Radiolabeled GRPR-antagonists for diagnostic imaging and treatment of GRPR-positive cancer
09839703 · 2017-12-12
Assignee
Inventors
- Theodosia Maina-Nock (Athens, GR)
- Berthold Artur Nock (Athens, GR)
- Marion de Jong Hendriks (Vlaardingen, NL)
Cpc classification
A61P1/04
HUMAN NECESSITIES
A61K51/088
HUMAN NECESSITIES
A61K51/08
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
A61P1/00
HUMAN NECESSITIES
A61P1/18
HUMAN NECESSITIES
A61P15/00
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to probes for use in the detection, imaging, diagnosis, targeting, treatment, etc. of cancers expressing the gastrin releasing peptide receptor (GRPR). For example, such probes may be molecules conjugated to detectable labels which are preferably moieties suitable for detection by gamma imaging and SPECT or by positron emission tomography (PET) or magnetic resonance imaging (MRI) or fluorescence spectroscopy or optical imaging methods.
Claims
1. A radiolabeled GRPR-antagonist of the general formula MC-S-P wherein: M is a radiometal and C is a metal chelator that stably binds M, or MC is a Tyr- or prosthetic group bound to a radiohalogen, wherein M is the radiohalogen; S is a ##STR00029## spacer covalently linked between C and P, wherein S is covalently attached to the N-terminus of P; and P is DPhe-Gln-Trp-Ala-Val-Gly-His-CO—NH—CH[CH.sub.2—CH(CH.sub.3).sub.2].sub.2 (SEQ ID NO: 1).
2. The radiolabeled GRPR-antagonist as claimed in claim 1, wherein M is selected from the group consisting of .sup.111In, .sup.99mTc, .sup.94mTc, .sup.67Ga, .sup.66Ga, .sup.68Ga, .sup.52Fe, .sup.69Er, .sup.72As, .sup.97Ru, .sup.203Pb, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.186Re, .sup.188Re, .sup.86Y, .sup.90Y, .sup.51Cr, .sup.52mMn, .sup.157Gd, .sup.177Lu, .sup.161Tb, .sup.169Yb, .sup.175Yb, .sup.105Rh, .sup.166Dy, .sup.166Ho, .sup.153Sm, .sup.149Pm, .sup.151Pm, .sup.172Tm, .sup.121Sn, .sup.177mSn, .sup.213Bi, .sup.142Pr, .sup.143Pr, .sup.198Au, .sup.199Au, .sup.123I, .sup.124I, .sup.125I, and .sup.18F.
3. The radiolabeled GRPR-antagonist as claimed in claim 1, wherein the metal chelator C is a metal chelator for di- and trivalent metals.
4. The radiolabeled GRPR-antagonist as claimed in claim 3, wherein the metal chelator for di- and trivalent metals is a DTPA, NOTA, DOTA, or TETA chelator or a derivative thereof, including bifunctional derivatives thereof.
5. The radiolabeled GRPR-antagonist as claimed in claim 1, wherein the metal chelator C is selected from the group consisting of: ##STR00030##
6. The radiolabeled GRPR-antagonist as claimed in claim 1, wherein the metal chelator C is a metal chelator for radionuclides of technetium or rhenium.
7. The radiolabeled GRPR-antagonist of claim 1, wherein C is a metal chelator for radionuclides of technetium or rhenium selected from the group consisting of acyclic tetraamine; cyclam; PnAO; tetradentate chelators containing donor atom sets selected from the group consisting of P2S2-, N2S2- and N3S—; derivatives of said tetradentate chelators; bifunctional derivatives of said tetradentate chelators; HYNIC/co-ligand-based chelators; and bi- and tridentate chelators that form organometallic complexes via tricarbonyl technology.
8. The radiolabeled GRPR-antagonist as claimed in claim 1, wherein C is a metal chelator for radionuclides of technetium or rhenium selected from the group consisting of: ##STR00031## ##STR00032## and bifunctional derivatives thereof.
9. A diagnostic and/or therapeutic composition, comprising the radiolabeled GRPR-antagonist as claimed in claim 1 and a therapeutically acceptable excipient.
10. The radiolabeled GRPR-antagonist of claim 1, wherein the antagonist is NeoBOMB-1: ##STR00033## (M-DOTA-(p-aminobenzylamine-diglycolic acid)-[DPhe.sup.6,His.sup.12—CO—NH—CH[(CH.sub.2—CH(CH.sub.3).sub.2].sub.2,des-Leu.sup.13,des-Met.sup.14]BB(6-14)).
