Improvement of pharmacokinetics of radioiodinated Tyr(3)-octreotide by conjugation with carbohydrates

Among a variety of other factors, the clearance kinetics and routes of excretion of radiopharmaceuticals are of crucial importance for early and high tumor/background ratios and thus signal intensity in diagnostic imaging by single photon emission tomography (SPECT) or positron emission tomography (PET). To overcome the unfavorable pharmacokinetics of radiohalogenated octreotide analogues, we evaluated three carbohydrated conjugates of Tyr(3)-octreotide (TOC). Glucose ([(125)I]Gluc-TOC), maltose ([(125)I]Malt-TOC), and maltotriose ([(125)I]Mtr-TOC) derivatives of [(125)I]TOC were synthesized via Maillard reaction and subsequent radioiodination. In cells transfected with sst1-sst5, I-Malt-TOC, and I-Mtr-TOC show sst-subtype binding profiles similar to I-TOC with high affinity for sst2. Comparative biodistribution studies 10, 30, and 60 min pi in nude mice bearing rat pancreatic tumor xenografts showed fast blood clearance for all glycosylated derivatives. Due to their markedly increased hydrophilicity, [(125)I]Gluc-TOC and [(125)I]Malt-TOC were mainly cleared via the kidneys, which led to a significant decrease in activity accumulation in liver and intestine (5.3 and 1.4 versus 10.6%ID/g for [(125)I]TOC in the liver, 1.7 and 1.0 versus 3.8%ID/g for [(125)I]TOC in the intestine 60 min pi). For all compounds, hydrophilicity and uptake in liver and intestines correlate. Uptake of the carbohydrate conjugates in the kidney was comparable. Compared to the parent compound, the accumulation of the carbohydrated compounds in sst-rich tissues (pancreas, adrenals) was increased by a factor of 1.5-3.5. While tumor uptake of [(125)I]TOC (6.7 +/- 2.6%ID/g), [(125)I]Malt-TOC (5.3 +/- 1.9%ID/g), and [(125)I]Mtr-TOC (4.9 +/- 2.2%ID/g) at 30 min postinjection was comparable, accumulation of [(125)I]Gluc-TOC was significantly increased (10.1 +/- 2.8%ID/g at 30 min pi). Somatostatin receptor specificity of tumor uptake was confirmed by pretreatment, competition, and displacement experiments in vivo using 0.8 mg TOC/kg and gamma-camera imaging. Glycosylation proved to be a powerful tool for the development of high affinity sst ligands with excellent excretion profiles and improved tumor accumulation.     

Development of novel 68Ga- and 18F-labeled GnRH-I analogues with high GnRHR-targeting efficiency

A large majority of tumors of the reproductive system express the gonadotropin releasing hormone receptor (GnRHR). Blockade and activation of this receptor with various antagonistic and agonistic analogues of native GnRH-I (pGlu(1)-His(2)-Trp(3)-Ser(4)-Tyr(5)-Gly (6)-Leu(7)-Arg(8)-Pro(9)-Gly(10)-NH2), respectively, has shown efficient suppression of tumor growth. In this study, the GnRH-receptor system has been evaluated with respect to its suitability as a target for in vivo peptide receptor targeting using radiolabeled GnRH-analogues, and in parallel, new (18)F- and (68Ga)-labeled GnRH analogues have been developed. In vitro radioligand binding assays performed with various GnRHR-expressing human cell lines using [(125)I]Triptorelin (D-Trp(6)-GnRH-I) as the standard radioligand revealed a very low level of GnRH receptor expression on the cell surface. Generally, total cellular activity was very low (approximately 3% of the applied activity), and only a small fraction (max. 40%) of cell-associated activity could be attributed to receptor-specific radioligand binding/internalization. However, substitution of fetal calf serum by NU serum in the culture medium led to increased and stable GnRHR-expression, especially in the ovarian cancer cell line EFO-27, thus allowing for a stable experimental setup for the evaluation of the new radiolabeled GnRH-I analogues. The new radiolabeled GnRH-I analogues developed in this study were all based on the D-Lys(6)-GnRH-I-scaffold. For (68)Ga-labeling, the latter was coupled with DOTA at D-Lys(6). To allow (18)F-labeling via chemoselective oxime formation, D-Lys(6)-GnRH-I was also conjugated with Ahx (aminohexanoic acid) or beta-Ala, which in turn was coupled with Boc-aminooxyacetic acid. (18)F-labeling via oxime formation with 4-[(18)F]fluorobenzaldehyde was performed using the Boc-protected precursors. Receptor affinities of [(68)Ga]DOTA-GnRH-I, D-Lys(6)-Ahx([(18)F]FBOA)-GnRH-I, and D-Lys(6)-betaAla([(18)F]FBOA)-GnRH-I (FBOA = fluorobenzyloxime acetyl) were determined using GnRHR-membrane preparations, and internalization efficiency of the new radioligands was determined in EFO-27 cells. Both quantities were highest for D-Lys(6)-Ahx([(18)F]FBOA)-GnRH-I (IC 50 = 0.50 +/- 0.08 nM vs 0.13 +/- 0.08 nM for Triptorelin; internalization: 86 +/- 16% of the internal reference [(125)I]Triptorelin), already substantially reduced in the case of the -betaAla([(18)F]FBOA)-derivative (IC 50 = 0.86 +/- 0.13 nM; internalization: 42 +/- 3% of [(125)I]Triptorelin), while the [(68)Ga]DOTA-analogue showed almost complete loss of binding affinity and ligand internalization (IC50 = 13.3 +/- 1.0 nM; internalization: 2.6 +/- 1.0% of [(125)I]Triptorelin). Generally, the lipophilic residue [(18)F]FBOA is much better tolerated as a modification of the D-Lys(6)-side chain, with receptor affinity of the respective analogues strongly depending upon spacer length between the D-Lys(6)-side chain and the [(18)F]FBOA-moiety. In summary, D-Lys(6)(Ahx-[(18)F]FBOA)-GnRH-I shows the highest potential for efficient GnRHR-targeting in vivo of the compounds investigated. Unfortunately, however, the very low cell surface expression of GnRH-receptors and thus very low radioligand uptake by GnRHR-positive tumor cells found in vitro was also confirmed by a preliminary biodistribution study in OVCAR-3 xenografted nude mice using the standard GnRHR radioligand [(125)I]Triptorelin. Tumor uptake was lower than blood activity concentration at 1 h p.i. (0.49 +/- 0.05 vs 0.96 +/- 0.13 for tumor and blood, respectively). These data seriously challenge the suitability of the GnRHR-system as a suitable target for in vivo peptide receptor imaging using radiolabeled GnRH-I derivatives, despite the availability of high-affinity radiolabeled receptor-ligands such as D-Lys(6)(Ahx-[(18)F]FBOA)-GnRH-I.     

