be used in
accordance with this invention.
[0286] In an alternative embodiment, any of the antiproliferative
agents listed in the Background or below or any other such known
agents can be used in combination with a TC- or IF-binding
carrier/therapeutic and/or agent to achieve a combination
therapeutic effect.
[0287] The antiproliferative agent can be bound through a covalent
bond, a dative bond, a coordination bond, complexation (such as
found in a bound antibody/epitope), or ionic bond. Covalent bonding
is preferred over ionic bonding, however, a tightly held ionic bond
may be suitable. Below are nonlimiting examples of how agents can
be attached to carriers. Other routine means are known to those
skilled in the art, and are assumed included within the scope of
the invention.
[0288] Free Amines or Amides
[0289] The following are non-limiting examples of antiproliferative
agents that contain an amine group or an amide group, and thus can
be linked to the TC or IF binding carrier through that functional
moiety, using standard chemical reactions for covalent bond
formation to a nitrogen atom. 5
[0290] Dacarbazine (DTIC; DIC; DTIC-Dome; Dacatic; Deticene; DTIE;
ICDMT; ICDT) 6
[0291] Thalidomide (K 17; Distaval; Softenon; Sedalis; Talimol;
Pantosediv; Neurosedyn; Kevadon; Contergan; Synovir; NEO; neosedyn;
neurodyn; nevrodyn; nibrol; pangul) 7
[0292] Carboxylic Acids
[0293] The following are non-limiting examples of antiproliferative
agents that contain a carboxylic acid, and thus can be linked to
the TC or IF binding carrier through that functional moiety, using
standard chemical reactions for covalent bond formation by
derivatization of a carboxylic acid. 8
[0294] Benzyl carboxylic acids: aspirin, diclofenac, indomethacin,
ketorolac, ketoprofen, naproxen, diflunisal, mefenamic acid,
ioxoprofen, tolmefenamic acid, indoprofen, pirprofen, fenoprofen,
zaltoprofen, sulindac, tolmetin, suprofen, flurbiprofen,
pranoprofen, niflumic acid, flufenamic acid, zomopirac, bromfenac,
fenclofenac, alcofenac, orpanoxin, etodolic acid, fleclozic acid,
amfenac, emfenamic acid, benoxaprofen, fluoxaprofen, carprofen,
isofezolac, aceloferac, fenpufen, fenclorac, meclofenamate, and
clindac. Second line agents include gold salts, penicillamine,
methotrexate, and antimalarials.
[0295] Free Hydroxyl/Mercapto Groups
[0296] The following are non-limiting examples of antiproliferative
agents that contain a free hydroxyl or thiol group, and thus can be
linked to the TC or IF binding carrier through that functional
moiety, using standard chemical reactions for covalent bond
formation by derivatization of a hydroxyl or thiol. 9
[0297] Anthralin (Dithranol, anthra-derm; Lasan; psoriacid-stift;
batridrol; psoriacide; chrysodermol; cignolin; cygnolin; dermaline;
derobin; DithraSal; Dithrocream; Psorin) 10
[0298] Biologics
[0299] The following are non-limiting examples of biologics that
can be attached using known chemistry for attachment of such
moieties, for example, through protein or peptide covalent bond
formation, complexation such as through an antibody/epitope
linkage, or other ionic or covalent bond formation.
[0300] Nonlimiting examples include: Cyclosporine (Neoral.RTM.),
Calcipotriene (a synthetic form of vitamin D.sub.3); Coal tar,
gliadel, Halichondrin B, navelbine, cyclodisone, Mixtozantrone,
alpha interferon, Interferon-alfa, interferon alfa-2b recombinant,
interferon alfa-2a recombinant (aldesleukin), L-Asparaginase,
filgrastim (neupogen, G-CSF), sagramostin (GM-CSF), interleukin-2,
rocarbazine, droloxifene roloxifene, estramucine phosphate sodium,
streptozoci, and goserelin acetate.
[0301] Miscelleneous
[0302] The following are non-limiting examples of antiproliferative
agents that do not have readily available functional groups to
derivatize for covalent attachment to the TC-binding or IF-binding
carrier or linker, but can be attached through a suitable ionic
bond with close salt formation, wherein the carrier or linker
contains an appropriate counterion. 11
[0303] VII. Pharmaceutically Acceptable Salt or Prodrug
Formulations
[0304] The term "pharmaceutically acceptable salt or prodrug" is
used throughout the specification to describe any pharmaceutically
acceptable form (such as an ester, mono-, di- or tri-phosphate
ester, salt of an ester or a related group) of a TC- or IF-binding
carrier, which, upon administration to a patient, provides the
active compound. Pharmaceutically acceptable salts include those
derived from pharmaceutically acceptable inorganic or organic bases
and acids. Suitable salts include those derived from alkali metals
such as potassium and sodium, alkaline earth metals such as calcium
and magnesium, among numerous other acids well known in the
pharmaceutical art. Pharmaceutically acceptable prodrugs refer to a
compound that is metabolized, for example hydrolyzed or oxidized,
in the host to form the compound of the present invention. Typical
examples of prodrugs include compounds that have biologically
labile protecting groups on a functional moiety of the active
compound. Prodrugs include compounds that can be oxidized, reduced,
aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed,
dehydrolyzed, alkylated, dealkylated, acylated, deacylated,
phosphorylated, dephosphorylated to produce the active compound.
The compounds of this invention possess activity against infectious
disease or are metabolized to a compound that exhibits such
activity.
[0305] In cases where compounds are sufficiently basic or acidic to
form stable nontoxic acid or base salts, administration of the
compound as a pharmaceutically acceptable salt may be appropriate.
Examples of pharmaceutically acceptable salts are organic acid
addition salts formed with acids, which form a physiological
acceptable anion, for example, tosylate, methanesulfonate, acetate,
citrate, malonate, tartarate, succinate, benzoate, ascorbate,
.alpha.-ketoglutarate and .alpha.-glycerophosphate. Suitable
inorganic salts may also be formed, including, sulfate, nitrate,
bicarbonate and carbonate salts.
[0306] Pharmaceutically acceptable salts may be obtained using
standard procedures well known in the art, for example by reacting
a sufficiently basic compound such as an amine with a suitable acid
affording a physiologically acceptable anion. Alkali metal (for
example, sodium, potassium or lithium) or alkaline earth metal (for
example calcium) salts of carboxylic acids can also be made.
[0307] Any of the TC- or IF-binding carriers described herein can
be administered as a prodrug to increase the activity,
bioavailability, stability or otherwise alter the properties of the
carrier. A number of prodrug ligands are known. In general,
alkylation, acylation or other lipophilic modification of the mono,
di or triphosphate of the G.sup.1 substituent on the five-membered
"sugar-ring" moiety will increase the stability of the carrier.
Examples of substituent groups that can replace one or more
hydrogens on the phosphate moiety are alkyl, aryl, steroids,
carbohydrates, including sugars, 1,2-diacylglycerol and alcohols.
Many are described in R. Jones and N. Bischofberger, Antiviral
Research, 27 (1995) 1-17. Any of these can be used in combination
with the disclosed carriers to achieve a desired effect.
[0308] The G.sup.1 substituent of the active carrier can also be
provided as a 5'-phosphoether lipid or a 5'-ether lipid, as
disclosed in the following references, which are incorporated by
reference herein: Kucera, L. S., N. Iyer, E. Leake, A. Raben,
Modest E. K., D. L. W., and C. Piantadosi. 1990. "Novel
membrane-interactive ether lipid analogs that inhibit infectious
HIV-1 production and induce defective virus formation." AIDS Res.
Hum. Retro Viruses. 6:491-501; Piantadosi, C., J. Marasco C. J., S.
L. Morris-Natschke, K. L. Meyer, F. Gumus, J. R. Surles, K. S.
Ishaq, L. S. Kucera, N. Iyer, C. A. Wallen, S. Piantadosi, and E.
J. Modest. 1991. "Synthesis and evaluation of novel ether lipid
nucleoside conjugates for anti-HIV activity." J. Med. Chem.
34:1408.1414; Hosteller, K. Y., D. D. Richman, D. A. Carson, L. M.
Stuhmiller, G. M. T. van Wijk, and H. van den Bosch. 1992. "Greatly
enhanced inhibition of human immunodeficiency virus type 1
replication in CEM and HT4-6C cells by 3'-deoxythymidine
diphosphate dimyristoylglycerol, a lipid prodrug of
3,-deoxythymidine." Antimicrob. Agents Chemother. 36:2025.2029;
Hosetler, K. Y., L. M. Stuhmiller, H. B. Lenting, H. van den Bosch,
and D. D. Richman, 1990. "Synthesis and antiretroviral activity of
phospholipid analogs of azidothymidine and other antiviral
nucleosides." J. Biol. Chem. 265:61127.
[0309] Nonlimiting examples of U.S. patents that disclose suitable
lipophilic substituents that can be covalently incorporated into
the TC- or IF-binding agent, preferably at the G1 position of the
carrier or lipophilic preparations, include U.S. Pat. Nos.
5,149,794 (Sep. 22, 1992, Yatvin et al.); 5,194,654 (Mar. 16, 1993,
Hostetler et al., 5,223,263 (Jun. 29, 1993, Hostetler et al.);
5,256,641 (Oct. 26, 1993, Yatvin et al.); 5,411,947 (May 2, 1995,
Hostetler et al.); 5,463,092 (Oct. 31, 1995, Hostetler et al.);
5,543,389 (Aug. 6, 1996, Yatvin et al.); 5,543,390 (Aug. 6, 1996,
Yatvin et al.); 5,543,391 (Aug. 6, 1996, Yatvin et al.); and
5,554,728 (Sep. 10, 1996; Basava et al.), all of which are
incorporated herein by reference. Foreign patent applications that
disclose lipophilic substituents that can be attached to the
carrier of the present invention, or lipophilic preparations,
include WO 89/02733, WO 90/00555, WO 91/16920, WO 91/18914, WO
93/00910, WO 94/26273, WO 96/15132, EP 0 350 287, EP 93917054.4,
and WO 91/19721.
[0310] VIII. Stereoisomerism and Polymorphism
[0311] Compounds of the present invention having a chiral center
may exist in and be isolated in optically active and racemic forms.
Some compounds may exhibit polymorphism. The present invention
encompasses racemic, optically-active, polymorphic, or
stereoisomeric form, or mixtures thereof, of a compound of the
invention, which possess the useful properties described herein.
The optically active forms can be prepared by, for example,
resolution of the racemic form by recrystallization techniques, by
synthesis from optically-active starting materials, by chiral
synthesis, or by chromatographic separation using a chiral
stationary phase or by enzymatic resolution.
[0312] IX. Transport Proteins
[0313] In humans, the average daily intake (in a Western diet) of
vitamin B 12 is about 4-5 g. Additional synthesis of cobalamin may
be produced in the ileum and the right colon, but in an unknown
amount. The total lumenal cobalamin that must be assimilated each
day in humans is estimated at 7-14 g, the sum of the dietary and
endogenous cobalamin. Intestinal epithelial cells possess carriers
and transporters that are highly efficient in the uptake of the
small products of digestion, such as vitamins, minerals and amino
acids. These mechanisms are necessary for the uptake of these
molecules, as the epithelial cell layer presents an almost
impenetrable barrier to peptides larger than five or six amino
acids in size. The cobalamins of the present invention are large
molecules that are not absorbed directly from the intestine, as
they are too big to diffuse across the intestinal wall. Therefore,
the absorption of the cobalamins is dependent upon transport
proteins. The uptake of vitamin B12 from the intestine to the blood
is perhaps the most complex uptake mechanism of all the vitamins,
involving at least four separate cobalamin binding proteins and
receptors.
