In vitro and in vivo metabolism of Lu-AMBA, a GRP-receptor binding compound, and the synthesis and characterization of its metabolites.

The metabolism of (177)Lu-AMBA (AMBA = DO3A-CH(2)CO-G-(4-aminobenzoyl)-QWAVGHLM-NH(2)), a radiotherapeutic compound in clinical development that binds to GRP and NMB receptors, was studied in vitro (mouse, rat and human plasma, mouse kidney homogenate) and in vivo (by analysis of mouse and rat plasma and urine following IV injection of (177)Lu-AMBA). The primary metabolites were Lu-DO3A-CH(2)CO-G-Abz4-R, where R = -Q-OH (A), -QW-OH (B), and -QWAVGH-OH (C). Minor amounts of (D) where R = -QWAVGHLM-OH and (E) -QWAVGHL-OH were also observed. Clearance of (177)Lu-AMBA and of radioactivity from mouse and rat blood was rapid in vivo. In mouse and rat urine, only metabolites Lu-A and Lu-B were found-no parent drug was excreted. Unmetalated ligands and (nat)Lu and (177)Lu complexes for Lu-AMBA metabolites A-E were synthesized, characterized by HPLC and MS, and used to perform in vitro competition and direct binding studies on GRP receptor-positive PC-3 (human prostate) cancer cells. Biodistribution studies with (177)Lu-labeled metabolites A-E were performed in PC-3 tumor-bearing mice and the results compared with intact (177)Lu-AMBA. IC(50) values for unmetalated metabolite ligands A-E were >400 nM in PC-3 cells in competition binding studies against (177)Lu-AMBA. No direct binding to PC-3 cells was observed with (177)Lu-labeled A-C, confirming IC(50) results. (177)Lu-labeled metabolites A-E showed no uptake in GRP-receptor positive tumor or pancreas in PC-3 tumor bearing mice. All metabolites were rapidly excreted via the renal route (approximately 78-87%) within 1 h. These results demonstrate that the tumor uptake observed with (177)Lu-AMBA is due to parent drug and not due to any of its identified metabolites.

Ranibizumab, a mAb against VEGF-A for the potential treatment of age-related macular degeneration and other ocular complications.

Genentech Inc and Novartis Ophthalmics AG have developed and launched the humanized anti-VEGF antibody fragment ranibizumab, a 48-kDa humanized antibody fragment that inhibits all forms of biologically active VEGF-A, for the treatment of age-related macular degeneration by intravitreal administration. Phase I to III clinical trials to confirm the role of ranibizumab in the treatment of choroidal neovascularization (phase II and III), diabetic macular edema (phase II and III), retinal venous occlusion (phase II and III), telangiectasia (phase I and II), central serous chorioretinopathy (phase I), polypoidal choroidal vasculopathy (phase I/II), conjunctival neoplasms (phase I) and von Hippel-Lindau syndrome (phase I) are ongoing.

177Lu-AMBA: Synthesis and characterization of a selective 177Lu-labeled GRP-R agonist for systemic radiotherapy of prostate cancer.