Meta-iodobenzylguanidine derivatives containing a second guanidine moiety

Radioiodinated meta-iodobenzylguanidine (MIBG) is used in the diagnosis and therapy of various neuroendocrine tumors. To investigate whether an additional guanidine function in the structure of MIBG will yield analogues that may potentially enhance tumor-to-target ratios, two derivatives-one with a guanidine moiety and another with a guanidinomethyl group at the 4-position of MIBG-were prepared. In the absence of any uptake-1 inhibiting conditions, the uptake of 4-guanidinomethyl-3-[(131)I]iodobenzylguanidine ([(131)I]GMIBG) by SK-N-SH cells in vitro was 1.7+/-0.1% of input counts, compared to a value of 40.3+/-1.4% for [(125)I[MIBG suggesting that guanidinomethyl group at the 4-position negated the biological properties of MIBG. On the other hand, 4-guanidino-3-[(131)I]iodobenzylguanidine ([(131)I]GIBG) had an uptake (5.6+/-0.3%) that was 12-13% that of [(125)I]MIBG (46.1+/-2.7%), and the ratio of uptake by control over DMI-treated (nonspecific) cultures was higher for [(131)I]GIBG (20.9+/-0.3) than [(125)I]MIBG itself (15.0+/-2.7). The exocytosis of [(131)I]GIBG and [(125)I]MIBG from SK-N-SH cells was similar. The uptake of [(131)I]GIBG in the mouse target tissues, heart and adrenals, as well as in a number of other tissues was about half that of [(125)I]MIBG. These results suggest that substitution of guanidine functions, especially a guanidinomethyl group, in MIBG structure may not be advantageous.     

Site-specifically traced drug release and biodistribution of a paclitaxel-antibody conjugate toward improvement of the linker structure

Tumor-directed drug delivery is a promising strategy in cancer treatment, and in this field, monoclonal antibodies constitute an important class of targeting vehicles. A critical issue in the design of targeting conjugates is the timing of the release of the cytotoxic payload, with the ideal situation being the release at the maximum tumor uptake of the targeting molecule. A site-specific radiolabeling technique was used to elucidate the biodistribution and in vivo drug release pattern of an antibody conjugate of paclitaxel (PTX, 1, Figure 1) in which the drug and the antibody moieties were connected by a succinate (SX) linker. In this new method, a metabolite of PTX, 3'-(4-hydroxyphenyl)paclitaxel (3'-OH-PTX, 2, Figure 1) was used as a tyrosine mimic for the synthesis of the drug site-labeled conjugate (DSL, [(125)I]-3'-OH-PTXSXC225). This was achieved by iodogen (125)I-labeling of 3'-OH-PTXSX and subsequent conjugation to C225. The antibody site-labeled conjugate (ASL, PTXSX-[(125)I]-C225) was prepared by direct radioiodination of PTXSXC225. Biodistribution of these compounds was studied in Balb/c nude mice bearing DU-145 human prostate carcinoma xenografts. While the 4 and 24 h tumor uptake (in percent injected dose per gram of tissue, %ID/g) for [(125)I]-3'-OH-PTXSXC225 were 3.3 +/- 1.5 and 1.7 +/- 0.6%ID/g, the PTXSX-[(125)I]-C225 showed tumor uptake values of 3.8 +/- 4.2 and 14.8 +/- 4.2%ID/g at these time points. This difference in the tumor uptake over time indicates an early cleavage of the drug with respect to the antibody tumor localization. This was further confirmed by an in vitro drug release kinetics study leading to a half-life of about 2 h for PTXSXC225 under physiological conditions. To increase the stability of the PTX-MAb bond, a new conjugate (PTXGLC225) with glutaric acid (GL) as the linker was synthesized. Under the same conditions, the PTXGLC225 showed a 16-fold increase in the half-life (t(1/2)) of the drug release. The effect of the increased t(1/2) of this compound on the antitumor activity of the conjugate was tested in a DU-145 human prostate tumor-implanted mouse model. In comparison to a previous similar experiment with PTXSXC225, better antitumor activity was observed for the PTXGLC225 conjugate as compared to controls. These results demonstrated the first time use of radioiodinated 3'-OH-PTX for in vivo tracing of a paclitaxel conjugate and application of the resulting information to the design of a therapeutically more useful PTX-MAb linker.     