[0314] Three distinct groups of transport proteins are involved in
the absorption and transport of cobalamins: intrinsic factor (IF),
haptocorrin (HC; also called R-protein; in which transcobalamin I
(TC-I) and transcobalamin III (TC-III) are members) and
transcobalamin II (TC-II). Both IF and TC II deficiencies lead to
abnormalities such as megaloblastic anemia and demyelinating
disorder of the nervous system. Each protein only has one subunit
and one binding site to cobalamin. IF is a 45 kDa (in humans) to 55
kDa (in hogs) plasma glycoprotein with 15% carbohydrate content.
HC's are 58 kDa (in humans) to 60 kDa (in rabbits) plasma
glycoproteins of 33-40% carbohydrate content with 16-19 sialic acid
residues. Human TC-II is a 43 kDa plasma protein (in humans) with
O% carbohydrate content. Each binding protein has a separate
affinity for cobalamin, as well as separate cell receptors.
Generally, cobalamin is initially bound by HC in the stomach,
followed by IF in the small intestine. An IF receptor is then
involved in the uptake of the IF-cobalamin complex by the
intestinal epithelial cell, leading to the proteolytic release of
cobalamin, and subsequent binding to TC-II.
[0315] Intrinsic factor (IF) and haptocorrin (HC; are the main
intestinal lumenal cobalamin binders. In particular IF is of
particular relevance to the field of oral peptide and protein
delivery. Therefore, IF is mainly produced in the gastric body and
medium sized ducts and HC is mainly produced in granulocytes, the
yolk sac, mammary glands, salivary acini and ducts. In general, in
plasma or serum, cobalamin is also bound to HC (derived from white
cells) or to TC-II. The former complex is taken up by the liver,
delivering free cobalamin to the intestinal lumen as the first limb
of an enterohepatic circulation.
[0316] IF is the most specific of the cobalamin-binding proteins.
Cyanocobalamin, hydoxy-cobalamin (HOCbl), methylcobalamin (MeCbl)
and adenyosylcobalamin (AdoCbl) bind to intrinsic factor with
similar affinities, thereby suggesting that the upper-axial ligand
of the cobalt does not influence the binding significantly.
However, after dietary release of vitamin B12, the affinity for the
cobalamin for IF is reduced, due to the low pH. Rather, the
released vitamin B.sub.12 is preferentially bound to salivary HC,
as HC may protect the vitamin from acid hydrolysis (possibly due to
the extensive glycosylation of HC).
[0317] HC comprises a group of immunologically identical proteins
secreted into many body fluids (plasma, milk, amniotic fluid,
saliva and gastric juices) from many types of cells (granulocytes,
mammary glands, yolk sac or visceral placental membranes, salivary
duct and acinar cells, and gastric mucosa of some species). These
proteins were known previously as R proteins (for rapid
electrophoresis), non-intrinsic factors or transcobalamin I and
III. They are characterized by different glycosylation processes
and account for much of the total bound cobalamin in the serum
(about 80% of bound cobalamin in serum). HC turns over very slowly
(t.sub.e,fra 1/2=10 days) and appears to serve as the major storage
protein for cobalamin and may also stabilize serum cobalamin
against transdermal photolysis (Allen, R. H. Prog Hematol. 1975, 9,
57-84).
[0318] Within the proximal small intestine, HC is degraded by
pancreatic enzymes, freeing cobalamin to combine with other
transport proteins, most notably IF. In contrast to HC's, the
IF-cobalamin complex is resistant to proteolytic digestion. Once
the cobalamin-transport protein is internalized via
receptor-mediated endocytosis, the cobalamin is cleaved from
transport protein via protease(s) and bound to transcobalamin II
(TC II). From there, the TC I-cobalamin complex is used for the
transport of absorbed cobalamin to peripheral tissues. Therefore,
TC-II is found in most tissues. Antibodies to TC II inhibit the
transport of cobalamins and block the proliferation of leukemic
cells in vitro (McLean, G. R. et al. Blood, 1997 , 89, 235-242). In
cow's milk, in particular, the major cobalamin binder is not HC,
but rather TC-II (Fedosov, S. N. et al. Biochemistry 1995, 34,
16082-16087 and Fedosov, S. N. et al. Biochim. Biophys. Acta. 1996,
1292, 113-119).
[0319] Early attempts to purify transport proteins utilized
conventional techniques such as ammonium sulfate fractionation, ion
exchange and size exclusion chromatography. These methods yielded a
product that was devoid of the other types transport proteins, and
in particular, separation of TC-II from TC-I was possible, but
contained other plasma proteins. The introduction of affinity
chromatography provided pure proteins even in extremely low
concentrations. Three main types of affinity columns have been used
to purify the transport proteins, in particular, columns containing
cobalamin coupled to different matrices. The first was a
monocarboxylic acid derivative of cobalamin linked to Sepharose
beads via a diamino-dipropylamine spacer arm (Allen, R. H. et al.
J. Biol Chem. 1972, 247, 7695-7701 and Allen, R. H. et al. J. Biol
Chem. 1973, 248, 3660-3669). However, it may be necessary to use a
chaotropic reagent to elute the protein from the matrix, possibly
resulting in a denatured transport protein, which may not be able
to renature. For instance, the elution of the bound protein from
Cohn fraction III of human plasma, a mixture that contains 27-40%
of the plasma TC-II, required the use of guanidine hydrochloride to
release the denatured TC-I1, which could not be renatured.
[0320] To avoid the use of chaotropic reagents, temperature- or
photolabile linkages between the cobalamin and the insoluble matrix
were developed (Nexo, E. "Cobalamin binding proteins," in Vitamin
B.sub.12 and B.sub.12-proteins, eds Krantler, B.; Arigoni, D. and
Golding, B. T.; Wiley & Sons, Ltd. 461-475). Matrices formed in
this manner are able to release the transport protein by
dissociating the cobalamin from the matrix, thus providing the
transport protein saturated with cobalamin, circumventing the
denaturant effect of chaotrophic agents.
[0321] In a preferred embodiment, for large scale purification of
transport protein, ion exchange chromatography or ammonium sulfate
fractionation is used prior to the purification of the transport
protein via an affinity column to concentrate the sample. In an
alternate embodiment, ion exchange or size exclusion chromatography
is used subsequent to the purification of the transport protein via
an affinity column.
[0322] X. Disorders Characterized by Abnormal Cellular
Proliferation Non-limiting examples of proliferative disorders
other than neoplasms that can be treated and/or imaged with a TC-
or IF-binding carrier linked to a therapeutic and/or diagnostic
agent include those in Table 1, as well as any others listed or
described in the Background of the Invention or otherwise in the
specification.
1TABLE 1 Organ System Disease/Pathology Dermatological Psoriasis
(all forms), acne vulgaris, acne rosacea, common warts, anogenital
(venereal) warts, eczema; lupus associated skin lesions;
dermatitides such as seborrheic dermatitis and solar dermatitis;
keratoses such as seborrheic keratosis, senile keratosis, actinic
keratosis, photo-induced keratosis, skin aging, including photo-
induced skin aging, keratosis follicularis, keloids and Prophylaxis
against keloid formation; leukoplakia, lichen, planus, keratitis,
contact dermatitis, eczema, urticaria, pruritus, hidradenitis, acne
inversa Cardiovascular Hypertension, vasculo-occlusive diseases
including Atherosclerosis, thrombosis and restenosis after
angioplasty; acute coronary syndromes such as unstable angina,
myocardial infarction, ischemic and non-ischemic cardiomyopathies,
post-MI cardiomyopathy and myo- cardial fibrosis, substance-induced
cardiomyopathy. Endocrine Insulin resistant states including
obesity, diabetes mellitus (types 1 & 2), diabetic retinopathy,
macular degeneration associated with diabetes, gestational
diabetes, impaired glucose tolerance, polycystic ovarian syndrome;
osteoporosis, osteopenia, accelerated aging of tissues and organs
including Werner's syndrome. Urogenital Endometriosis, benign
prostatic hyperplasia, leiomyoma, Polycystic kidney disease,
diabetic nephropathy. Pulmonary Asthma, chronic obstructive
pulmonary disease (COPD), reactive Airway disease, pulmonary
fibrosis, pulmonary hypertension. Connective Immunological
Rheumatoid arthritis, Raynaud's tissue/joints phenomenon/disease,
Sjogren's Syndrome, systemic sclerosis, systemic lupus
erythematosus, vasculitides, ankylosing spondylitis,
osteoarthritis, reactive arthritis, psoriatic arthritis,
fibromyalgia. Other Fibrocystic breast disease, fibroadenoma,
chronic fatigue syndrome.
[0323] Nonlimiting examples of neoplastic diseases or malignancies
treatable and/or diagnosable with the TC- or IF-binding carrier
linked to a therapeutic and/or diagnostic agent are listed in Table
2.
2TABLE 2 Organ System Malignancy/Cancer type Skin Basal cell
carcinoma, melanoma, squamous cell carcinoma; cutaneous T cell
lymphoma; Kaposi's sarcoma. Hema- Acute leukemia, chronic leukemia
and myelodysplastic tological syndromes. Urogenital Prostatic,
renal and bladder carcinomas, anogenital carcinomas including
cervical, ovarian, uterine, vulvar, vaginal, and those associated
with human papilloma virus infection. Neuro- Gliomas including
glioblastomas, astrocytoma, ependymoma, logical medulloblastoma,
oligodendroma; meningioma, pituitary adenoma, neuroblastoma,
craniopharyngioma. Gastro- Colon, colorectal, gastric, esophageal,
mucocutaneous intestinal carcinomas. Breast Breast cancer including
estrogen receptor and progesterone Receptor positive or negative
subtypes, soft tissue tumors. Metastasis Metastases resulting from
the neoplasms. Skeletal Osteogenic sarcoma, malignant fibrou
histeocytoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma,
myeloma. Diffuse Lymphoma (non-Hodgkin's or Hodgkin's), sickle cell
anemia. Tumors Other Angiomata, angiogenesis associated with the
neoplasms.
[0324] XI. Synthetic Techniques
[0325] Various synthetic techniques are known for preparing the
compounds of the present invention. For example, compounds wherein
the residue of a therapeutic and/or diagnostic agent is directly
linked to the 6-position of a compound of formula I (i.e. in which
X is L-T and L is a direct bond) can be prepared by reducing a
corresponding Co (III) compound of formula I to form a nucleophilic
Co (I) compound and treating this Co (I) compound with a residue of
a therapeutic and/or diagnostic agent (or a derivative thereof)
comprising a suitable leaving group, such as a halide. Similarly,
compounds wherein X is L-T and L is other than a direct bond can be
prepared by preparing a nucleophilic Co (I) species as described
herein above, and reacting it with a linker comprising a suitable
leaving group, such as a halide. Peptides and amino acids can be
attached to the 6-position by reducing a corresponding Co (III)
compound of formula I to form a nucleophilic Co (I) compound, and
treating the Co (I) compound with a suitable alkylating agent
comprising an amino acid or peptide.