UNLABELLED: Gastrin-releasing peptide receptors (GRP-R) are upregulated in many cancers, including prostate, breast, and lung. We describe a new radiolabeled bombesin (BBN) analog for imaging and systemic radiotherapy that has improved pharmacokinetics (PK) and better retention of radioactivity in the tumor. METHODS: DO3A-CH2CO-G-4-aminobenzoyl-Q-W-A-V-G-H-L-M-NH2 (AMBA) was synthesized and radiolabeled. The human prostate cancer cell line PC-3 was used to determine the binding (Kd), retention, and efflux of 177Lu-AMBA. Receptor specificity was determined by in vitro autoradiography in human tissues. PK and radiotherapy studies were performed in PC-3 tumor-bearing male nude mice. RESULTS: 177Lu-AMBA has a high affinity for the GRP-R (Kd, 1.02 nmol/L), with a maximum binding capacity (Bmax) of 414 fmol/10(6) cells (2.5 x 10(5) GRP-R/cell). Internalization was similar for 177Lu-AMBA (76.8%), 177Lu-BBN8 (72.9%), and 125I-[Tyr4]-BBN (74.9%). Efflux was markedly lower for 177Lu-AMBA (2.9%) compared with 177Lu-BBN8 (15.9%) and 125I-[Tyr4]-BBN (46.1%). By receptor autoradiography, Lu-AMBA binds specifically to GRP-R (0.8 nmol/L) and to the neuromedin B receptor (NMB-R) (0.9 nmol/L), with no affinity for the bb3 receptor (>1,000 nmol/L). 177Lu-AMBA was renally excreted (55 %ID 1 h [percentage injected dose at 1 h]); tumor uptake at 1 and 24 h was 6.35 %ID/g and 3.39 %ID/g, respectively. One or 2 doses of 177Lu-AMBA (27.75 MBq/dose) significantly prolonged the life span of PC-3 tumor-bearing mice (P < 0.001 and P < 0.0001, respectively) and decreased PC-3 tumor growth rate over controls. When compared using World Health Organization criteria, mice receiving 2 doses versus 1 dose of 177Lu-AMBA demonstrated a shift away from stable/progressive disease toward complete/partial response; by RECIST (Response Evaluation Criteria in Solid Tumors), median survival increased by 36% and time to progression/progression-free survival increased by 65%. CONCLUSION: 177Lu-AMBA binds with nanomolar affinity to GRP-R and NMB-R, has low retention of radioactivity in kidney, demonstrates a very favorable risk-benefit profile, and is in phase I clinical trials.

p53 transgenic mice are highly susceptible to 1, 2-dimethylhydrazine-induced uterine sarcomas.

p53 Transgenic mice were crossed with C57BL/6J mice to investigate whether a germ-line mutation in the p53 gene predisposes for tumorigenesis in mice. (C57BL/6J x UL53-3) F(1) mice were treated with 1,2-dimethylhydrazine (DMH), a colon carcinogen. The presence of a mutant p53 led to an increased incidence or multiplicity of uterine sarcomas, colon carcinomas, lung adenomas, and hepatomas in DMH-treated mice. The most significant effect of mutant p53 was the increased incidence of uterine sarcomas, which were found in approximately 90% of p53(val135/wt) mice but only seen in approximately 10% of p53(wt/wt) mice. After examination of 15 known p53 downstream target genes in uterine sarcomas and normal uteri, we found that expression of the Reprimo gene was significantly increased in normal uteri of p53(wt/wt) mice but not in either normal uterus or uterine sarcomas of p53(val135/wt) mice. In DMH-treated animals, long-term treatment with this chemopreventive agent, piroxicam, reduced colon carcinoma incidence and multiplicity in both p53(val135/wt) or p53(wt/wt) mice but did not affect the formation of uterine sarcomas, lung adenomas, or hepatomas. These results demonstrate a tissue-specific enhancement of tumorigenesis in multiple organs by the mutant p53 transgene and additionally support the utility of (C57BL/6J x UL53-3) F(1) mice for chemoprevention studies.

Farnesyltransferase inhibitors are potent lung cancer chemopreventive agents in A/J mice with a dominant-negative p53 and/or heterozygous deletion of Ink4a/Arf.