Pharmacological evaluation of an [(123)I] labelled imidazopyridine-3-acetamide for the study of benzodiazepine receptors

In vitro binding of the iodinated imidazopyridine, N',N'-dimethyl-6-methyl-(4'-[(123)I]iodophenyl)imidazo[1,2-a]pyridine-3-acetamide [(123)I]IZOL to benzodiazepine binding sites on brain cortex, adrenal and kidney membranes is reported. Saturation experiments showed that [(123)I]IZOL, bound to a single class of binding site (n(H)=0.99) on adrenal and kidney mitochondrial membranes with a moderate affinity (K(d)=30 nM). The density of binding sites was 22+/-6 and 1.2+/-0.4 pmol/mg protein on adrenal and kidney membranes, respectively. No specific binding was observed in mitochondrial-synaptosomal membranes of brain cortex. In biodistribution studies in rats, the highest uptake of [(123)I]IZOL was found 30 min post injection in adrenals (7.5% ID/g), followed by heart, kidney, lung (1% ID/g) and brain (0.12% ID/g), consistent with the distribution of peripheral benzodiazepine binding sites. Pre-administration of unlabelled IZOL and the specific PBBS drugs, PK 11195 and Ro 5-4864 significantly reduced the uptake of [(123)I]IZOL by 30% (p<0.05) in olfactory bulbs and by 51-86% (p<0.01) in kidney, lungs, heart and adrenals, while it increased by 30% to 50% (p<0.01) in the rest of the brain and the blood. Diazepam, a mixed CBR-PBBS drug, inhibited the uptake in kidney, lungs, heart, adrenals and olfactory bulbs by 32% to 44% (p<0.01) but with no effect on brain uptake and in blood concentration. Flumazenil, a central benzodiazepine drug and haloperidol (dopamine antagonist/sigma receptor drug) displayed no effect in [(123)I]IZOL in peripheral organs and in the brain. [(123)I]IZOL may deserve further development for imaging selectively peripheral benzodiazepine binding sites.     

Biological evaluation of ring- and side-chain-substituted m-iodobenzylguanidine analogues

A number of ring- and side-chain-substituted m-iodobenzylguanidine analogues were evaluated for their lipophilicity, in vitro stability, uptake by SK-N-SH human neuroblastoma cells in vitro, and biodistribution in normal mice. As expected, the lipophilicity of m-iodobenzylguanidine increased when a halogen was introduced onto the ring and decreased with the addition of polar hydroxyl, amino, and nitro substitutents. Most of the derivatives showed reasonable stability up to 24 h in PBS at 37 degrees C. While N(1)-hydroxy-N(3)-3-[(131)I]iodobenzylguanidine and 3,4-dihydroxy-5-[(131)I]iodobenzylguanidine generated a more nonpolar product in addition to the free iodide, 3-[(131)I]iodo-4-nitrobenzylguanidine decomposed to a product more polar than the parent compound. The specific uptake of 4-chloro-3-[(131)I]iodobenzylguanidine, 3-[(131)I]iodo-4-nitrobenzylguanidine, and N(1)-hydroxy-N(3)-3-[(131)I]iodobenzylguanidine by SK-N-SH human neuroblastoma cells in vitro, relative to that of m-[(125)I]iodobenzylguanidine, was 117 +/- 10%, 50 +/- 4%, and 12 +/- 2%, respectively. The specific uptake of the known m-iodobenzylguanidine analogues 4-hydroxy-3-[(131)I]iodobenzylguanidine and 4-amino-3-[(131)I]iodobenzylguanidine was 80 +/- 4% and 66 +/- 4%, respectively. None of the other m-iodobenzylguanidine derivatives showed any significant specific uptake by SK-N-SH cells. Heart uptake of 4-chloro-3-[(131)I]iodobenzylguanidine in normal mice was higher than that of m-[(125)I]iodobenzylguanidine at later time points (11 +/- 1% ID/g versus 3 +/- 1% ID/g at 24 h; p < 0.05) while uptake of 3-[(131)I]iodo-4-nitrobenzylguanidine and of N(1)-hydroxy-N(3)-3-[(131)I]iodobenzylguanidine in the heart was lower than that of m-iodobenzylguanidine at all time points. In accordance with the in vitro results, none of the other novel m-iodobenzylguanidine derivatives showed any significant myocardial or adrenal uptake in vivo.     