[0326] Coupling of L-T to the ribose moiety at K or G.sup.1 may be
accomplished by activating the natural OH at either K or G.sup.1
with a suitable reagent such as succinic anhydride, to yield a
reactive group such as a carboxylate. This technique is described
in detail in Toraya, Bioinorg. Chem. 4:245-255, 1975.
[0327] Coupling of L-T to M can be accomplished using techniques
described in detail in Jacobsen, Anal. Biochem. 113:164-171,
1981.
[0328] The residue of vitamin B.sub.12 or its analog can be
prepared by any suitable means known in the art. For example, a
monocarboxylic acid or dicarboxylic acid of cobalamin can be
prepared as disclosed in U.S. Pat. No. 5,739,313. These compounds
can be prepared by the mild acid hydrolysis of cyanocobalamin,
which has been shown to yield a mixture of mono-, a dicarboxylic
acid and one tricarboxylic acid. These carboxylic acids are derived
from the propionamide side chains designated b, d- and e-, as
discussed hereinabove, which are more susceptible to hydrolysis
than the amide groups on acetamide side chains a-, c- and g-. The
b-, d-, and e-monocarboxylic acids can be separated by column
chromatography. L. Anton et al., J. Amer. Chem. Soc.,102, 2215
(1980). See, also, J B. Armitage et al., L Chem. Sot., 3349 (1953);
K. Bernhauer, Biochem. Z., 344, 289 (1966); H. P. C. Hogenkamp et
al., Biochemistry, 14, 3707 (1975); and L. Ellenbogen, in
"Cobalamin," Biochem. and Pathophysiol, B. Babior, ed., Wiley, N.Y.
(1975) at chapter 5.
[0329] Additional compounds, intermediates, and synthetic
preparations thereof are disclosed, for example, in Hogenkamp, H.
et al., Synthesis and Characterization of nido-Carborane-Cobalamin
Conjugates, Nucl. Med. & Biol., 2000, 27, 89-92; Collins, D.,
et al., Tumor Imaging Via Indium 111-Labeled
DTPA-Adenosylcobalamin, Mayo Clinic Proc., 1999, 74:687-691.
[0330] XII. Diagnostic Compositions and Administration
[0331] Preferred modes of administration of the TC- or IF-binding
carriers and therapeutic and/or diagnostic agents are parenteral,
intravenous, intradermal, intra-articular, intra-synovial,
intrathecal, intra-arterial, intracardiac, intramuscular,
subcutaneous, intraorbital, intracapsular, intraspinal,
intrasternal, topical, transdermal patch, via rectal, vaginal or
urethral suppository, peritoneal, percutaneous, nasal spray,
surgical implant, internal surgical paint, infusion pump, or via
catheter. In one embodiment, the agent and carrier are administered
in a slow release formulation such as an implant, bolus,
microparticle, microsphere, nanoparticle or nanosphere. For
standard information on pharmaceutical formulations, see Ansel, et
al., Pharmaceutical Dosage Forms and Drug Delivery Systems, Sixth
Edition, Williams & Wilkins (1995).
[0332] The TC- or IF-binding carriers/therapeutic and/or diagnostic
agents can, for example, be administered intravenously or
intraperitoneally by infusion or injection. Solutions of the
substance can be prepared in water, optionally mixed with a
nontoxic surfactant. Dispersions can also be prepared in glycerol,
liquid polyethylene glycols, triacetin and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0333] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the substance which are adapted for the
extemporaneous preparation of sterile injectable or infusible
solutions or dispersions, optionally encapsulated in liposomes. In
all cases, the ultimate dosage form must be sterile, fluid and
stable under the conditions of manufacture and storage. The liquid
carrier or vehicle can be a solvent or liquid dispersion medium
comprising, for example, water, normal saline, ethanol, a polyol
(for example, glycerol, propylene glycol, liquid polyethylene
glycols, and the like), vegetable oils, nontoxic glyceryl esters,
and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the formation of liposomes, by the
maintenance of the required particle size in the case of
dispersions or by the use of surfactants. The prevention of the
action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, benzyl alcohol, sorbic acid, thimerosal and
the like. In many cases, it will be preferable to include isotonic
agents, for example, sugars, buffers or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin.
[0334] Sterile injectable solutions are prepared by incorporating
the substance in the required amount in the appropriate solvent
with various of the other ingredients enumerated above, as
required, followed by filter sterilization. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and the freeze
drying techniques, which yield a powder of the active ingredient
plus any additional desired ingredient present in the previously
sterile-filtered solutions.
[0335] Injectable solutions are particularly advantageous for local
administration of the therapeutic composition. In particular,
parenchymal injection can be used to deliver the therapeutic
composition directly to a tumorous growth. Intra-articular
injection is a preferred alternative in cases of arthritis where
the practitioner wishes to treat one or only a few (such as 2-6)
joints. Additionally, the therapeutic compounds are injected
directly into lesions (intra-lesion administration) in appropriate
cases. Intradermal administration is an alternative for dermal
lesions.
[0336] The therapeutic compound is optionally administered
topically by the use of a transdermal therapeutic system (see,
Barry, Dermatological Formulations, (1983) p. 181 and literature
cited therein). Transdermal drug delivery (TDD) has several
advantages over oral delivery. When compared to oral delivery, TDD
avoids gastrointestinal drug metabolism, reduces first pass effects
and provides a sustained release of drugs for up to seven days
(Elias, et al. Percutaneous Absorption: Mechanisms-Methodology-Drug
Delivery; Marcel Dekker, NY: 1, 1989). This method is especially
useful with many therapeutic proteins that are susceptible to
gastrointestinal degradation and exhibit poor gastrointestinal
uptake. When compared to injections, TDD eliminates the associate
pain and the possibility of infection. While such topical delivery
systems have been designed largely for transdermal administration
of low molecular weight drugs, by definition they are capable of
percutaneous delivery. They can be readily adapted to
administration of the therapeutic compounds of the invention by
appropriate selection of the rate-controlling microporous membrane.
Topical application can also be achieved by applying the compound
of interest, in a cream, lotion, ointment, or oil based carrier,
directly to the skin. Typically, the concentration of therapeutic
compound in a cream, lotion or oil is 1-2%.
[0337] For drug targeting to lung tissue, the therapeutic compound
is formulated into a solution, suspension, aerosol or particulate
dispersion appropriate for application to the pulmonary system. The
therapeutic agent may be inhaled via nebulizer, inhalation capsule,
inhalation aerosol, nasal solution, intratracheal as a solution via
syringe, or endotracheal tube as an aerosol or via as a nebulizer
solution. Aerosols are prepared using an aqueous aerosol, liposomal
preparation or solid particles containing the compound. A
nonaqueous (e.g. fluorocarbon propellant) suspension could be used.
Sonic nebulizers are preferred because they minimize exposing the
therapeutic compound to shear, which can result in degradation of
the compound.
[0338] Delivery of the cobalamin conjugates of the instant
invention by the mucosal route also offers an attractive
administration alternative. The prototype formulation for nasal
solutions will contain the vitamin B.sub.12 conjugate dissolved in
a suitable aqueous or non-aqueous solvent such as propylene glycol,
an antioxidant and aromatic oils as flavoring agents. The
formulation may also contain suitable propellant(s).
[0339] For ophthalmic applications, the therapeutic compound is
formulated into solutions, suspensions and ointments appropriate
for use in the eye. For opthalmic formulations, see Mitra (ed.),
Ophthalmic Drug Delivery Systems, Marcel Dekker, Inc., New York,
New York (1993), and also Havener, W. H., Ocular Pharmacology, C.
V. Mosby Co., St. Louis (1983).
[0340] Useful dosages of the compounds of formula I can be
determined by comparing their in vitro activity, and in vivo
activity in animal models. Methods for the extrapolation of
effective dosages in mice, and other animals, to humans are known
to the art; for example, see U.S. Pat. No. 4,938,949. The amount of
the substance required for use in treatment, prophylaxis and/or
diagnosis will vary not only with the particular salt selected but
also with the route of administration, the nature of the condition
being treated and the age and condition of the patient and will be
ultimately at the discretion of the attendant physician or
clinician.
[0341] In general, however, a suitable dose for nuclear medicine
(using a radioactive therapeutic and/or diagnostic agent) will be
in the range of from about 0.1 .mu.g/patient to about 1000
.mu.g/patient, from about 0.5 to about 500 .mu.g/patient, or from 1
.mu.g/patient to about 100 .mu.g/patient.
[0342] A suitable dose for imaging medicine (using a paramagnetic
therapeutic and/or diagnostic agent) will be in the range of from
about 0.1 mg/patient to about 100 mg/patient, from about 0.5 to
about 50 mg/patient, or from 1 mg/patient to about 10
mg/patient.
[0343] For therapeutic applications, a suitable dose will be in the
range of from about 0.05 picograms/kilogram to about 100 mg/kg,
from about 10 to about 75 mg/kg of body weight per day, such as 3
to about 50 mg per kilogram body weight of the recipient per day,
preferably in the range of 6 to 90 mg/kg/day, most preferably in
the range of 15 to 60 mg/kg/day. The substance is conveniently
administered in unit dosage form; for example, containing 5 to 1000
mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of
active ingredient per unit dosage form.
[0344] Ideally, the substance should be administered to achieve
peak plasma concentrations of from about 0.05 to about 100 .mu.M,
preferably, about 1 to 50 .mu.M, most preferably, about 2 to about
30 .mu.M. This may be achieved, for example, by the intravenous
injection of a 0.005 to 10% solution of the substance, optionally
in saline, or orally administered as a bolus containing about
0.5-250 mg of the substance. Desirable blood levels may be
maintained by continuous infusion to provide about 0.01-5.0
mg/kg/hr or by intermittent infusions containing about 0.4-15 mg/kg
of the substance.
[0345] The substance may conveniently be presented in a single dose
or as divided doses administered at appropriate intervals, for
example, as two, three, four or more sub-doses per day.
[0346] The cobalamin conjugates may be administered orally in
combination with a pharmaceutically acceptable vehicle such as an
inert diluent or an edible carrier. They may be enclosed in hard or
soft shell gelatin capsules, may be compressed into tablets, or may
be incorporated directly with the food of the patient's diet. For
oral therapeutic administration, the substance may be combined with
one or more excipients and used in the form of ingestible tablets,
buccal tablets, troches, capsules, elixirs, suspensions, syrups,
wafers, and the like. Such compositions and preparations should
contain at least 0.1% of the substance. The percentage of the
compositions and preparations may, of course, be varied and may
conveniently be between about 2 to about 60% of the weight of a
given unit dosage form. The amount of substance in such
therapeutically useful compositions is such that an effective
dosage level will be obtained.
[0347] Tablets, troches, pills, capsules, and the like may also
contain the following: binders such as gum tragacanth, acacia, corn
starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, fructose, lactose or aspartame or
a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring may be added. When the unit dosage form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier, such as a vegetable oil or a polyethylene glycol. Various
other materials may be present as coatings or to otherwise modify
the physical form of the solid unit dosage form. For instance,
tablets, pills or capsules may be coated with gelatin, wax, shellac
or sugar and the like. A syrup or
[0348] elixir may contain the active compound, sucrose or fructose
as a sweetening agent, methyl and propylparabens as preservatives,
a dye and flavoring such as cherry or orange flavor. Of course, any
material used in preparing any unit dosage form should be
pharmaceutically acceptable and substantially non-toxic in the
amounts employed. In addition, the substance may be incorporated
into sustained-release preparations and devices.