11. A diagnostic and/or therapeutic composition, comprising the radiolabeled GRPR-antagonist as claimed in claim 10 and a therapeutically acceptable excipient.
12. The radiolabeled GRPR-antagonist of claim 1, wherein the antagonist is NeoBOMB-2: ##STR00034## (M—N.sub.4-(p-aminobenzylamine-diglycolic acid)-[DPhe.sup.6,His.sup.12—CO—NH—CH[(CH.sub.2—CH(CH.sub.3).sub.2].sub.2,des-Leu.sup.13,des-Met.sup.14]BB(6-14)).
13. A diagnostic and/or therapeutic composition, comprising the radiolabeled GRPR-antagonist as claimed in claim 12 and a therapeutically acceptable excipient.
14. The radiolabeled GRPR-antagonist of claim 1, wherein M is .sup.111In, .sup.177Lu, .sup.67Ga, .sup.68Ga, .sup.99mTc, .sup.186Re, or .sup.188Re.
15. The radiolabeled GRPR-antagonist of claim 10, wherein M is .sup.111In, .sup.177Lu, .sup.67Ga, or .sup.68Ga.
16. The radiolabeled GRPR-antagonist of claim 12, wherein M is .sup.99mTc, .sup.186Re, or .sup.188Re.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
DETAILED DESCRIPTION
(9) The research leading to the invention has unexpectedly revealed an alternative route for effective in vivo targeting of somatostatin-positive tumors, namely the use of somatostatin receptor antagonists. Most surprisingly and against their inability to internalize, such analogs have shown a much higher uptake and retention in animal xenografts and a very rapid washout from background tissues.
(10) A tentative explanation for the higher tumor uptake of somatostatin receptor antagonists is their ability to bind to a significantly higher number of the overall somatostatin receptor population available on the cell-membrane of cancer cells than their internalizing agonistic counterparts.
(11) According to the invention, GRPR-antagonists are chemically modified to accommodate a diagnostic and/or therapeutic radionuclide that they stably bind. After administration in a human or an animal subject they serve as a molecular vehicle to transfer a radiodiagnostic signal and/or a radiotoxic load on the primary GRPR.sup.+-tumor and its metastases.
(12) More specifically, it was found according to the invention that administration of certain novel GRPR-antagonist-based radioligands unexpectedly resulted in an unprecedentedly high and specific uptake and a remarkably prolonged retention of human GRPR.sup.+-xenografts in mice in contrast to [.sup.99mTc]Demobesin 1. Furthermore, these agents showed significantly higher metabolic stability after injection in mice, compared to [.sup.99mTc]Demobesin 1.
(13) The GRPR-antagonists of the invention have important structural differences in relation to the original [.sup.99mTc]Demobesin 1 motif. Firstly, their labeling with a wide range of bi- and trivalent radiometals, but also with .sup.99mTc and .sup.186/188Re, is made possible by coupling of suitable bifunctional chelators at their N-terminus in addition to tetraamine-related frameworks. In this way, radiodiagnostic imaging is possible with SPECT and PET with gamma and positron-emitters while labeling with beta-, Auger and alpha emitters is feasible as well, opening the opportunity for therapeutic applications. Then, their metabolic stability and pharmacokinetic profile, especially in terms of tumor-retention has largely improved, as demonstrated by preclinical biodistribution results in female SCID mice bearing human PC-3 xenografts presented at length.
(14) More specifically, the structure of new analogs comprises the following parts:
(15) a) The chelator attached to the N-terminus—this can be either an acyclic or a cyclic tetraamine, HYNIC, N.sub.3S-chelators and derivatives thereof, linear or cyclic polyamines and polyaminopolycarboxylates like DTPA, EDTA, DOTA, NOTA, NOTAGA, TETA and their derivatives, a.o. In addition, a suitable group, such a prosthetic group or a Tyr, for labeling with radiohalogens, can be introduced at this position;
(16) b) The radionuclide—this may be i) a gamma emitter, such as .sup.99mTc, .sup.111In, .sup.67Ga, .sup.131I, .sup.125I, a.o., suitable for imaging with a conventional gamma-camera, a SPECT or an hybrid SPECT/CT or SPECT/MRI system; ii) a positron emitter, such as .sup.68Ga, .sup.66Ga, .sup.64Cu, .sup.86Y, .sup.44Sc, .sup.124I, .sup.18F, a.o., suitable for imaging with a PET or a hybrid PET/CT or PET/MRI system, or iii) a beta, Auger or alpha emitter, such as .sup.186Re, .sup.188Re, .sup.90Y, .sup.177Lu, .sup.111In, .sup.67Cu, .sup.212Bi, .sup.175Yb, .sup.47Sc, .sup.131I, .sup.125I, etc., suitable for radionuclide therapy;
(17) c) The spacer between the chelator and the peptide motif, which may vary in length, type and lipophilicity and may include PEGx (x=0-20), natural and unnatural amino acids, sugars, alkylamino residues or combinations thereof;
(18) d) The peptide chain, with strategic amino acid replacements undertaken with D-amino acids, unnatural amino acids and other suitable residues.