Radioiodine and 211At-labeled guanidinomethyl halobenzoyl octreotate conjugates: potential peptide radiotherapeutics for somatostatin receptor-positive cancers

Derivatives of the somatostatin analogues octreotide and octreotate labeled with radioiosotopes are used in the diagnosis and therapy of somatostatin receptor (SSTR)-positive tumors. A method has been devised to synthesize {N-(4-guanidinomethyl-3-iodobenzoyl)-Phe1-octreotate (GMIBO). Receptor binding assay and scatchard analysis yielded a Kd of 4.83 +/- 0.19 nM for this peptide. Derivatives of this peptide labeled with radioiodine ([*I]GMIBO) and the alpha-particle-emitting radiohalogen 211At N-(3-[211At]astato-4-guanidinomethylbenzoyl)-Phe1-octreotate; [211At]AGMBO} were prepared in a single step from a tin precursor in radiochemical yields of 30-35% and 15-20%, respectively. Paired-label internalization assays performed with the SSTR-positive D341 Med human medulloblastoma cell line demonstrated that [125I]GMIBO and [211At]AGMBO were specifically internalized 20-40% more than Nalpha-(1-deoxy-D-fructosyl)-[131I]I-Tyr3-octreotate ([131I]I-Glu-TOCA), the radioiodinated octreotide derivative previously shown to exhibit maximum internalization in this cell line. Uptake of [131I]GMIBO in D341 Med subcutaneous xenografts in a murine model (8.34 +/- 1.82 versus 8.10 +/- 2.23% ID/g at 1h) and SSTR-expressing normal tissues was comparable to that of [125I]I-Glu-TOCA and was shown to be specific. However, the uptake of [131I]GMIBO also was substantially higher in liver (16.9 +/- 3.15 versus 1.39 +/- 0.45% ID/g at 1 h) and in kidneys (44.33 +/- 6.47 versus 3.44 +/- 0.68% ID/g at 1h) compared to that of [125I]I-Glu-TOCA. These data suggest that these novel peptide conjugates retain their specificity for SSTR both in vitro and in vivo; however, because of their higher accumulation in normal tissues they would be best applied in settings amenable to loco-regional administration such as medulloblastoma neoplastic meningitis.     

Synthesis and characterization of styrylchromone derivatives as beta-amyloid imaging agents

Several promising agents have been synthesized and evaluated for in vivo imaging probes of beta-amyloid plaques in Alzheimer's disease (AD) brain. Recently, we have developed flavone derivatives, which possess the basic structure of the 2-phenylchromone, as useful candidates for amyloid imaging agents. In an attempt to further develop novel tracers, we synthesized and evaluated a series of 2-styrylchromone derivatives, which replace the 2-phenyl substituent of flavone backbone with the 2-styryl. A series of radioiodinated styrylchromone derivatives were designed and synthesized. The binding affinities for amyloid plaques were assessed by in vitro binding assay using pre-formed synthetic Abeta(1-40) aggregates. The new series of styrylchromone derivatives showed high binding affinity to Abeta aggregates at the K(d) values of 32.0, 17.5 and 8.7nM for [(125)I]6, [(125)I]9, and [(125)I]12, respectively. In biodistribution studies using normal mice, [(125)I]6 and [(125)I]9 examined in normal mice displayed high brain uptakes with 4.9 and 2.8%ID/g at 2min post injection. The radioactivity washed out from the brain rapidly (1.6 and 1.0%ID/g at 60min post injection for [(125)I]6 and [(125)I]9, respectively). But [(125)I]12 did not show marked brain uptake, and the washout rate from the brain was relatively slow throughout the time course (1.1 and 1.4%ID/g at 2 and 30min post injection, respectively). Although additional modifications are necessary to improve the brain uptake and rapid clearance of non-specifically bound radiotracer, the styrylchromone backbone may be useful as a backbone structure to develop novel beta-amyloid imaging agents.     

Radiosynthesis,in vitro and in vivo evaluation of 123I-labeled anandamide analogues for mapping brain FAAH

Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for terminating the signaling of endocannabinoids, including anandamide. This paper is the first report of the synthesis, [¹²³I]-labeling and in vitro and in vivo evaluation of anandamide analogues as potential metabolic trapping radioligands for in vivo evaluation of brain FAAH. N-(2-Iodoethyl)linoleoylamide (2) and N-(2-iodoethyl)arachidonylamide (4) were synthesized with good yields (75% and 86%, respectively) in a two steps procedure starting from their respective acids. In vitro analyses, performed using recombinant rat FAAH and [³H]-anandamide, demonstrated interaction of 2 and 4 with FAAH (IC₅₀ values of 5.78 μM and 3.14 μM, respectively). [¹²³I]-2 and [¹²³I]-4 were synthesized with radiochemical yields of 21% and 12%, respectively, and radiochemical purities were >90%. Biodistribution studies in mice demonstrated brain uptake for both tracers (maximum values of 1.23%ID/g at 3 min pi for [¹²³I]-2 and 0.58%ID/g at 10 min pi for [¹²³I]-4). However, stability studies demonstrated the sensitivity of both tracers to dehalogenation.     

Synthesis and evaluation of glycosylated octreotate analogues labeled with radioiodine and 211At via a tin precursor