[0349] Sublingual tablets are designed to dissolve very rapidly.
Examples of such formulations include ergotamine tartrate,
isosorbide dinitrate, isoproterenol HCl. The formulation of these
tablets contain, in addition to the drug, a limited number of
soluble excipients, usually lactose and powdered sucrose, but
occasionally dextrose and mannitol.
[0350] The process of making sublingual tablets involves moistening
the blended powder components with an alcohol-water solvent system
containing approximately 60% alcohol and 40% water.
[0351] In addition to the cobalamin conjugate, the prototype
formulation for sublingual tablets may contain a binder such as
povidone or HPMC, diluents such as lactose, mannitol, starch or
cellulose, a disintegrant such as pregelatinized or modified
starch, lubricants such as magnesium stearate, stearic acid or
hydrogenated vegetable oil, a sweetener such as saccharin or
sucrose and suitable flavoring and coloring agents.
[0352] It is preferred that the TC- or IF-binding carrier and the
therapeutic and/or diagnostic agent be administered parenterally,
not orally, to increase bioavailability and delivery to
proliferative tissue.
[0353] The TC- or IF-binding carrier and the therapeutic and/or
diagnostic agent can be administered in the course of surgical or
medical treatment, prophylaxis and/or diagnosis of the afflicted
site. For example, the TC- or IF-binding carrier and therapeutic
and/or diagnostic agent can be positioned directly at the site of
proliferation during the course of surgery either by painting the
formulation onto the surface of the afflicted area (with or without
a carrier matrix) or by depositing a bolus of material in a
suitable matrix that is released into the afflicted area over time.
In another embodiment, the TC- or IF-binding carrier and the
therapeutic and/or diagnostic agent are administered directly into
the proliferative mass via injection or catheter.
[0354] In yet another embodiment, a TC- or IF-binding carrier
attached to a radiodiagnostic can be used in lymphoscintigraphy, to
identify the sentinel or closest lymph node to an afflicted site of
hyperproliferation for investigation of the existence or possible
spread of disease, for example, as occurs in metastasis. In this
embodiment, the TC-binding or IF-binding agent and radiodiagnostic
are administered, preferably via injection, to a site
circumferential to the afflicted area in the skin. The
radiodiagnostic is preferentially absorbed into the sentinal node
or proliferating cells due to the presence of the TC-binding or
IF-binding agent, and then is monitored in its normal course of
travel in the lymphatic system to the closest lymph node. Colored
dye is sometimes also added to help identify the sentinel node. The
node is then removed and checked for the presence of abnormally
proliferating cells.
[0355] In one embodiment, the agent and carrier are administered in
a slow release formulation such as an implant, bolus,
microparticle, microsphere, nanoparticle or nanosphere. Nonlimiting
examples of sustained release compositions include semi-permeable
polymer matrices in the form of shaped articles, e.g., films,
microcapsules or microspheres. Sustained release matrices include,
for example, polylactides (U.S. Pat. No. 3,773,919), copolymers of
L-glutamic acid and y-ethyl-L-glutamate (Sidman et al., Biopolymers
22:547-556, 1983), or poly-D-(-)-3-hydroxybut- yric acid (EP
133,988). Sustained release compositions also include one or more
liposomally entrapped compounds of formula I. Such compositions are
prepared by methods known per se, e.g., as taught by Epstein et al.
Proc. Natl. Acad. Sci. USA 82:3688-3692, 1985. Ordinarily, the
liposomes are of the small (200-800 .ANG.) unilamellar type in
which the lipid content is greater than about 30 mol % cholesterol,
the selected proportion being adjusted for the optimal therapy.
[0356] A number of sustained-release implants are known in the art.
Most implants are "matrix" type, and comprise an active compound
dispersed in a matrix of a carrier material. The carrier material
may be either porous or non-porous, solid or semi-solid, and
permeable or impermeable to the active compound. Matrix devices are
typically biodegradable, i.e., they slowly erode after
administration. Alternatively, matrix devices may be nondegradable,
and rely on diffusion of the active compound through the walls or
pores of the matrix. Matrix devices are preferred for the
applications contemplated herein.
[0357] Thus, in one embodiment the invention provides a surgical
implant for localized delivery of an anti-proliferative agent
comprising the cobalamin conjugate of the present invention, and a
biodegradable binder. The implant preferably is capable of
releasing and delivering the cobalamin conjugate to substantially
all of an area of clear margin that results from a surgical
resection, and is also preferably capable of releasing the
cobalamin conjugate at a substantially constant rate. In another
embodiment the invention provides a method of delivering a
therapeutic and/or diagnostic agent to an area of clear margin
following a surgical resection comprising (i) providing an implant
comprising a TC- or IF-binding carrier linked to a therapeutic
and/or diagnostic agent and a biodegradable binder; and (ii)
placing the implant into a void created by surgical resection.
[0358] The surgical implant can exhibit a variety of forms. In one
embodiment the implant is a bolus, comprising a viscous and
deformable material capable of being shaped and sized before or
during implantation to complement a void created by a surgical
resection, and sufficiently deformable upon implantation to contact
substantially all of an area of clear margin. The surgical implant
can also comprising a plurality of capsules that can be poured into
the void created by a surgical resection. These capsules will
contain the cobalamin conjugate and a suitable binder. Because they
are flowable, they can be poured into the void created by a
surgical lumpectomy, and thereby contact substantially all of the
areas of clear margin.
[0359] Many suitable compositions for the implant are known and can
be used in practicing the invention. Such compositions are
described in, for example, Chasin et al., Biodegradable Polymers as
Drug Delivery Systems, Marcel Dekker Inc., NY, ISBN 0-8247-8344-1.
Preferable compositions are pharmaceutically acceptable,
biodegradable, and meet the particular release profile
characteristics that are required to achieve the administration
regime involved.
[0360] The implant typically comprises a base composition that acts
as a matrix to contain and hold the contents of the implant
together. The base composition can, in turn, comprise one or more
constituents. Examples of base compositions include polymers and
copolymers of anhydrides, orthoester, lactic acid, glycolic acid,
dioxonane, trimethylene carbonate, .epsilon.-caprolactone,
phosphazene and glyceryl monostearate.
[0361] In one embodiment the base composition for the matrix
comprises a polyanhydride, which can be synthesized via the
dehydration of diacid molecules by melt condensation. Degradation
times can be adjusted from days to years according to the
hydrophobicity of the monomer selected. The materials degrade
primarily by surface erosion and possess excellent in vivo
compatibility. In one embodiment the polyanhydride is formed from
sebasic acid and hexadecandioic acid (poly(SA-HDA anhydride).
Wafer-like implants using this base composition have been approved
for use in brain cancer, as Giadel.RTM., by Guilford
Pharmaceuticals.
[0362] The implant optionally can comprise erosion and
biodegradation enhancers that facilitate the erosion of the matrix,
the dissolution of the core composition, or the uptake of the core
composition via metabolic processes. Particularly suitable erosion
and biodegradation enhancers are biodegradable in biological
fluids, and biocompatible. Hydrophilic constituents are typical,
because they are capable of enhancing the erosion of the implant in
the presence of biological fluids. For example, K. Juni et al.,
Chem. Pharm. Bull., 33, 1609 (1985) disclose that the release rate
of bleomycin from polylactic acid microspheres is greatly enhanced
by incorporating fatty acid esters into the microspheres. Other
exemplary hydrophilic constituents are described, for example, in
Wade & Weller, Handbook of pharmaceutical Excipients (London:
Pharmaceutical Press; Washington D.C.: American Pharmaceutical
Ass'n 1995), and include the polyethylene glycols ("PEGs"),
propylene glycol ("PG"), glycerin, and sorbitol.
[0363] Surfactants further enhance the erosion of the matrix and
the release of the drug. Surfactants are generally capable of
increasing the wettability and the solubility of the base
composition in biological fluids, and thereby causing the
disintegration and erosion of the implant. Surfactants can also
help to break down the core composition matrix when, for example,
the method of forming the dosage form has reduced the solubility of
any of the constituents. Surfactants can also improve the uptake of
the dosage forms into the bloodstream. Suitable surfactants
include, for example, glyceryl based surfactants such as glyceryl
monooleate and glyceryl monolaurate, poloxamers such as Pluronic
F127, and polysorbates such as polyoxyethylene sorbitan monooleate
("Tween 80").
[0364] The implant could also include components that retard the
rate at which the implant erodes or biodegrades (erosion and/or
biodegradation retardants). Hydrophobic constituents are a
particularly suitable class of components for retarding the rate at
which the outer layer biodegrades. Suitable hydrophobic
constituents are described, for example, in the Handbook of
Pharmaceutical Excipients, the disclosure from which being hereby
incorporated by reference. Exemplary hydrophobic constituents
include peanut oil, olive oil and castor oil.
[0365] Any proportions or types of constituents can be chosen that
effectively achieve a desired release profile, and thereby carry
out the prescribed administration regime. The most desirable base
compositions generally release the drug substantially continuously,
and biodegrade completely shortly after substantially all of the
drug has been effectively released. The amount of drug included in
the dosage forms is determined by the total amount of the drug to
be administered, and the rate at which the drug is to be delivered.
The total amount of the drug to be delivered is determined
according to clinical requirements, and in keeping with the
considerations that typically inform drug dosage determinations in
other contexts. The surgical implant also can contain one or more
other drugs having therapeutic efficacy in the intended
applications, such as an antibiotic, an analgesic or an
anesthetic.
[0366] XIII. Controlled Release Formulations
[0367] The TC- or IF-binding carrier and therapeutic and/or
diagnostic agent is optionally administered in a controlled release
formulation, which can be a degradable or nondegradable polymer,
hydrogel, organogel, or other physical construct that modifies the
bioabsorption, half life or biodegradation of the TC- or IF-binding
carrier/therapeutic and/or diagnostic agent. The controlled release
formulation can be a material that is painted or otherwise applied
onto the afflicted site, either internally or externally. In one
embodiment, the invention provides a biodegradable bolus or implant
that is inserted into the pocket created by surgical resection of a
tumor, or directly into the tumor itself. In another example, the
controlled release formulation can be applied to a psoriatic
lesion, eczema, atopic dermatitis, lichen planus, wart, pemphigus
vulgaris, actinic keratosis, basal cell carcinoma or squamous cell
carcinoma. The controlled release formulation can likewise be
applied to a blood vessel to treat or prevent restenosis,
retinopathies or atherosclerosis. The controlled release
formulation with appropriated selected therapeutic and/or
diagnostic agent can be used to coat a transplanted organ or tissue
to prevent rejection. It can alternatively be implanted or
otherwise applied near the site of rheumatoid arthritis.
[0368] The field of biodegradable polymers has developed rapidly
since the synthesis and biodegradability of polylactic acid was
first reported in 1966 by Kulkarni et al. "Polylactic acid for
surgical implants," Arch. Surg., 93, 839. Several other polymers
are now known to biodegrade, such as polyanhydrides and
polyorthoesters, which take advantage of labile backbone linkages
(see: Domb et al. Macromolecules, 22, 3200, 1989; and Heller et al.