(19) e) The C-terminus, wherein the both Leu.sup.13 and Met.sup.14-NH.sub.2 in the native BBN sequence have been omitted. Terminal His.sup.12 is present as the amidated or ester form, whereby amides or esters may be represented by several mono- and di-alkylamides, aromatic amides or mixed alkyl-aryl amides, or alkyl and/or aryl esters.
(20) The invention thus relates to GRPR-antagonists of the general formula
MC-S-P
wherein:
MC is a metal chelate, which comprises: at least one (radio)metal (M) and a chelator (C) which stably binds M; alternatively MC may represent a Tyr- or a prosthetic group carrying a (radio)halogen.
S is an optional spacer covalently linked between the N-terminal of P and C and may be selected to provide a means for (radio)halogenation;
P is a GRP receptor peptide antagonist of the general formula:
Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6-Xaa.sub.7-CO—Z
wherein:
(21) Xaa.sub.1 is not present or is selected from the group consisting of amino acid residues Asn, Thr, Phe, 3-(2-thienyl)alanine (Thi), 4-chlorophenylalanine (Cpa), α-naphthylalanine (α-Nal), β-naphthylalanine (β-Nal), 1,2,3,4-tetrahydronorharman-3-carboxylic acid (Tpi), Tyr, 3-iodo-tyrosine (o-I-Tyr), Trp, pentafluorophenylalanine (5-F-Phe) (all as L- or D-isomers);
(22) Xaa.sub.2 is Gln, Asn, His
(23) Xaa.sub.3 is Trp, 1,2,3,4-tetrahydronorharman-3-carboxylic acid (Tpi)
(24) Xaa.sub.4 is Ala, Ser, Val
(25) Xaa.sub.5 is Val, Ser, Thr
(26) Xaa.sub.6 is Gly, sarcosine (Sar), D-Ala, β-Ala
(27) Xaa.sub.7 is His, (3-methylhistidine (3-Me)His
(28) Z is selected from —NHOH, —NHNH.sub.2, —NH-alkyl, —N(alkyl).sub.2r or —O-alkyl
(29) or
(30) ##STR00013##
(31) wherein X is NH (amide) or O (ester) and R1 and R2 are the same or different and selected from a proton, a (substituted)alkyl, a (substituted) alkyl ether, an aryl, an aryl ether or an alkyl-, halogen, hydroxyl or hydroxyalkyl substituted aromatic group.
(32) Z is preferably selected from one of the following formulae, wherein X is NH or O:
(33) ##STR00014## ##STR00015##
Preferably, R1 is the same as R2.
(34) In the GRPR-antagonists of the invention P is preferably selected from the group consisting of:
(35) DPhe-Gln-Trp-Ala-Val-Gly-His-CO—NH—CH[CH.sub.2—CH(CH.sub.3).sub.2].sub.2 (SEQ ID NO:1);
(36) DPhe-Gln-Trp-Ala-Val-Gly-His-CO—O—CH[CH.sub.2—CH(CH.sub.3).sub.2].sub.2 (SEQ ID NO:2);
(37) DPhe-Gln-Trp-Ala-Val-Gly-His-CO—NH—CH(CH.sub.2—CH.sub.2—CH.sub.2—CH.sub.3).sub.2 (SEQ ID NO:3);
(38) DTyr-Gln-Trp-Ala-Val-Gly-His-CO—NH—CH[CH.sub.2—CH(CH.sub.3).sub.2].sub.2 (SEQ ID NO: 4).