Carbohydration of N-terminus and substitution of a threonine for the threoninol residue at the C-terminus of Tyr3-octreotide (TOC) has resulted in improved pharmacokinetics and tumor targeting of its radioiodinated derivatives. Yet, these peptides are very susceptible to in vivo deiodination due to the similarity of monoiodotyrosine (MIT) to thyroid hormone. The goal of this work was to develop octreotate analogues containing both a sugar moiety and a nontyrosine prosthetic group on which a radioiodine or 211At can be introduced. Solid-phase synthesis and subsequent modifications delivered an iodo standard of the target peptide N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-(3-iodobenzoyl)-Lys0-octreotate (GIBLO) and the corresponding tin precursor N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-[(3-tri-n-butylstannyl)benzoyl]-Lys0-octreotate (GTBLO). GIBLO displaced [125I]TOC from somatostatin receptor subtype 2 (SSTR2)-positive AR42J rat pancreatic tumor cell membranes with an IC50 of 0.46 +/- 0.05 nM suggesting that GIBLO retained affinity to SSTR2. GTBLO was radiohalogenated to [131I]GIBLO and N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-(3-[211At]astatobenzoyl)-Lys0-octreotate ([211At]GABLO) in 21.2 +/- 4.9% and 46.8 +/- 9.5% radiochemical yields, respectively. From a paired-label internalization assay using D341 Med medulloblastoma cells, the maximum specific internalized radioactivity from [131I]GIBLO was 1.78 +/- 0.8% of input dose compared to 9.67 +/- 0.43% for N(alpha)-(1-deoxy-D-fructosyl)-[125I]iodo-Tyr3-octreotate ([125I]I-Gluc-TOCA). Over a 4 h period, the extent of internalization of [131I]GIBLO and [211At]GABLO was similar in this cell line. In D341 Med murine subcutaneous xenografts, the uptake of [125I]I-Gluc-TOCA at 0.5, 1 and 4 h was 21.5 +/- 4.0% ID/g, 18.8 +/- 7.7% ID/g, and 0.9 +/- 0.4% ID/g, respectively. In comparison, these values for [131I]GIBLO were 6.9 +/- 1.2% ID/g, 4.7 +/- 1.4% ID/g, and 0.8 +/- 0.5% ID/g. Both in vitro and in vivo catabolism studies did not suggest the severance of the lys0 along with its appendages from the peptide. Taken together, although GIBLO maintained affinity to SSTR2, its tumor uptake both in vitro and in vivo was substantially lower than that of I-Gluc-TOCA suggesting other factors such as net charge and overall geometry of the peptide may be important.     

Somatostatin receptors (sst2) are coupled to Go and modulate GTPase activity in the rabbit retina

The role of somatostatin and its mechanism of action in the retina remains an important target for investigation. Biochemical and pharmacological studies were engaged to characterize the somatostatin receptors in the rabbit retina, and their coupling to G-proteins. The ability of selective ligands to inhibit [125I]Tyr11-somatostatin-14 binding to rabbit retinal membranes was examined. The sst2 analogues SMS201-995, MK678, and BIM23014, displayed IC50 values of 0.28 +/- 0.12, 0.04 +/- 0.01 and 1.57 +/- 0.39 nm, respectively. The sst1 analogue CH275 moderately displaced the [125I]Tyr11-somatostatin-14 binding, while selective analogues for sst3, sst4 and sst5 had minimal effect. Immunoblotting and/or immunohistochemistry studies revealed the presence of the pertussis toxin sensitive Gi1/2, and Go proteins, as well as Gs. Somatostatin-14 and MK678 stimulated GTPase activity in a concentration-dependent manner with EC50 values of 42.8 +/- 16.8 and 70.0 +/- 16.5 nm, respectively, thus supporting the functional coupling between the receptor and the G-proteins. CH275 stimulated the GTPase activity moderately, in agreement with its binding profile. The antisera raised against Goalpha and Gi1/2alpha inhibited the somatostatin-induced high-affinity GTPase activity, but only anti-Goalpha inhibited the MK678 stimulation of the enzyme. These results suggest that somatostatin mediates its actions in the rabbit retina by interacting mainly with sst2 receptors that couple to Goalpha.     

Study of fluxes at low concentrations of l-tri-iodothyronine with rat liver cells and their plasma-membrane vesicles. Evidence for the accumulation of the hormone against a gradient

1. Influx and efflux of l-tri-[(125)I]iodothyronine with isolated rat liver parenchymal cells and their plasma-membrane vesicles were studied by a rapid centrifugation technique. 2. At 23 degrees C and in the concentration range that included the concentration of free l-tri-iodothyronine in rat plasma (3-5pm) influx into cells was saturable; an apparent K(t) value of 8.6+/-1.6pm was obtained. 3. At 5pm-l-tri-[(125)I]iodothyronine in the external medium the ratios of the concentrations inside to outside in cells and plasma-membrane vesicles were 38:1 and 366:1 respectively after 7s of incubation. At equilibrium (60s at 23 degrees C) uptake of l-tri-[(125)I]iodothyronine by cells was linear with the hormone concentration, whereas that by plasma-membrane vesicles exhibited an apparent saturation with a K(d) value of 6.1+/-1.3pm. 4. Efflux of l-tri-[(125)I]iodothyronine from cells equilibrated with the hormone (5-123pm) was constant up to 21 s; the amount that flowed out was 17.7+/-3.8% when cells were equilibrated with 5pm-hormone. When plasma-membrane vesicles were equilibrated with l-tri-[(125)I]iodothyronine (556-1226pm) 66.8+/-5.8% flowed out after 21 s. 5. From a consideration of the data on efflux from cells and binding of l-tri-[(125)I]iodothyronine to the liver homogenate, as studied by the charcoal-adsorption and equilibrium-dialysis methods, it appears that 18-22% of the hormone exists in the free form in the cell. 6. Vinblastine and colchicine diminished the uptake of l-tri-[(125)I]iodothyronine by cells but not by plasma-membrane vesicles; binding to the cytosol fraction was not affected. Phenylbutazone, 6-n-propyl-2-thiouracil, methimazole and corticosterone diminished the uptake by cells, plasma-membrane vesicles and binding to the cytosol fraction to different extents. 7. These results suggest that at low concentrations of l-tri-[(125)I]iodothyronine rat liver cells and their plasma-membrane vesicles accumulated the hormone against an apparent gradient by a membrane-mediated process. Contribution of cytoplasmic proteins to uptake by plasma-membrane vesicles was negligible. The amount of l-tri-[(125)I]iodothyronine required to achieve half-maximal uptake agrees with that occurring in the free form in the blood, conferring physiological importance to the transporting system in the plasma membrane of the liver cell.     