Biodegradable Polymers as Drug Delivery Systems, Dekker, N.Y.:
1990). Several polymers which degrade into naturally occurring
materials have also been described, such as crosslinking gelatin,
hyaluronic acid (della Valle et al. U.S. Pat. No. 4,987,744 and
U.S. Pat. No. 4,957,744) and polyaminoacids (Miyake et al., 1974),
which spurred the usage of polyesters by Holland et al. Controlled
Release, 4, 155, 1986 and alph-hydroxy acids (i.e. lactic acid and
glycolic acid), which remain the most widely used biodegradable
materials for applications ranging from closure devices (sutures
and staples) to drug delivery systems (Smith et al. U.S. Pat. No.
4,741,337; Spilizeqski et al. J. Control. Rel., 2, 197, 1985).
[0369] These polymers can be tailored to degrade at a desired rate
and with a desired kinetics by selecting the appropriate monomers,
method of preparation and molecular weight. Differences in
crystallinity of the monomer can alter the polymeric degradation
rate. Due to the relatively hydrophobic nature of most polymers,
actual mass loss can begin with the oligomeric fragments that are
small enough to be water soluble; hence, even the initial molecular
weight can influence the degradation rate.
[0370] Hydrogels can be used in controlled release formulations.
Such polymers are formed from macromers with a polymerizable,
non-degradable, region that is separated by at least one degradable
region. For example, the water soluble, non-degradable, region can
form the central core of the macromer and have at least two
degradable regions which are attached to the core, such that upon
degradation, the non-degradable regions (in particular a
polymerized gel) are separated. Specifically, as disclosed in U.S.
Pat. No. 5,626,863 to Hubbell et al., the macromers are
PEG-oligoglycolyl-acrylates, with the appropriate end caps to
permit rapid polymerization and gelation. Acrylates can be
polymerized readily by several initiating systems such as eosin
dye, ultraviolet or visible light. The polyethyleneglycol (PEG) is
highly hydrophilic and biocompatible. The oligoglycolic acid is a
poly(a-hydroxy acid) which can be readily degraded by hydrolysis of
the ester linkage into glycolic acid, a nontoxic metabolite. Other
chain extensions include polylactic acid, polycaprolactone,
polyorthoesters, polyanhydrides and polypeptides. This entire
network can be gelled into a biodegradable network that can be used
to entrap and homogeneously disperse water-soluble drugs for
delivery at a controlled rate. Further, the gel can entrap
particulate suspensions of water-insoluble drugs. (See also: U.S.
Pat. No. 4,591,496 to Cohen et al. (Process for Making Systems for
the Controlled Release of Macromolecules); U.S. Pat. No. 5,545,442
to Van Savage et al. (Method for Using a Radiation Cured Drug
Release Controlling Membrane); U.S. Pat. No. 5,330,768 to Park et
al. (Controlled Drug Delivery Using Polymer/Pluronic Blends); U.S.
Pat. No. 5,122,367 to Ron et al. (Polyanhydride Bioerodible
Controlled Release Implants for Administration of Stabilized Growth
Hormone); U.S. Pat. No. 5,545,409 to Laurencin et al. (Delivery
System for Controlled Release of Bioactive Factors); U.S. Pat. No.
5,629,009 to Laurencin et al. (Delivery System for Controlled
Release of Bioactive Factors).
[0371] Alternatively, delivery of biologically active substances,
both in vitro and in vivo, via encapsulation has been well
described in the prior art. U.S. Pat. No. 4,352,883 to Lim et al.
entitled "Encapsulation of Biological Material" discloses the
encapsulation of proteins within a membrane by suspending the
protein in an aqueous medium containing a water-soluble gum that
can be reversibly gelled to form the suspension into droplets.
These droplets can be gelled further into discrete,
shape-retaining, water insoluble temporary capsules with the aid of
a solution of multivalent cations. The temporary capsules then can
be further wrapped by an ionically cross-linking surface layer to
form a semipermeable membrane around the capsules that is permeable
to small molecules but impermeable to larger molecules.
Microencapsulations of glycoproteins have also been well described.
U.S. Pat. No. 4,324,683 to Lim et al. entitled "Encapsulation of
Labile Biological Material" encapsulates a glycoprotein by a
two-step interfacial polymerization process to form capsules with
well-controlled porosity. The microcapsules serve to protect the
active substances from attack by microorganisms and from any
immunological response. U.S. Pat. No. 5,718,921 to Mathiowitz et
al. (Microspheres Comprising Polymer and Drug Dispersed There
Within) discloses a method to encapsulate relatively
temperature-labile drugs into a microsphere.
[0372] Several methods have been developed to reversibly
encapsulate biologically active substances. One that can be applied
both to in vitro and in vivo studies has been described in U.S.
Pat. No. 4,900,556 by Wheatley et al. entitled "System for Delayed
and Pulsed Release of Biologically-Active Substances." In this
disclosed system, the biologically-active substance can be released
either at a constant rate over a period of time or in discrete
pulses. The biologically active materials are entrapped within
liposomes encapsulated within semipermeable microcapsules or
permeable polymeric matrix. Release of the desired materials is
governed by the permeability of both the liposome and the
surrounding matrix (the matrix integrity is directly proportional
to the liposome integrity); the permeability of the liposome can be
engineered by modifying the composition and the method for making
the liposome to produce liposome that are sensitive to specific
stimuli such as temperature, pH or light. For example, by including
a phospholipase that degrades the liposome within some or all of
the liposomes or the surrounding matrix, the liposome can be
destabilized and broken down over a period of time. Other systems
have been developed, e.g. U.S. Pat. No. 4,933,185 by Wheatley et
al., which utilize a core made up of a polymer (such as an
ionically cross-linked polysaccharide with calcium alginate or
chitin) around which there is an ionically bound skin (such as a
polycationic skin of poly-L-lysine) whose integrity is dependent on
the core polymer. With an impermeable skin, when the core polymer
can be degraded by enzymes (such as alginase from the bacteria,
chitinase or hydrolase), there is a sudden release of biologically
active substance from the core. Alternatively, the skin can be
partially permeable for a gradual release of drug upon degradation
of the core.
[0373] Nanoparticles are especially useful in the delivery of drugs
parenterally or intravenously such that the delivery device is
small with a long circulating half-life. A number of injectable
drug delivery systems have been investigated, including
microcapsules, microparticles, liposomes and emulsions. The major
obstacle for these delivery systems is the rapid clearance of the
materials from the blood stream by the macrophages of the
reticuloendothelial system (RES). For example, polystyrene
particles as small as sixty nanometers in diameter are cleared from
the blood within two to three minutes. Liposomal drug delivery
systems have also been extensively studied for this application
because they were expected to freely circulate in the blood.
Coating of the liposomes with poly(ethylene glycol) (PEG) increased
the half-life of the carriers due to PEG's hydrophobic chains which
reduced its protein absorption and thus its RES uptake. U.S. Pat.
No. 5,543,158 to Gref et al. (Biodegradable Injectable
Nanoparticles) describes a carrier system specifically targeted
towards carriers suitable for intravenous delivery with a
controlled release mechanism with modified polyglycols.
[0374] U.S. Pat. No. 5,626,862, U.S. Pat. No. 5,651,986 and U.S.
Pat. No. 5,846,565 to Brem et al. (Controlled Local Delivery of
Chemotherapeutic Agents for Treating Solid Tumors) discloses the
use of these carriers for the specific delivery of chemotherapeutic
agents to increase bioavailability. Therefore, the devices act as
reservoirs that release drugs over an extended period of time while
at the same time preserves the bioactivity and bioavailability of
the agent. U.S. Pat. No. 5,286,763 to Gerhard et al. (Bioerodible
Polymers for Drug Delivery in Bone) further discloses that
bioerodible polymers can be used to deliver chemotherapeutic agents
directly into the bone. Cohen et al. U.S. Pat. No. 5,562,099
(Polymeric Microparticles Containing Agents for Imaging) discusses
the usage of these carriers as contrast agents. The polymeric
microparticle is filled with contrast agents for enhanced
imaging.
[0375] Books describing methods of controlled delivery that are
appropriate for the delivery of the TC- or IF-binding
carriers/therapeutic and/or diagnostic agents of the present
invention include: Robert S. Langer, Donald L. Wise, editors;
Medical applications of controlled release (Volumes 1 and 2); Boca
Raton, Fla.: CRC Press, 1984; and William J. M. Hrushesky, Robert
Langer, and Felix Theeuwes, editors; Temporal control of drug
delivery (series); New York: New York Academy of Sciences,
1991.
[0376] Nonlimiting examples of U.S. Patents that describe
controlled release formulations are: U.S. Pat. No. 5,356,630 to
Laurencin et al. (Delivery System for Controlled Release of
Bioactive Factors); ; U.S. Pat. No. 5,797,898 to Santini, Jr. et
al. (Microchip Drug Delivery Devices); U.S. Pat. No. 5,874,064 to
Edwards et al. (Aerodynamically Light Particles for Pulmonary Drug
Delivery); U.S. Pat. No. 5,548,035 to Kim et al. (Biodegradable
Copolymer as Drug Delivery Matrix Comprising Polyethyleneoxide and
Aliphatic Polyester Blocks); U.S. Pat. No. 5,532,287 to Savage et
al. (Radiation Cured Drug Release Controlling Membrane); U.S. Pat.
No. 5,284,831 to Kahl et al. (Drug Delivery Porphyrin Composition
and Methods); U.S. Pat. No. 5,741,329 to Agrawal et al. (Methods of
Controlling the pH in the Vicinity of Biodegradable Implants); U.S.
Pat. No. 5,820,883 to Tice et al. (Methods for Delivering Bioactive
Agents into and Through the Mucosally-Associated Lymphoid Tissues
and Controlling Their Release);U.S. Pat. No. 5,955,068 to Gouin et
al. (Biodegradable polyanhydrides Derived from Dimers of Bile
Acids, and Use Thereof as Controlled Drug Release Systems); U.S.
Pat. No. 6,001,395 to Coombes et al. (Polymeric Lamellar Substrate
Particles for Drug Delivery);
[0377] U.S. Pat. No. 6,013,853 to Athanasiou et al. (Continuous
Release Polymeric Implant Carriers); U.S. Pat. No. 6,060,582 to
Hubbell et al. (Photopolymerizable Biodegradable Hydrogels as
Tissue Contacting Materials and Controlled Release Carriers); U.S.
Pat. No. 6,113,943 to Okada et al. (Sustained-Release Preparation
Capable of Releasing a Physiologically Active Substance); and PCT
Publication No. WO 99/59548 to Oh et al. (Controlled Drug Delivery
System Using the Conjugation of Drug to Biodegradable Polyester);
U.S. Pat. No. 6,123,861 (Fabrication of Microchip Drug Delivery
Devices);
[0378] U.S. Pat. No. 6,060,082 (Polymerized Liposomes Targeted to M
cells and Useful for Oral or Mucosal Drug Delivery); U.S. Pat. No.