Mutations in the Kras2 gene are seen in both human and mouse lung adenocarcinomas. The protein product (p21ras) encoded by the Kras2 gene must be post-translationally modified at a terminal CAAX motif in order to be biologically active. In this study, we systematically investigated the chemopreventive efficacy of two different farnesyltransferase inhibitors (FTIs): one is a peptidomimetic (FTI-276) and the other is an imidazole (L778-123). Both FTIs are designed to inhibit the post-translational modification of p21ras proteins with a terminal CAAX motif. In a complete chemoprevention study, where the inhibitor was administered before carcinogen was given, and throughout the study, FTI-276 treatment significantly reduced both the tumor multiplicity by 41.7% (P<0.005), and the total tumor volume by 79.4% (P<0.0001). In the late treatment study, where mice were treated with an inhibitor 12 to 20 weeks after carcinogen administration, FTI-276 treatment resulted in a 60% reduction in tumor multiplicity and 58% reduction in tumor volume. Next, we examined the chemopreventive efficacy of a new FTI, L-778,123, on lung tumor development in A/J mice and transgenic mice with a dominant-negative p53 mutation and/or heterozygous deletion of Ink4a/Arf. Treatment of mice with L-778,123 for a period of 10 weeks from 20 weeks to 30 weeks post carcinogen initiation resulted in an approximately 50% decrease in tumor multiplicity in wild-type mice and mice with a dominant-negative p53 mutation and/or heterozygous deletion of the Ink4a/Arf tumor suppressor genes. Interestingly, tumor volume was decreased approximately 50% in wild-type mice and in mice with an Ink4a/Arf heterozygous deletion, while tumor volume was decreased approximately 75% in animals with a dominant-negative p53 and in mice with both a p53 mutation and heterozygous deletion of Ink4a/Arf. This result suggests that FTI exhibited a significantly (P<0.05) more efficacious chemopreventive effect in animals with alterations of p53 and Ink4a/Arf as contrasted with wild-type mice. Thus, FTIs are potent lung chemopreventive agents in both A/J mice and transgenic mice harboring a dominant-negative p53 and heterozygous deletion of Ink4a/Arf. In fact, L-778,123 is more effective in inhibiting primary lung progression in mice with a p53 mutation and/or an Ink4a/Arf deletion than in wild-type animals.

Automated synthesis, characterization and biological evaluation of [(68)Ga]Ga-AMBA, and the synthesis and characterization of (nat)Ga-AMBA and [(67)Ga]Ga-AMBA.

Ga-AMBA (Ga-DO3A-CH(2)CO-G-[4-aminobenzoyl]-QWAVGHLM-NH(2)) is a bombesin-like agonist with high affinity for gastrin releasing peptide receptors (GRP-R). Syntheses for (nat)Ga-AMBA, [(67)Ga]Ga-AMBA and [(68)Ga]Ga-AMBA were developed. The preparation of HPLC-purified and Sep-Pak purified [(68)Ga]Ga-AMBA were fully automated, using the built-in radiodetector of the Tracerlab FX F-N synthesizer to monitor fractionated (68)Ge/(68)Ga generator elution and purification. The total synthesis time, including the fractional elution of the generator, was 20 min for Sep-Pak purified material and 40 min for HPLC-purified [(68)Ga]Ga-AMBA. Both [(67)Ga]Ga-AMBA and [(177)Lu]Lu-AMBA showed comparable high affinity for GRP-R in the human prostate cancer cell line PC-3 in vitro (k(D)=0.46+/-0.07; 0.44+/-0.08 nM), high internalization (78; 77%) and low efflux from cells at 2 h (2.4+/-0.7; 2.9+/-1.8%). Biodistribution results in PC-3 tumor-bearing male nude mice showed comparable uptake for [(177)Lu]Lu-, [(111)In]In-, [(67)Ga]Ga- and [(68)Ga]Ga-AMBA.

In vitro binding evaluation of 177Lu-AMBA, a novel 177Lu-labeled GRP-R agonist for systemic radiotherapy in human tissues.