Novel imaging agents for β-amyloid plaque based on the N-benzoylindole core

We report the synthesis and evaluation of a series of N-benzoylindole derivatives as novel potential imaging agents for β-amyloid plaques. In vitro binding studies using Aβ(1-40) aggregates versus [(125)I]TZDM showed that all these derivatives demonstrated high binding affinities (K(i) values ranged from 8.4 to 121.6 nM). Moreover, two radioiodinated compounds [(125)I]7 and [(125)I]14 were prepared. Autoradiography for [(125)I]14 displayed intense and specific labeling of Aβ plaques in the brain sections of AD model mice (C57, APP/PS1) with low background. In vivo biodistribution in normal mice exhibited sufficient initial brain uptake for imaging (2.19% and 2.00%ID/g at 2 min postinjection for [(125)I]7 and [(125)I]14, respectively). Although additional modifications are necessary to improve brain uptake and clearance from the brain, the N-benzoylindole may be served as a backbone structure to develop novel β-amyloid imaging probes.     

Influence of different spacers on the biological profile of a DOTA-somatostatin analogue

Radiolabeled somatostatin analogues have been successfully used for targeted radiotherapy and for imaging of somatostatin receptor (sst1-5)-positive tumors. Nevertheless, these analogues are subject to improving their tumor-to-nontarget ratio to enhance their diagnostic or therapeutic properties, preventing nephrotoxicity. In order to understand the influence of lipophilicity and charge on the pharmacokinetic profile of [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)]-somatostatin-based radioligands such as [DOTA,1-Nal3]-octreotide (DOTA-NOC), different spacers (X) based on 8-amino-3,6-dioxaoctanoic acid (PEG2), 15-amino-4,7,10,13-tetraoxapentadecanoic acid (PEG4), N-acetyl glucosamine (GlcNAc), triglycine, beta-alanine, aspartic acid, and lysine were introduced between the chelator DOTA and the peptide NOC. All DOTA-X-NOC conjugates were synthesized by Fmoc solid-phase synthesis. The partition coefficient (log D) at pH = 7.4 indicated that higher hydrophilicity than [111In-DOTA]-NOC was achieved with the introduction of the mentioned spacers, except with triglycine and beta-alanine. The high affinity of [InIII-DOTA]-NOC for human sst2 (hsst2) was preserved with the structural modifications, while an overall drop for hsst3 affinity was observed, except in the case of [InIII-DOTA]-beta-Ala-NOC. The new conjugates preserved the good affinity for hsst5, except for [InIII-DOTA]-Asn(GlcNAc)-NOC, which showed decreased affinity. A significant 1.2-fold improvement in the specific internalization rate in AR4-2J rat pancreatic tumor cells (sst2 receptor expression) at 4 h was achieved with the introduction of Asp as a spacer in the parent compound. In sst3-expressing HEK cells, the specific internalization rate at 4 h for [111In-DOTA]-NOC (13.1% +/- 0.3%) was maintained with [111In-DOTA]-beta-Ala-NOC (14.0% +/- 1.8%), but the remaining derivatives showed <2% specific internalization. Biodistribution studies were performed with Lewis rats bearing the AR4-2J rat pancreatic tumor. In comparison to [111In-DOTA]-NOC (2.96% +/- 0.48% IA/g), the specific uptake in the tumor at 4 h p.i. was significantly improved for the 111In-labeled sugar analogue (4.17% +/- 0.46% IA/g), which among all the new derivatives presented the best tumor-to-kidney ratio (1.9).     

99mTc-labeling and in vitro and in vivo evaluation of HYNIC- and (Nalpha-His)acetic acid-modified [D-Glu1]-minigastrin

Gastrin/CCK-2 receptors are overexpressed in a number of tumors such as medullary thyroid cancer (MTC) and small cell lung cancer (SCLC). Recently [D-Glu1]-minigastrin (MG) has been radiolabeled with 131I, 111In, and 90Y and evaluated in patients. This study describes the labeling and evaluation of MG with technetium-99m using two different labeling approaches: HYNIC as bifunctional coupling agent and (Nalpha-His)Ac as tridentate ligand for 99mTc(CO3) labeling. Labeling was perfomed at high specific activities using Tricine and EDDA as coligands for HYNIC-MG and [99mTc(OH2)3(CO)3]+ for (Nalpha-His)Ac-MG. Stability experiments were carried out by reversed phase HPLC analysis in PBS, serum, histidine, and cysteine solutions, as well as rat liver and kidney homogenates. Receptor binding and internalization experiments were performed using CCK-2 receptor positive AR42J rat pancreatic tumor cells. Biodistribution experiments were carried out in nude mice carrying AR42J tumors by injection of 99mTc-labeled peptide with or without coinjection of 50 microg of minigastrin I human (MGh). HYNIC-MG and (Nalpha-His)Ac-MG could be radiolabeled at high specific activities (>1 Ci/micromol). For HYNIC-MG, high labeling yields (>95%) were achieved using Tricine and EDDA as coligands. Stability experiments of all 99mTc-labeled conjugates revealed a high stability of the label in PBS and serum as well as toward challenge with histidine and cysteine. Incubation in kidney homogenates resulted in a rapid degradation of all conjugates with <10% intact peptide after 60 min at 37 degrees C, with no considerable differences between the radiolabeled conjugates; a somewhat lower degradation rate was seen in liver homogenates. Protein binding varied considerably with lowest levels for 99mTc-EDDA/HYNIC-MG. Competition experiments of unlabeled conjugates on AR42J membranes versus [125I-Tyr12]-gastrin I showed high CCK-2 receptor affinity for all conjugates under study. Internalization behavior was very rapid for all radiolabeled conjugates in the order of 99mTc-(Nalpha-His)Ac-MG > 99mTc-EDDA/HYNIC-MG > 99mTc-Tricine/HYNIC-MG. In tumor-bearing nude mice the highest tumor-uptake was observed with 99mTc-EDDA/HYNIC-MG (8.1%ID/g) followed by 99mTc-Tricine/HYNIC-MG (2.2%ID/g) and 99mTc-(Nalpha-His)Ac-MG (1.2%ID/g) which correlated with kidney uptake (101.0%ID/g, 53.8%ID/g, 1.8%ID/g respectively). In this series of compounds 99mTc-EDDA/HYNIC-MG with its very high tumor/organ ratios except for kidneys seems to be the most promising agent to target CCK-2 receptors. Despite promising properties concerning receptor binding, internalization, and in vitro stability, 99mTc-(Nalpha-His)Ac-MG showed low tumor uptake in vivo.     