6,041,253 (Effect of Electric Field and Ultrasound for Transdermal
Drug Delivery); U.S. Pat. No. 6,018,678 (Transdermal protein
delivery or measurement using low-frequency sonophoresis); U.S.
Pat. No. 6,007,845 Nanoparticles And Microparticles Of Non-Linear
Hydrophilic-Hydrophobic Multiblock Copolymers; U.S. Pat. No.
6,004,534 Targeted Polymerized Liposomes For Improved Drug
Delivery; U.S. Pat. No. 6,002,961 Transdermal Protein Delivery
Using Low-Frequency Sonophoresis; U.S. Pat. No. 5,985,309
Preparation Of Particles For Inhalation; U.S. Pat. No. 5,947,921
Chemical And Physical Enhancers And Ultrasound For Transdermal Drug
Delivery; U.S. Pat. No. 5,912,017 Multiwall Polymeric Microspheres;
U.S. Pat. No. 5,911,223 Introduction Of Modifying Agents Into Skin
By Electroporation; U.S. Pat. No. 5,874,064 Aerodynamically Light
Particles For Pulmonary Drug Delivery; U.S. Pat. No. 5,855,913
Particles Incorporating Surfactants For Pulmonary Drug Delivery;
U.S. Pat. No. 5,846,565 Controlled Local Delivery Of
Chemotherapeutic Agents For Treating Solid Tumors; U.S. Pat. No.
5,837,752 Semi-Interpenetrating Polymer Networks; U.S. Pat. No.
5,814,599 Transdermal Delivery Of Encapsulated Drugs; U.S. Pat. No.
5,804,178 Implantation Of Cell-Matrix Structure Adjacent Mesentery,
Omentum Or Peritoneum Tissue; U.S. Pat. No. 5,797,898 Microchip
Drug Delivery Devices; U.S. Pat. No. 5,770,417 Three-Dimensional
Fibrous Scaffold Containing Attached Cells For Producing
Vascularized Tissue In Vivo; U.S. U.S. Pat. No. 5,770,193
Preparation Of Three-Dimensional Fibrous Scaffold For Attaching
Cells To Produce Vascularized Tissue In Vivo; U.S. Pat. No.
5,762,904 Oral Delivery Of Vaccines Using Polymerized Liposomes;
U.S. Pat. No. 5,759,830 Three-Dimensional Fibrous Scaffold
Containing Attached Cells For Producing Vascularized Tissue In
Vivo; U.S. Pat. No. 5,749,847 Delivery Of Nucleotides Into
Organisms By Electroporation; U.S. Pat. No. 5,736,372 Biodegradable
Synthetic Polymeric Fibrous Matrix Containing Chondrocyte For In
Vivo Production Of A Cartilaginous Structure; U.S. Pat. No.
5,718,921 Microspheres Comprising Polymer And Drug Dispersed There
Within; U.S. Pat. No. 5,696,175 Preparation Of Bonded Fiber
Structures For Cell Implantation; U.S. Pat. No. 5,667,491 Method
For Rapid Temporal Control Of Molecular Transport Across Tissue;
U.S. Pat. No. 5,654,381 Functionalized Polyester Graft Copolymers;
U.S. Pat. No. 5,651,986 Controlled Local Delivery Of
Chemotherapeutic Agents For Treating Solid Tumors; U.S. Pat. No.
5,629,009 Delivery System For Controlled Release Of Bioactive
Factors; U.S. Pat. No. 5,626,862 Controlled Local Delivery Of
Chemotherapeutic Agents For Treating Solid Tumors; U.S. U.S. Pat.
No. 5,593,974 Localized Oligonucleotide Therapy; U.S. Pat. No.
5,578,325 Nanoparticles And Microparticles Of Non-Linear
Hydrophilic-Hydrophobic Multiblock Copolymers; U.S. Pat. No.
5,562,099 Polymeric Microparticles Containing Agents For Imaging;
U.S. Pat. No. 5,545,409 Delivery System For Controlled Release Of
Bioactive Factors; U.S. Pat. No. 5,543,158 Biodegradable Injectable
Nanoparticles; U.S. Pat. No. 5,514,378 Biocompatible Polymer
Membranes And Methods Of Preparation Of Three Dimensional Membrane
Structures; U.S. Pat. No. 5,512,600 Preparation Of Bonded Fiber
Structures For Cell Implantation; U.S. Pat. No. 5,500,161 Method
For Making Hydrophobic Polymeric Microparticles; U.S. Pat. No.
5,487,390 Gas-filled polymeric microbubbles for ultrasound imaging;
U.S. Pat. No. 5,399,665 Biodegradable polymers for cell
transplantation; U.S. Pat. No. 5,356,630 Delivery system for
controlled release of bioactive factors; U.S. Pat. No. 5,330,768
Controlled drug delivery using polymer/pluronic blends; U.S. Pat.
No. 5,286,763 Bioerodible polymers for drug delivery in bone; U.S.
Pat. No. 5,149,543 lonically cross-linked polymeric microcapsules;
U.S. Pat. No. 5,128,420 Method of making hydroxamic acid polymers
from primary amide polymers; U.S. Pat. No. 5,122,367 Polyanhydride
bioerodible controlled release implants for administration of
stabilized growth hormone; U.S. Pat. No. 5,100,668 Controlled
release systems containing heparin and growth factors; U.S. Pat.
No. 5,019,379 Unsaturated polyanhydrides; U.S. Pat. No. 5,010,167
Poly(amide-and imide-co-anhydride) for biological application; U.S.
Pat. No. 4,948,587 Ultrasound enhancement of transbuccal drug
delivery; U.S. Pat. No. 4,946,929 Bioerodible articles useful as
implants and prostheses having predictable degradation rates; U.S.
Pat. No. 4,933,431 One step preparation of poly(amide-anhydride);
U.S. Pat. No. 4,933,185 System for controlled release of
biologically active compounds; U.S. Pat. No. 4,921,757 System for
delayed and pulsed release of biologically active substances; U.S.
Pat. No. 4,916,204 Pure polyanhydride from dicarboxylic acid and
coupling agent; U.S. Pat. No. 4,906,474 Bioerodible polyanhydrides
for controlled drug delivery; U.S. Pat. No. 4,900,556 System for
delayed and pulsed release of biologically active substances; U.S.
Pat. No. 4,898,734 Polymer composite for controlled release or
membrane formation; U.S. Pat. No. 4,891,225 Bioerodible
polyanhydrides for controlled drug delivery; U.S. Pat. No.
4,888,176 Controlled drug delivery high molecular weight
polyanhydrides; U.S. Pat. No. 4,886,870 Bioerodible articles useful
as implants and prostheses having predictable degradation rates;
U.S. Pat. No. 4,863,735 Biodegradable polymeric drug delivery
system with adjuvant activity; U.S. Pat. No. 4,863,611
Extracorporeal reactors containing immobilized species; U.S. Pat.
No. 4,861,627 Preparation of multiwall polymeric microcapsules;
U.S. Pat. No. 4,857,311 Polyanhydrides with improved hydrolytic
degradation properties; U.S. Pat. No. 4,846,786 Bioreactor
containing suspended, immobilized species; U.S. Pat. No. 4,806,621
Biocompatible, bioerodible, hydrophobic, implantable polyimino
carbonate article; U.S. Pat. No. 4,789,724 Preparation of anhydride
copolymers; U.S. Pat. No. 4,780,212 Ultrasound enhancement of
membrane permeability; U.S. Pat. No. 4,779,806 Ultrasonically
modulated polymeric devices for delivering compositions; U.S. Pat.
No. 4,767,402 Ultrasound enhancement of transdermal drug delivery;
U.S. Pat. No. 4,757,128High molecular weight polyanhydride and
preparation thereof; U.S. Pat. No. 4,657,543 Ultrasonically
modulated polymeric devices for delivering compositions; U.S. Pat.
No. 4,638,045 Non-peptide polyamino acid bioerodible polymers; U.S.
Pat. No. 4,591,496 Process for making systems for the controlled
release of macromolecules.
[0379] The invention will now be illustrated by the following
non-limiting Examples.
EXAMPLES
[0380] Poly-L-lysine hydrobromide (MW 500-2000) and (MW 1000-4000),
adenosine, 1-ethyl-3(3'-dimethylaminopropyl) carbodiimide,
trifluoroacetic acid, trifluoroacetic anhydride,
2,2,2-trifluoroethylamin- e hydrochloride and
1-hydroxybenzotriazole were purchased from Sigma Chemical Co.
Sephodex-G-10 was purchased from Pharmacia Biotech, Inc. Thin layer
chromatography (TLC) silica gel plates were obtained from Eastman
Kodak Co. Solvents and other reagents were obtained in the highest
purity available. Cyanocobalamin-b, d and e monocarboxylic acid and
the b,d-dicarboxylic acid were prepared as described before (Anton,
D. L., Hogenkamp, H. P. C., Walker, T. E. and Matwiyoff, N. A.,
Carbon-13 nuclear magnetic resonance studies of the monocarboxylic
acids of cyanocobalamin. Assignments of the b-, d-, and
e-monocarboxylic acids, J. Am. Chem. Soc., 102, 2215-2219 (1980)).
5' chloro-5'-deoxyadenosine was synthesized by the method of
Kikugawa and Ichino (Kikugawa, K. and Ichino, M., Tetrahedron
Lett., 87 (1971)). The adenosylcobalamin-monocarb- oxylic acid was
prepared as described before (Hogenkamp, H. P. C., Chemical
synthesis and properties of analogs of adenosylcobalamin,
Biochemistry, 13, 2736-2740 (1974)).
Example 1
[0381] Cyanotrifluoroethylamidocobalamin (4, 5 and 6, FIG. 3).
[0382] Separate reaction mixtures containing 600 mg (.about.4 mmol)
of the b, d and e-cyanocobalamin monocarboxylic acids (compounds 1,
2, 3, FIG. 3), hydroxybenzo-triazole 540 mg (4 mmol),
1-ethyl-3(3'-dimethylaminoprop- yl) carbodiimide 768 mg (4 mmol)
and 2,2,2-trifluoroethylamino hydrochloride 678 mg (5 mmol) were
dissolved in 50 mL water and the pH adjusted to 6.8 with 1N NaOH.
The progress of the reactions was monitored by TLC using
2-propanol-NH.sub.4OH-water (7:1:2) as the solvent. After 2 hours
incubation at room temperature, the mixtures were extracted into
92% aqueous phenol. The phenol layers were extensively washed with
water to remove the water-soluble reagents. One volume of acetone
and three volumes of ether were then added to the phenol phases and
the desired fluorocobalamins were back extracted into water. The
aqueous phases were extracted three times with ether to remove
residual phenol. The solutions were concentrated on a rotary
evaporator and the fluorocobalamins crystallized from aqueous
acetone. Yields 4, 600 mg; 5, 540 mg; 6, 470 mg.
Example 2
[0383] Adenosyltrifluoroethylamidocobalamins (8 and 9, FIG. 3).
[0384] Separately the b- and e-cyanotrifluoroethylamidocobalamins
(compounds 5, 6) 500 mg (.times.0.33 mmol) were reduced with sodium
borohydride to their cobalt (I) forms, which in turn were reacted
with 5' chloro-5'-deoxyadenosine as described before (Hogenkamp, H.