(39) The radionuclide, a metal M or a halogen, is suitable for diagnostic or therapeutic use, in particular for imaging or radionuclide therapy and preferably selected from the group consisting of .sup.111In, .sup.33mIn, .sup.99mTc, .sup.94mTc, .sup.67Ga, .sup.66Ga, .sup.68Ga, .sup.52Fe, .sup.69Er, .sup.72As, .sup.97Ru, .sup.203Pb, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.186Re, .sup.188Re, .sup.86Y, .sup.90Y, .sup.51Cr, .sup.52mMn, .sup.157Gd, .sup.177Lu, .sup.161Tb, .sup.169Yb, .sup.175Yb, .sup.105Rh, .sup.166Dy, .sup.166Ho, .sup.153Sm, .sup.149Pm, .sup.151Pm, .sup.172Tm, .sup.121Sn, .sup.177mSn, .sup.213Bi, .sup.142Pr, .sup.143Pr, .sup.198Au, .sup.199Au, .sup.123I, .sup.124I, .sup.125I, .sup.18F a.o.
(40) The metal chelator C is preferably a metal chelator for di- and trivalent metals, and is in particular a DTPA-, NOTA-, DOTA-, or TETA-based chelator or a mono- or bifunctional derivative thereof.
(41) Preferably, the metal chelator C is selected from the group consisting of:
(42) ##STR00016## ##STR00017##
(43) When the metal chelator C is a metal chelator for technetium or rhenium, it is preferably selected from acyclic tetraamine-, cyclam-, PnAO-, or tetradentate chelators containing P.sub.2S.sub.2-, N.sub.2S.sub.2- and N.sub.3S-donor atom sets and mono- and bifunctional derivatives thereof, or HYNIC/co-ligand-based chelators, or bi- and tridentate chelators forming organometallic complexes via the tricarbonyl technology.
(44) Suitable examples of C are:
(45) ##STR00018## ##STR00019##
(46) The spacer S is linked between P and C by covalent bonds and may be selected to provide a means for using a radiohalogen, such as (radio)iodination. The spacer is preferably selected from the group consisting of:
(47) a) aryl containing residues of the formulae:
(48) ##STR00020##
wherein PABA is p-aminobenzoic acid, PABZA is p-aminobenzylamine, PDA is phenylenediamine and PAMBZA is p-(aminomethyl)benzylamine;
b) dicarboxylic acids, ω-aminocarboxylic acids, α,ω-diaminocarboxylic acids or diamines of the formulae:
(49) ##STR00021##
wherein DIG is diglycolic acid and IDA is iminodiacetic acid;
c) PEG spacers of various chain lengths, in particular PEG spacers selected from the formulae:
(50) ##STR00022##
d) α- and β-amino acids, single or in homologous chains of various chain lengths or heterologous chains of various chain lengths, in particular:
(51) ##STR00023##
GRP(1-18), GRP(14-18), GRP(13-18), BBN(1-5), or [Tyr.sup.4]BBN(1-5); or
e) combinations of a, b and c.
(52) GRPR-antagonists of the invention are preferably selected from the group consisting of compounds of the formulae:
(53) ##STR00024##
wherein MC and P are as defined above.
(54) It is understood that specific chemical structures disclosed herein are illustrative examples of various embodiments of the invention and that GRPR-antagonists of the general formula: MC-S-P are not limited to the structures of examples provided.
(55) The invention further relates to a therapeutic composition, comprising a GRPR-antagonist as claimed and a therapeutically acceptable excipient.
(56) The invention also relates to the GRPR-antagonists as claimed for use as a medicament. The medicament is preferably a diagnostic or therapeutic agent for diagnosing or treating primary and/or metastatic GRPR.sup.+ cancers, such as prostate cancer, breast cancer, small cell lung cancer, colon carcinoma, gastrointestinal stromal tumors, gastrinoma, renal cell carcinomas, gastroenteropancreatic neuroendocrine tumors, oesophageal squamous cell tumors, neuroblastomas, head and neck squamous cell carcinomas, to name some of the few, as well as in vasculature of ovarian, endometrial and pancreatic tumors.
(57) The invention will be further illustrated in the Examples that follows and which are not intended to limit the invention in any way.
EXAMPLE
(58) Introduction
(59) Compounds of the invention were made and tested as described below. The following disclosed embodiments are merely representative of the invention which may be embodied in various forms. Thus, specific structural, functional, and procedural details disclosed in the following examples are not to be interpreted as limiting.