Nepsilon-(3-[*I]Iodobenzoyl)-Lys5-Nalpha-maleimido-Gly1-GEEEK ([*I]IB-Mal-D-GEEEK): a radioiodinated prosthetic group containing negatively charged D-glutamates for labeling internalizing monoclonal antibodies

Novel methods are needed for the radiohalogenation of cell-internalizing proteins and peptides because rapid loss of label occurs after lysosomal processing when these molecules are labeled using conventional radioiodination methodologies. We have developed a radiolabeled prosthetic group that contains multiple negatively charged D-amino acids to facilitate trapping of the radioactivity in the cell after proteolysis of the labeled protein. N(epsilon)-(3-[(125)I]iodobenzoyl)-Lys(5)-N(alpha)-maleimido-Gly(1)-GEEEK ([(125)I]IB-Mal-D-GEEEK) was synthesized via iododestannylation in 90.3 +/- 3.9% radiochemical yields. This radioiodinated agent was conjugated to iminothiolane-treated L8A4, an anti-epidermal growth factor receptor variant III (EGFRvIII) specific monoclonal antibody (mAb) in 54.3 +/- 17.7% conjugation yields. In vitro assays with the EGFRvIII-expressing U87MGDeltaEGFR glioma cell line demonstrated that the internalized radioactivity for the [(125)I]IB-Mal-D-GEEEK-L8A4 conjugate increased from 14.1% at 1 h to 44.7% at 24 h and was about 15-fold higher than that of directly radioiodinated L8A4 at 24 h. A commensurately increased tumor uptake in vivo in athymic mice bearing subcutaneous U87MGDeltaEGFR xenografts (52.6 +/- 14.3% injected dose per gram versus 17.4 +/- 3.5% ID/g at 72 h) also was observed. These results suggest that [(125)I]IB-Mal-d-GEEEK is a promising reagent for the radioiodination of internalizing mAbs.     

In Vivo Differences between Two Optical Isomers of Radioiodinated o-iodo-trans-decalinvesamicol for Use as a Radioligand for the Vesicular Acetylcholine Transporter

Purpose

To develop a superior VAChT imaging probe for SPECT, radiolabeled (-)-OIDV and (+)-OIDV were isolated and investigated for differences in their binding affinity and selectivity to VAChT, as well as their in vivo activities.

Procedures

Radioiodinated o-iodo-trans-decalinvesamicol ([¹²⁵I]OIDV) has a high binding affinity for vesicular acetylcholine transporter (VAChT) both in vitro and in vivo. Racemic [¹²⁵I]OIDV was separated into its two optical isomers (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV by HPLC. To investigate VAChT binding affinity (Ki) of two OIDV isomers, in vitro binding assays were performed. In vivo biodistribution study of each [¹²⁵I]OIDV isomer in blood, brain regions and major organs of rats was performed at 2,30 and 60 min post-injection. In vivo blocking study were performed to reveal the binding selectivity of two [¹²⁵I]OIDV isomers to VAChT in vivo. Ex vivo autoradiography were performed to reveal the regional brain distribution of two [¹²⁵I]OIDV isomers and (-)-[¹²³I]OIDV for SPECT at 60 min postinjection.

Results

VAChT binding affinity (Ki) of (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV was 22.1 nM and 79.0 nM, respectively. At 2 min post-injection, accumulation of (-)-[¹²⁵I]OIDV was the same as that of (+)-[¹²⁵I]OIDV. However, (+)-[¹²⁵I]OIDV clearance from the brain was faster than (-)-[¹²⁵I]OIDV. At 30 min post-injection, accumulation of (-)-[¹²⁵I]OIDV (0.62 ± 0.10%ID/g) was higher than (+)-[¹²⁵I]OIDV (0.46 ± 0.07%ID/g) in the cortex. Inhibition of OIDV binding showed that (-)-[¹²⁵I]OIDV was selectively accumulated in regions known to express VAChT in the rat brain, and ex vivo autoradiography further confirmed these results showing similar accumulation of (-)-[¹²⁵I]OIDV in these regions. Furthermore, (-)-[¹²³I]OIDV for SPECT showed the same regional brain distribution as (-)-[¹²⁵I]OIDV.