P. C., Chemical synthesis and properties of analogs of
adenosylcobalamin, Biochemistry, 13, 2736-2740 (1974)). The
reaction mixtures were acidified to a pH of 3 with 1N HCl and
applied to separate columns of A6 50.times.2 (200-400 mesh, pH
3.0). The columns were washed with water and the desired
adenosylcobalamins eluted with 0.1 M sodium acetate pH 6A. After
desalting by extraction into phenol as described above, both 8 and
9 were isolated as red powders. Yields 315 mg and 320 mg
respectively.
Example 3
[0385] Cyano-bis-trifluoroethylamidocobalamin (7, FIG. 4).
[0386] A reaction mixture containing cyanocobalamin-b,
d-dicarboxylic acid (540 mg, .about.0.36 mmol) was reacted with
2,2,2-trifluoroethylamine hydrochloride 678 mg (5 mmol) as
described above, compound 7 was crystallized from aqueous acetone.
Yield 630 mg.
Example 4
[0387] Cyano-b-trifluoroacetamido butylamide cobalamin (10, FIG.
5).
[0388] Cyanocobalamin-b-(9-aminobutyl) amide (600 mg, .about.0.4
mmol) was prepared as described by Collins, D. A. and Hogenkamp, H.
P. C., Transcobalamin II receptor imaging via radiolabeled
diethylenetriaminepentaacetate cobalamin analogs, J. Nucl. Med.,
38, 717-723 (1997), hydroxybenzotriazole 540 mg (4 mmol),
1-ethyl-3(3'-dimethylamino-propyl) carbodiimide 768 mg (4 mmol) and
sodium trifluoroacetate (680 mg, 5 mmol) were dissolved in 50 mL
water and the pH adjusted to 6.2 with IN NaOH. After incubation at
room temperature for 5 hours, the reaction mixture was desalted as
described above. The resulting aqueous solution was purified by
column chromatography (A6, 50.times.2, 200-400 mesh, pH 3.0) and
the pass through collected. The solution was concentrated and
compound 10 was crystallized from aqueous acetone. Yield 315
mg.
Example 5
[0389] Cyanotrifluoroacetyl polylysine cobalamin (11, FIG. 6).
[0390] Poly-L-lysine hydrobromide (MW 500-2000) 500 mg,
cyanocobalamin-b-carboxylic acid 300 mg (.about.0.2 mmol),
hydroxybenzotriazole (338 mg, 2.5 mmol) and
1-ethyl-3(3'-dimethylaminopro- pyl) carbodiimide 480 mg (2.5 mmol)
were dissolved in 10 mL of water and the pH adjusted to 6.5 with IN
NaOH. After incubation at room temperature for 4 hr, the reaction
mixture was purified by chromatography (Sephodex G-10, 40.times.3
cm), which was eluted with water. The red eluents that also reacted
with ninhydrin were pooled and freeze dried. The freeze-dried
preparation was dissolved in mL saturated sodium bicarbonate and
reacted with 1 mL of trifluoroacetic anhydride for 1 hr. The
preparation was again purified by chromatography and lyophilized to
yield 490 mg of compound 11 as a fluffy powder.
Example 6
Cyanocobalamin-b-(4-aminobutyl)amide.
[0391] A mixture containing cyanocobalamin-b-carboxylic acid (1.0
g, 0.6 mmol), hydroxybenzotriazole (0.81 g, 6 mmol) and
1,4-diaminobutane dihydrochloride (4.8 g, 30 mmol) in 100 mL of
water was adjusted to pH 7.8.
1-Ethyl-3-(3'-dimethylaminopropyl)carbodiimide (1.26 g, 6.6 mmol)
was then added, the pH was adjusted to 6.4 and the reaction stirred
at room temperature for 24 h. TLC on silica gel using
n-butanol-acetic acid water (5:2:3) showed the reaction to be
complete. Cyanocobalamin-b-(4-ami- nobutyl)amide was extracted into
92% aqueous phenol and the phenol layer was washed several times
with equal volumes of water. To the phenol extract were added 3
volumes of diethylether and 1 volume of acetone. The desired
cobalamin was removed from the organic phase by several extractions
with water. The combined aqueous layers were extracted three times
with diethylether to remove residual phenol, concentrated to
approximately 20 mL in vacuo and crystallized from aqueous acetone.
Yield 955 mg, 92%.
Example 7
[0392] Cyanocobalamin-b-(4-aminobutyl)amide DTPA.
[0393] Cyanocobalamin-b-(4-aminobutyl) amide (500 mg), 0.3 mmol)
was dissolved in 30 mL saturated sodium bicarbonate and treated
with solid DTPA dianhydride (1.2 g, 3.4 mmol). The progress of the
reaction was monitored by TLC on PEI plates using n-butanol-acetic
acid-water (5:2:3) as the solvent. After 30 min incubation at room
temperature a second 1.2 g of the dianhydride was added. After two
additional additions of dianhydride with adjustments of the pH to
8.2 the reaction mixture was incubated overnight.
Cyanocobalamin-DPTA product was then extracted into 92% aqueous
phenol, desalted, purified by AG-1.times.2 200-400 mesh in acetate
form, eluting with 1.0 M acetic acid and washed with water. The
preparation was evaporated to dryness in vacuo and isolated as a
glass. Yield 460 mg, 77%. The cyanocobalamin-DTPA adduct behaves as
a polyanion on paper electrophoresis in 0.1 M sodium phosphate
buffer pH 7.1.
Example 8
[0394] Methylcobalamin-b-(4-aminobutyl)amide.
[0395] Methylcobalamin-b-carboxylic acid (1.0 g, 0.6 mmol) was
reacted with diaminobutane dihydrochloride as described above for
the cyano derivative. The cobalamin was purified by extraction
through phenol (see above) and the resulting aqueous solution was
concentrated in vacuo. This solution was chromatographed on AG1-X2
200-400 mesh in the acetate form (20.times.2.5 cm) and the pass
through collected. The pass through was concentrated to
approximately 20 mL and the desired cobalamin crystallized from
aqueous acetone. Yield 920 mg, 88%. Unreacted
methylcobalamin-b-carboxylic acid was eluted with 1 M acetic acid,
concentrated and crystallized from aqueous acetone. Yield 60 mg,
6%.
Example 9
[0396] Methylcobalamin-b-(4-aminobutyl)amide DTPA.
[0397] Methylcobalamin-b-(4-aminobutyl)amide (500 mg, 0.3 mmol) was
dissolved in 30 mL saturated sodium bicarbonate and reacted with
solid DTPA dianhydride as described above. The methyl
cobalamin-DTPA product was extracted into 92% aqueous phenol,
desalted, purified by AG-1.times.2 200-400 mesh in acetate form,
eluting with 1.0 M acetic acid and washed with water, evaporated to
dryness in vacuo and isolated as a glass. Yield 600 mg, 96%.
Example 10
[0398] Adenosylcobalamin-b-(4-aminobutyl)amide.
[0399] Adenosylcobalamin-b-carboxylic acid (500 mg, 0.3 mmol) was
reacted with diaminobutane dihydrochloride (2.4 mg, 15 mmol) as
described above. The cobalamin was purified by extraction through
phenol (see above). The resulting aqueous solution was concentrated
in vacuo and applied to AG-50.times.2, 200-400 mesh, in the
hydrogen form (20.times.25 cm). The column was washed thoroughly
with water to remove hydroxybenzotriazole and the desired cobalamin
eluted with 1 M ammonium hydroxide. After an additional extraction
through phenol, adenosylcobalamin-b-(4-aminobutyl)a- mide was
isolated as a glass. Yield 366 mg, 77%.
Example 11
[0400] Adenosylcobalamin-b-(4-aminobutyl)amide DTPA.
[0401] Adenosylcobalamin-b-(4-aminobutyl)amide (366 mg, 0.23 mmol)
was dissolved in 30 mL saturated sodium bicarbonate and treated
with solid DTPA dianhydride (1.0 g, 2.8 mmol) as described above.
The cobalamin was purified through phenol (see above). The
resulting aqueous solution was concentrated, extracted into 92%
aqueous phenol, desalted, purified by AG-1.times.2 200-400 mesh in
acetate form, eluting with 0.5 M acetic acid and washed with water.
The solution was evaporated to dryness in vacuo and
adenosylcobalamin-b-(4-aminobutyl)amide DTPA isolated as a glass.
Yield 400 mg, 80%.
Example 12
[0402] Chelation of Radionuclides.
[0403] Under dim light, 1000 .mu.g of methyl-, adenosyl-, and
cyanocobalamin-b-(4-aminobutyl)amide-DTPA were separately dissolved
in 200 .mu.L of normal saline. Next, 500 .mu.Ci of Indium-111 or
250 .mu.Ci of Gadolinium-153 were added to the cobalamin-DTPA
solutions. The reactions were carried out at room temperature and
room air. For the chelation of technetium, the dissolved cobalamin
DTPA complexes were separately placed into sealed 2 mL vials. Next,
200 .mu.L of stannous chloride solution (1000 .mu.g/mL normal
saline) was added to each vial. The vials were purged with nitrogen
gas for 5 minutes. After this time, 1-5 mCi of Technetium-99m was
added to the N.sub.2 purged vials. Each vial underwent further
nitrogen purging for 5 minutes. All chelation reactions were mixed
gently for 5 minutes.
[0404] Control mixtures of 1000 .mu.g of cyanocobalamin were
dissolved in 200 .mu.L of normal saline. Cyanocobalamin was mixed
with Tc-99m at room temperature and room air, as well as within
nitrogen purged vials containing 200 .mu.L of the described
stannous chloride solution. Additionally, the cobalamin-DTPA
complexes underwent Tc-99m labeling in open vials at room air in
the absence of the stannous chloride.
Example 13
[0405] Interaction with Intrinsic Factor and Transcobalamin
Proteins.
[0406] Under dim light, 1000 .mu.g of the non-labeled methyl-,
adenosyl-, and cyanocobalamin-b-(4-aminobutyl)amide-DTPA, as well
as 1000 .mu.g of cyanocobalamin and DTPA (Sigma, St. Louis, Mo.
63178), were separately dissolved in 10 mL of normal saline at room
temperature. Each of the five 1000 .mu.g/10 mL samples was stored
in sealed, aluminum-wrapped 10 mL vials to prevent exposure to
light. No buffers were added to the solutions. The pH of the
solutions, measured by a Beckman 40 pH meter (Beckman Instruments,
Fullerton, Calif.): Cyanocobalamin 5.75, DTPA=3.78; cyano, methyl
and adenosylcobalamin-DTPA analogues were 5.75, 6.10, and 6.19,
respectively.
[0407] To assess in vitro binding to Intrinsic Factor (IF) and
Transcobalamins (TC), the intrinsic factor blocking antibody (IFBA)
and Unsaturated vitamin B.sub.12 Binding Capacity (UBBC) assays
were performed with serum randomly obtained from five patients
being evaluated for pernicious anemia at the Mayo Clinic. The IFBA
and UBBC assays were first performed for clinical purposes as
previously described by V. F. Fairbanks et al., Mayo Clin. Proc.,
58, 203 (1983); Intrinsic Factor Blocking Antibody (.sup.57Co)
Radioassay-Package insert, Diagnostic Products Corp.; D. Grossowicz
et al., Proc. Exp. Biol., 109, 604 (1962) and C. Gottlieb et al.,
Blood, 25, 6 (1965).