(60) Materials and Methods
(61) Radiolabeling and QC
(62) Labeling with .sup.111In
(63) Indium (In-111) chloride in 50 mM HCl was purchased from Mallinckrodt Medical B. V., Petten, The Netherlands, at an activity concentration of 10-20 mCi/mL. In general, DOTA-peptide conjugates of the present invention were radiolabeled with Indium-111 at specific activities of 0.1-0.2 mCi In-111/nmol DOTA-peptide conjugate. Briefly, 3-15 nmol of DOTA-peptide conjugate dissolved in water was mixed with 2.5-12.5 μL of 1.0 M pH 4.6 sodium acetate buffer, 1-5 μL of 0.1 M sodium ascorbate in water and 30-150 μL of .sup.111InCl.sub.3 (0.3-3.0 mCi). The radiolabeling reaction mixture was incubated in a boiling water bath for 20 to 30 min. For quality control a 2 μL aliquot of the radiolabeling solution was quenched with 28 μL of an acetate buffered solution of Na.sub.2-EDTA (5 mM, pH 4.6). After a successful radiolabeling (more than 95% peptide-bound radioactivity) Na.sub.2-EDTA (0.1 M, pH 4.6) was added to the radiolabeling solution to a final concentration of 1 mM.
(64) Labeling with .sup.67Ga
(65) Gallium (Ga-67) chloride was obtained either in dilute HCl at an activity concentration of 498-743 mCi/mL from Nordion, Wesbrook Mall, Vancouver, Canada or at an activity concentration of 80 mCi/mL from Mallinckrodt Medical B. V., Petten, The Netherlands.
(66) In general, DOTA-peptide conjugates of the present invention were radiolabeled with Gallium-67 at specific activities of 0.1-0.2 mCi Ga-67/nmol DOTA-peptide conjugate. Briefly, 3-15 nmol of DOTA-peptide conjugate dissolved in water was mixed with 50-125 μL of 1.0 M pH 4.0 sodium acetate buffer and 5-15 μL of .sup.67GaCl.sub.3 (0.5-3.0 mCi. The radiolabeling reaction mixture was incubated in a boiling water bath for 30 min. For HPLC quality control a 2 μL aliquot of the radiolabeling solution was quenched with 28 μL of an acetate buffered solution of Na.sub.2-EDTA (5 mM, pH 4.0). After a successful labeling (more than 95% peptide-bound radioactivity) Na.sub.2-EDTA (0.1 M, pH 4.0) was added to the radiolabeling solution to a final concentration of 1 mM.
(67) Labeling with .sup.177Lu
(68) Lutetium (Lu-177) chloride in 50 mM HCl was purchased from IDB Radiopharmacy, The Netherlands, at an activity concentration of 100 mCi/mL.
(69) In general, DOTA-peptide conjugates of the present invention were radiolabeled with Lutetium-177 to a specific activity of up to 0.5 mCi Lu-177/nmol DOTA-peptide conjugate. Briefly, 3-15 nmol of DOTA-peptide conjugate dissolved in water was mixed with 4-16 μL of 1.0 M pH 4.6 sodium acetate buffer and 15-75 μL of .sup.67GaCl.sub.3 (1.5-7.5 mCi). Radiolysis was minimized by the addition of 5 μl of gentisic acid (80 mM) dissolved in 0.2 M sodium ascorbate. The reaction mixture was incubated in a boiling water bath for 30 min. For HPLC quality control a 2 μL aliquot of the radiolabeling solution was quenched with 28 μL of an acetate buffered solution of Na.sub.2-EDTA (5 mM, pH 4.6). After a successful radiolabeling (more than 95% peptide-bound radioactivity) Na.sub.2-EDTA (0.1 M, pH 4.6) was added to the radiolabeling solution to a final concentration of 1 mM.
(70) Labeling with .sup.99mTc
(71) Tetraamine-coupled peptides were dissolved in 50 mM acetic acid/EtOH 8/2 v/v to a final 1 mM peptide concentration. Each bulk solution was distributed in 50 μL aliquots in Eppendorf tubes and stored at −20° C. Labeling was conducted in an Eppendorf vial, wherein the following solutions were consecutively added: i) 0.5 M phosphate buffer pH 11.5 (50 μL), ii) 0.1 M sodium citrate (5 μL, iii) [.sup.99mTc]NaTcO.sub.4 generator eluate (415 mL, 10-20 mCi), iv) peptide conjugate stock solution (15 μL, 15 nmol) and v) freshly made SnCl.sub.2 solution in EtOH (30 μg, 15 μL). After reaction for 30 min at ambient temperature, the pH was brought to ˜7 by adding 1 M HCl (10 μL).