Conclusion

These results suggest that radioiodinated (-)-OIDV may be a potentially useful tool for studying presynaptic cholinergic neurons in the brain.     

Design, synthesis, and biological evaluation of an antagonist-bombesin analogue as targeting vector

The gastrin releasing peptide receptor (GRP-R) is overexpressed on a number of tumors and cancer cell lines including pancreas, prostate, breast, gastrointestinal, and small cell lung cancer (SCLC). Radiolabeled bombesin (BBN) analogues have exhibited high binding affinity and specificity to the GRP-R. A bombesin analogue with an antagonist targeting vector at the C-terminus, DOTA-aminohexanoyl-[D-Phe(6), Leu-NHCH 2CH 2CH3(13), des Met(14)] BBN[6-14] (1, "Bomproamide"), has been synthesized and displays high binding affinity (IC50 = 1.36 +/- 0.09 nM) against (125)I-Tyr (4)-BBN in in vitro competitive assays using PC-3 cells. Maximum internalization of (111)In-1 reached 14% in PC-3 cells after 45 min of incubation. Rapid (0.25 h PI) and high (12.21 +/- 3.2%ID/g) pancreatic uptake of (111)In-1 was observed in healthy CF-1 mice, and 90% of the activity was blocked by coinjection of 100 mug of BBN. Rapid (0.25 h PI) and high uptake (6.90 +/- 1.06%ID/g) was observed in PC-3 prostate cancer xenografts in SCID mice, as well as visualized clearly in a SPECT/CT study. These results support the use of a bombesin construct with an antagonist C-terminal vector as a candidate of choice for specific in vivo imaging of tumors overexpressing GRP-receptors.     

Evaluation of chemical, physical, and biologic properties of tumor-targeting radioiodinated quinazolinone derivative

Our group is developing a novel technology, enzyme-mediated cancer imaging and therapy (EMCIT), that aims to entrap radioiodinated compounds within solid tumors for noninvasive tumor detection and therapy. In this approach, a water-soluble, radioiodinated prodrug is hydrolyzed in vivo to a highly water-insoluble compound by an enzyme overexpressed extracellularly by tumor cells. We have synthesized and characterized the water-soluble prodrug, 2-(2'-phosphoryloxyphenyl)-6-[(125)I]iodo-4-(3H)-quinazolinone [(125)I]5, which is readily hydrolyzed by alkaline phosphatase, an enzyme expressed by many tumor cell lines, to a water-insoluble drug, 2-(2'-hydroxyphenyl)-6-[(125)I]iodo-4-(3H)-quinazolinone [(125)I]1. In the course of our study, we discovered that ammonium 2-(2'-phosphoryloxyphenyl)-6-tributylstannyl-4-(3H)-quinazolinone, an intermediate in the radioiodination of the prodrug, exists as two isomers (3 and 4) whose radioiodination leads, respectively, to [(125)I]6 and [(125)I]5. These prodrugs have different in vitro and in vivo biologic activities. Compound 6 is not hydrolyzed by alkaline phosphatase (ALP), whereas 5 is highly soluble (mg/mL) in aqueous solution and is rapidly dephosphorylated in the presence of ALP to 1, a water-insoluble molecule (ng/mL). Mouse biodistribution studies indicate that [(125)I]6 has high uptake in kidney and liver and [(125)I]5 has very low uptake in all normal organs. Compounds 3 and 6 are converted, respectively, to 4 and 5 after incubation in DMSO. The stability of 5 in human serum is high. The minimum ALP concentration needed to hydrolyze 5 is much greater than the ALP level in the blood of patients with cancer, and the latter should not affect the pharmacokinetics of the compound. Incubation of 5 with viable human and mouse tumor-cell lines--but not with normal human cells and mouse tissues--leads to its hydrolysis and the formation of large crystals of 1. We expect that 5 will also be hydrolyzed in vivo by tumor cells that express phosphatase activity extracellularly and anticipate the specific precipitation of radioiodinated 1 within tumor cell clusters. This should lead to high tumor-to-normal-tissue ratios and enable imaging (SPECT/PET) and radionuclide therapy of solid tumors.     

Synthesis and evaluation of [123I] labeled iodovinyl amino acids syn-, anti-1-amino-3-[2-iodoethenyl]-cyclobutane-1-carboxylic acid, and 1-amino-3-iodomethylene-cyclobutane-1-carboxylic acid as potential SPECT brain tumor imaging agents

syn- and anti-1-amino-3-[2-iodoethenyl]-cyclobutane-1-carboxylic acid (syn-, anti-IVACBC 16, 17) and their analogue 1-amino-3-iodomethylene-cyclobutane-1-carboxylic acid (gem-IVACBC 18) were synthesized and radioiodoinated with [(123)I] in 34-43% delay-corrected yield. All these amino acids entered 9L gliosarcoma cells primarily via L-type transport in vitro with high uptake of 8-10% ID/1 x 10(6) cells. Biodistribution studies of [(123)I]16, 17 and 18 in rats with 9L gliosarcoma brain tumors demonstrated high tumor to brain ratios (4.7-7.3:1 at 60 min post-injection). In this model, syn-, anti-, and gem-[(123)I]IVACBC are promising radiotracers for SPECT brain tumor imaging.