[0408] Next, the serum from the same five patients underwent
modified IFBA and UBBC assays. Specifically, 1 .mu.L of the five
previously described solutions was separately incubated with
purified IF or serum, to potentially saturate all IF and TC-binding
sites. After incubation for 20 minutes at room temperature and for
another 20 minutes at 4.degree. C., 500 .mu.l of the stock (1000
.mu.g/l) Cobalt-57-cyanocobalamin (Mallinckrodt Medical, Inc., St.
Louis, Mo. 63134) solution was added and the usual IFBA and UBBC
protocols were then followed. All supernatant activity was counted
for four minutes on a gamma counter (Micromedix 10/20, Huntsville,
Ala. 35805). The results are shown in Table 3.
[0409] The IFBA assay demonstrated that DTPA does not significantly
bind to IF (values less than the negative reference), whereas
cyanocobalamin and the cobalamin-DTPA analogs do, in varying
degrees, competitively inhibit Co-57 cyanocobalamin from binding to
intrinsic factor. By using the counts of the Clinical run divided
into the counts of the five solutions, the efficacy of binding to
intrinsic factor can be estimated. The averaged percent binding of
the five solutions to IF was: cyanocobalamin=92.5%;
methyl-cobalamin-b-(4-aminobutyl)-amide-DTPA=63.2%;
cyanocobalamin-b-(4-aminobutyl)-amide-DTPA=52.9%;
adenosylcobalamin-b-(4-- aminobutyl)-amide-DTPA=41.0% and 0.8% for
DTPA. This is in contrast to the disclosure in Houts (U.S. Pat. No.
4,465,775) that the (b)-monocarboxylic acid of vitamin B.sub.12 and
its radioiodinated derivative exhibit very low binding to IF.
[0410] Likewise the averaged percent binding of the five solutions
to the transcobalamin proteins was: cyanocobalamin=100%,
methylcobalamin-b-(4-am- inobutyl)amide-DTPA=94.0%,
adenosylcobalamin-b-(4-aminobutyl)amide-DTPA=90- .4%,
cyanocobalamin-b-(4-aminobutyl)amide-DTPA=66.4% and 3.6% for
DTPA.
[0411] Thus, the attachment of DTPA to vitamin B.sub.12 does alter
its binding to the carrier proteins. As expected, non-labeled
cyanocobalamin had the greatest affinity for IF and the
transcobalamin proteins. Methylcobalamin-b-(4-aminobutyl)amide-DTPA
was next, followed by adenosylcobalamin-b-(4-aminobutyl)amide-DTPA,
and finally cyano-cobalamin-b-(4-aminobutyl)amide-DTPA. There was
also some nonspecific binding of DTPA to the carrier proteins (0.8%
and 3.6%).
Example 14
[0412] Cyano-and Adenosyl Cobalamin-Spermine
[0413] Cyanocobalamin-b-monocarboxylic acid or adenosyl cobalamin-b
monocarboxylic acid were separately reacted with 5 fold excess of
spermine in the presence of a 10 fold excess of
hydroxybenzotriazole and 1-ethyl-3(3'-dimethylaminopropyl)
carbodiimide. The progress of the reaction was monitored by TLC on
silica gel plates using isopropanol-ammonium hydroxide-water
(7:1:2) as the solvent. After incubation at room (for 24 h)
temperature (in the dark) the reaction mixtures were extracted into
90% aqueous phenol. The phenol phase was washed with water
(7.times.) to remove the water soluble reagents. Finally the
cobalamins were back extracted into water after the addition of one
volume of acetone and three volumes of ether. The aqueous solutions
were evaporated to dryness and stirred with acetone to remove
traces of hydroxybenzotriazole. The cobalamins were isolated as red
powders.
Example 15
[0414] Cyano-and Adenosyl Cobalamin-Spermine DTPA Conjugate
[0415] The cobalamin-spermine derivatives were separately dissolved
in sodium bicarbonate (saturated aqueous solution) and reacted with
ten fold excess of DTPA bis-anhydride. The reaction was followed by
TLC on PEI plates using the same solvent. A second ten fold excess
of the dianhydride was added after 2 hr. After incubation for 24
hr. at room temperature the reaction mixtures were extracted with
aqueous phenol as described above, the phenol phases were
extensively washed with water. The desired products were isolated
as red powders as described above. UV-visible specters copy showed
the correct spectra for the cyano-and adenosyl cobalamin
derivatives.
Example 16
[0416] Cyanocobalamin-Poly L-Lysine-DTPA Conjugate.
[0417] Poly-L-lysine hydrobromide (Sigma no. PO879), degree of
polymerization .about.11 units, molecular weight range 1000-4000
(500 mg) was dissolved in water (20 mL).
Cyanocobalamin-b-monocarboxylic acid (150 mg, .about.100 .mu.mol),
1-ethyl-3 (3'-dimethylaminopropyl) carbodiimide (480 mg, 2.5 mmol)
and hydroxybenzotriazole (338 mg, 2.5 mmol) were added. The pH of
the mixture was adjusted and maintained at approximately 8 with 1 N
sodium hydroxide. The progress of the reaction was monitored by
thin layer chromatography on silica gel sheets using isopropanol:
ammonium hydroxide: water (7:1:2) as the developing agent.
[0418] Upon completion of the reaction, the mixture was applied to
a column of Sephadex G-10 (3.times.40 cm). The column was eluted
with water and 2 mL fractions were collected. The red eluents that
reacted with ninhydrin were pooled and freeze dried (i.e.,
lyophilized).
[0419] Recovery of the cyanocobalamin-poly-L-lysine complex (about
70%) was obtained. The cyanocobalamin-poly-L-lysine complex was
dissolved in water (10 mL) and a saturated solution of sodium
bicarbonate (10 mL) and DTPA bisanhydride (Sigma) (375 mg, 1 mmol)
were added.
[0420] The progress of the reaction was monitored by TLC as
described above. The cyanocobalamin-poly-L-lysine-DTPA conjugate
was purified on Sephadex G-10 as described above. The final product
was freeze dried and isolated as a red powder.
Example 17
[0421] Cyanocobalamin-Poly L-Lysine-DTPA Conjugate.
[0422] Poly-L-lysine (Sigma no. 8954) degree of polymerization
.about.8 units, molecular weight range 500-2000 (500 mg) was
dissolved in water (20 mL). Cyanocobalamin-b-monocarboxylic acid
(150 mg, .about.100 .mu.mols), 1-ethyl-3 (3'-dimethylaminopropyl)
carbodiimide (480 mg, 2.5 mmol) and hydroxybenzotriazole (338 mg,
2.5 mmol) were added. The pH of the mixture was adjusted and
maintained at approximately 8 with 1 N sodium hydroxide. The
progress of the reaction was monitored by thin layer chromatography
on silica gel sheets using isopropanol-ammonium hydroxide-water
(7:1:2) as the developing agent.
[0423] Upon completion of the reaction, the mixture was applied to
a column of Sephadex G-10 (3.times.40 cm). The column was eluted
with water and 2 mL fractions were collected. The red eluents that
reacted with ninhydrin were pooled and freeze dried (i.e.,
lyophilized).
[0424] Recovery of the cyanocobalamin-poly-L-lysine complex (about
70%) was obtained. The cyanocobalamin-poly-L-lysine complex was
dissolved in water (10 mL) and a saturated solution of sodium
bicarbonate (10 mL) and DTPA bisanhydride (Sigma) (375 mg, 1 mmol)
were added.
[0425] The progress of the reaction was monitored by TLC as
described above. The cyanocobalamin-poly-L-lysine-DTPA conjugate
was purified on Sephadex G-10 as described above. The final product
was freeze dried and isolated as a red powder.
Example 18
[0426] Imaging Data.
[0427] In vitro unsaturated B.sub.12 binding capacity (UBBC) has
demonstrated that cyanocobalamin-poly-L-lysine,
cyanocobalamin-poly-L-lys- ine-polyDTPA compounds have in vitro
biological activity that is 92% and 43.4% when compared to
cyanocobalamin. Comparison of cyanocobalamin-DTPA to cyanocobalamin
was 66.4% (Transcobalamin II receptor imaging via radiolabeled
diethylene-triaminepentaacetate cobalamin analogs, J. Nucl. Med.,
38, 717-723 (1997); also described in U.S. Pat. No. 5,739,313). The
specific activity has been increased from 300 Ci in the cobalamin
mono-DTPA compounds to 4.5 mCi with the cobalamin
poly-L-lysine-polyDTPA complex (D. A. Collins, H. P. C. Hogenkamp,
M W Gebard, Tumor Imaging Indium-111-labeled
DTPA-adenosylcobalamin, Mayo Clinic Proceedings, 1999; 74; 687-691;
Biodistribution of Radiolabeled Adenosylcobalamin in Humans, Review
of 30 patents submitted to Mayo Clinic Proceedings). This should
improve tumor-to-background ratio, which can be evaluated in murine
tumor models. Attachment of either the adenosyl and methyl group as
the beta ligand should improve the biological activity as it did
with the cyanocobalamin mono-DTPA compound (Transcobalamin II
receptor imaging via radiolabeled diethylene-triaminepentaacetate
cobalamin analogs, J. Nucl. Med., 38, 717-723 (1997); also
described in U.S. Pat. No. 5,739,313).
Example 19
[0428] Proposed Synthesis of Daunorubicin- and
Doxorubicin-Cobalamin Conjugates (FIG. 7).
[0429] Modification of the carbohydrate moiety (daunosamine) of
daunorubicin (12) with L-leucine can be accomplished by reacting
daunorubicin HCl (0.5 g) in 100 mL borate buffer pH=10 (containing
KCl) with L-leucine-carboxyanhydride (1 mmole in 5 mL acetone) at
0.degree. C. under nitrogen. After reaction for 5 minutes at
0.degree. C., the mixture can be acidified to pH 3.5 with
H.sub.2SO.sub.4, stirred for 15 minutes and adjusted to pH=7 to
give the desired L-leucyl daunorubicin (13). Reaction of (13) with
a cobalamin-mono or dicarboxylic acid in the presence of a
water-soluble carbodiimide and hydroxybenzo-triazole will yield the
daunorubicin-cobalamin conjugate (14). These conjugates can be
isolated via the usual phenol extraction, extensive washing of the
phenol phase with water and finally displacing the
cobalamin-conjugates from the phenol phase into water by the
addition of acetone and diethyl ether.
[0430] Modification of doxorubicin should be similar (Ger. Patent
1,813,518, Jul. 10, 1969; Chem Abstracts, 71, 91866 (1969)). D.
Deprez-Decampaneere, M Mosquelier, R. Bourain and A. Trosect, Curr.
Chemother. Proc., Int. Congr. Chemother., 10th, p. 1242 (1978) have
found that N-(L-leucyl) daunorubicin but not the D isomer was
hydrolyzed in vivo to regenerate daunorubicin. See, "Doxorubicin,
Anticancer Antibiotics," Federico Arcamone, Medicinal Chemistry,
Vol. 17, Academic Press, 1981.
[0431] The invention has been described with reference to various
specific and preferred embodiments and techniques. However, it
should be understood that many variations and modifications may be
made while remaining within the spirit and scope of the
invention.
* * * * *
References