(72) Quality Control
(73) HPLC analyses were conducted on a Waters Chromatograph (Waters, Vienna, Austria) efficient with a 600 solvent delivery system; the chromatograph was coupled to twin detection instrumentation, comprising a photodiode array UV detector (either Waters model 996 or model 2998) and a Gabi gamma detector from Raytest (RSM Analytische Instrumente GmbH, Germany). Data processing and chromatography were controlled via the Millennium or Empower 2 Software (Waters, USA). A XBridge Shield RP18 column (5 μm, 4.6×150 mm, Waters, Ireland) coupled to the respective 2-cm guard column was eluted at 1 ml/min flow rate with a linear gradient system starting from 10% B and advancing to 70% B within 60 min, with solvent A=0.1% aqueous trifluoroacetic acid and solvent B=acetonitrile.
(74) Metabolic Study in Mice
(75) Radioligand Injection and Blood Collection
(76) A bolus containing the radioligand in normal saline (100-150 μL, ≈3 nmol, 200-500 μCi) was injected in the tail vein of Swiss albino mice. Animals were kept for 5 min in a cage with access to water and were then euthanized promptly by cardiac puncture while under a mild ether anesthesia. Blood (500-900 μL) was collected from the heart with a syringe and transferred in a pre-chilled Eppendorf tube on ice.
(77) Plasma Separation and Sample Preparation
(78) Blood was centrifuged to remove blood cells (10 min, 2000 g/4° C.). The plasma was collected, mixed with acetonitrile (MeCN) in a 1/1 v/v ratio and centrifuged again (10 min, 15000 g/4° C.). Supernatants were concentrated to a small volume (gentle N.sub.2-flux at 40° C.), diluted with saline (≈400 μL) and filtered through a Millex GV filter (0.22 μm).
(79) HPLC Analysis for Radiometabolite Detection
(80) Aliquots of plasma samples (prepared as described above) were loaded on a Symmetry Shield RPM column which was eluted at a flow rate of 1.0 mL/min with the following gradient: 100% A to 90% A in 10 min and from 90% A to 60% for the next 60 min (A=0.1% aqueous TFA (v/v) and B=MeCN). Elution of radiocomponents was monitored by a gamma detector. For .sup.99mTc-radiopeptides, ITLC-SG analysis was performed in parallel using acetone as the eluent to detect traces of TcO.sub.4.sup.− release (TcO.sub.4.sup.− Rf=1.0).
(81) Studies in GRPR.sup.+-Tumor Bearing Mice
(82) Tumor Induction
(83) A ≈150 μL bolus containing a suspension of 1.5×10.sup.7 freshly harvested human PC-3 cells in normal saline was subcutaneously injected in the flanks of female SCID mice. The animals were kept under aseptic conditions and 2-3 weeks later developed well-palpable tumors at the inoculation site (80-150 mg).
(84) Biodistribution and Calculation of Results
(85) On the day of the experiment, the selected radiopeptide was injected in the tail vein of tumor-bearing mice as a 100 μL bolus (1-2 μCi, 10 pmol total peptide; in saline/EtOH 9/1 v/v). Animals were sacrificed in groups of four under a mild ether anesthesia by cardiac puncture at predetermined time points pi (postinjection). Additional sets of three to four animals were co-injected with excess [Tyr.sup.4]BBN (≈40 nmol) along with test radiopeptide and were sacrificed at 4 h pi (blocked animals). Samples of blood and tissues of interest were immediately collected, weighed and measured for radioactivity in a γ-counter. Stomach and intestines were not emptied of their contents, but measured as collected. Biodistribution data were calculated as percent injected dose per gram tissue (% ID/g) using the Microsoft Excel program with the aid of suitable standards of the injected dose.
(86) Results
(87) The results of the various illustrative tests are described herebelow by referring to the corresponding figure. Specific structural, functional, and procedural details disclosed in the following results are not to be interpreted as limiting.
(88)
(89) ##STR00025##
(90)
(91)
(92) ##STR00026##
(93)
(94)
(95) ##STR00027##
(96)
(97)
(98) ##STR00028##
(99)