Members of the gastrin-releasing peptide (GRP) family and its analogs bombesin (BBN) have been implicated in the biology of several human cancers including prostate, breast, colon and lung. To date, three mammalian GRP/BBN receptor subtypes have been cloned and characterized: the neuromedin B receptor (NMBR), the GRP receptor (GRPR) and the BBN-receptor subtype 3 (BB(3)). The fourth BBN receptor subtype, BB(4), has only been identified in amphibian and at present no mammalian equivalent of this receptor has been described. GRPR analogs have been used as carriers to deliver drugs, radionuclides and cytotoxins to target various cancer types that are GRPR positive. We investigated the in vitro binding properties of (177)Lu-AMBA, a novel radiolabelled BBN analog currently undergoing clinical trial as systemic radiotherapy for hormone refractory prostate cancer (HRPC) patients. Pharmacological analyses of the (177)Lu-AMBA was determined using in vitro binding studies using membrane target system containing specific receptor subtypes. We investigated the distribution of binding sites for (177)Lu-AMBA by receptor autoradiography on human neoplastic and non-neoplastic tissues. Pharmacological characterizations of (177)Lu-AMBA shows, high affinity towards NMB and GRP receptors, while little or no affinity towards BB(3) receptor. Among the 40 different types of non-neoplastic tissues tested seven of them showed limited but specific binding of (177)Lu-AMBA. Fourteen of 17 primary prostate cancers, six of 13 primary breast cancers expressed binding sites for (177)Lu-AMBA. Furthermore, no apparent differences in (177)Lu-AMBA-binding sites expression were observed between matched pairs (primary vs. secondary) of prostate and breast cancer tissues. These data represent the molecular basis for clinical applications of (177)Lu-AMBA for diagnosis and treatment of GRP-R and NMB-R positive tumors.

177Lu-AMBA biodistribution, radiotherapeutic efficacy, imaging, and autoradiography in prostate cancer models with low GRP-R expression.

UNLABELLED: (177)Lu-DO3A-CH(2)CO-G-4-aminobenzoyl-Q-W-A-V-G-H-L-M-NH(2) ((177)Lu-AMBA) is a radiolabeled bombesin derivative that is bound and internalized by cells expressing the G-protein-coupled gastrin-releasing peptide receptor (GRP-R) and is currently in phase I clinical trials. In previous radiotherapy studies with PC-3 xenografted mice, (177)Lu-AMBA treatment significantly increased survival and reduced tumor growth rates. The PC-3 tumor cell line has an elevated expression of GRP-Rs (2.5 x 10(5)/cell), whereas LNCaP--a prostate cancer metastatic cell line representing the early androgen-sensitive stage of prostate cancer--and DU145--an androgen-insensitive metastatic line--express lower receptor numbers (5.9 x 10(3) and 1.2 x 10(4)/cell, respectively). Because of tumor heterogeneity, the high number of receptors in the PC-3 line may not represent the clinical situation, and little definitive work on the GRP-R status of primary prostate tumors and metastases exists. We sought to evaluate the tumor binding and imaging potential of (177)Lu-AMBA in low GRP-R models of prostate cancer and determine how reduced expression affects (177)Lu-AMBA radiotherapy efficacy. METHODS: The LNCaP and DU145 cell lines were used to determine the binding (K(d)), retention, and efflux of (177)Lu-AMBA. Biodistribution radiotherapy, imaging, and autoradiography studies were performed in LNCaP, DU145, or PC-3 tumor-bearing male nude mice. Immunohistochemistry was used to determine the proliferative state in LNCaP and DU145 models and the vascular phenotype of LNCaP radiotherapy tumors. RESULTS: (177)Lu-AMBA binds to GRP-R in these cell lines with high affinity (K(d) of LNCaP, 0.65 +/- 0.2 nM; K(d) of DU145, 0.53 +/- 0.1 nM). The uptake of (177)Lu-AMBA is at least 10-fold less in LNCaP and DU145 cell lines than it is in the PC-3 cell line. Autoradiography identifies activity concentrated in areas of viable tumor tissue, and gamma-images of (177)Lu-AMBA identify tumors in vivo. Despite having lower uptake, (177)Lu-AMBA demonstrated radiotherapeutic efficacy and decreased proliferation in the LNCaP and DU145 xenografts; in the LNCaP model, (177)Lu-AMBA normalized the phenotype of microvasculature, reducing tumoral blood pooling. CONCLUSION: (177)Lu-AMBA is a single radiolabeled agent that combines targeted radiotherapy after imaging dosimetry with the potential for single-agent or multimodality therapy for prostate cancer.