The Effects of the Spacer on Radiochemical and Biological Properties of New Radiolabeled Bombesin(7-14) Derivative

Objective: The aim of this study was to develop 99mTc-[HYNIC-X-D-Phe13]-BBN(7-14)NH2 derivatives using two different tripeptidic spacer groups (X=GGG and X=SSS) in order to improve its pharmacokinetics, in vitro stability, specific binding, and affinity. Background: Bombesin (BBN), a 14-aminoacid amphibian peptide homolog of mammalian gastrinreleasing peptide (GRP), has demonstrated the ability to bind with high affinity and specificity to GRP receptor, which is overexpressed on a variety of human cancers. Methods: Peptide conjugates labeled with 99mTc using tricine-EDDA and radiochemical purity was assessed by TLC and HPLC. The stability of radio conjugates was evaluated in the presence of saline and human serum. Affinity, internalization, and also dissociation Constant was evaluated using MDAMB- 231 and PC-3 cell line. Biodistribution study was performed in BALB/C mice. Results: Labeling yield of ˃95% was obtained. The change introduced in the BBN sequence increased plasma stability. In vitro blocking studies showed that binding and internalization of both radiolabeled peptides are mediated by their receptors on the surface of MDA-MB-231 and PC-3 cells. Biodistribution results demonstrated a rapid blood clearance, with predominantly renal excretion. Specific binding in GRP receptor-positive tissues, such as pancreas was confirmed with a blocking study. Conclusions: The introduction of the spacer sequence between chelator and BBN(7-14) led to improved bidistribution. Analog with tri-Gly spacer is the more promising radiopeptide for targeting GRP receptors than Ser conjugates. : Therefore, these analogs can be considered as a candidate for the identification of bombesin-positive tumors.

Clinical translation of nanomaterials for early detection of genetic abnormalities in fetus and retinopathy in neonates and adults

Nuclear medicine covers a wide variety of radionuclides to meet demands of disease. In the current study, first we have looked at the application of mono-amine mono-amide ligands for Re(V), 99mTc(V), and 186Re(V) with respect to bombesin for receptor targeting in the pancreas. While procedures for synthesizing 99mTc complexes is similar to other reported procedures, rhenium complexes were synthesized using [ReO(citrate)2] - as the starting material, simplifying purification and isolation. Further studies for the 222-MAMA-BBN complex set included biodistribution studies, which determined that the 99mTc-BBN complex binds to GRP receptors in the pancreas, [about]3% ID/g. The 323-MAMA complex and derivatives were investigated to determine if the 222- or the 323-MAMA backbone provide: an easier preparation, a better framework for chelating given metals, and better transport as a targeting receptor. It is found that, in comparative studies, the 222-MAMA derivates are more preferred in chelation. However, in either case, once the metal is chelated, there is no conversion of products upon the addition of a more preferred ligand system. Another avenue of target therapy being pursued is the study of 105Rh. We are specifically looking at the study of chelation with tetrathioether complexes, to rhodium(III) to translate to the radiotracer scale. Three product isomers are formed in the reaction of rhodium, using SnCl2, with 222-S4-diAcOH. The carboxylate arm can either be free dangling, one bound to the metal, or both (removing bound chlorides respectively); all of these isomers can be easily separated using HPLC. These three species will be avoided when translated to the ligand bombesin analog. Future research in this area will be done with the 105Rh radiotracer for biological applications.

Synthesis design and nuclear medicine applications for radiometal, beta and gamma emission, chelated complexes

Nuclear medicine covers a wide variety of radionuclides to meet demands of disease. In the current study, first we have looked at the application of mono-amine mono-amide ligands for Re(V), 99mTc(V), and 186Re(V) with respect to bombesin for receptor targeting in the pancreas. While procedures for synthesizing 99mTc complexes is similar to other reported procedures, rhenium complexes were synthesized using [ReO(citrate)2] as the starting material, simplifying purification and isolation. Further studies for the 222-MAMA-BBN complex set included biodistribution studies, which determined that the 99mTc-BBN complex binds to GRP receptors in the pancreas, [about]3 percent ID/g. The 323-MAMA complex and derivatives were investigated to determine if the 222- or the 323-MAMA backbone provide: an easier preparation, a better framework for chelating given metals, and better transport as a targeting receptor. It is found that, in comparative studies, the 222-MAMA derivates are more preferred in chelation. However, in either case, once the metal is chelated, there is no conversion of products upon the addition of a more preferred ligand system. Another avenue of target therapy being pursued is the study of 105Rh. We are specifically looking at the study of chelation with tetrathioether complexes, to rhodium(III) to translate to the radiotracer scale. Three product isomers are formed in the reaction of rhodium, using SnCl2, with 222-S4-diAcOH. The carboxylate arm can either be free dangling, one bound to the metal, or both (removing bound chlorides respectively); all of these isomers can be easily separated using HPLC. These three species will be avoided when translated to the ligand bombesin analog. Future research in this area will be done with the 105Rh radiotracer for biological applications.

High expression of gastrin-releasing peptide receptors in the vascular bed of urinary tract cancers: promising candidates for vascular targeting applications

Tumoral gastrin-releasing peptide (GRP) receptors are potential targets for diagnosis and therapy using radiolabeled or cytotoxic GRP analogs. GRP-receptor overexpression has been detected in endocrine-related cancer cells and, more recently, also in the vascular bed of selected tumors. More information on vascular GRP-receptors in cancer is required to asses their potential for vascular targeting applications. Therefore, frequent human cancers (n=368) were analyzed using in vitro GRP-receptor autoradiography on tissue sections with the 125I-[Tyr4]-bombesin radioligand and/or the universal radioligand 125I-[d-Tyr6, β-Ala11, Phe13, Nle14]-bombesin(6–14). GRP-receptor expressing vessels were evaluated in each tumor group for prevalence, quantity (vascular score), and GRP-receptor density. Prevalence of vascular GRP-receptors was variable, ranging from 12% (prostate cancer) to 92% (urinary tract cancer). Different tumor types within a given site had divergent prevalence of vascular GRP-receptors (e.g. lung: small cell cancer: 0%; adenocarcinoma: 59%; squamous carcinoma: 83%). Also the vascular score varied widely, with the highest score in urinary tract cancer (1.69), moderate scores in lung (0.91), colon (0.88), kidney (0.84), and biliary tract (0.69) cancers and low scores in breast (0.39) and prostate (0.14) cancers. Vascular GRP-receptors were expressed in the muscular vessel wall in moderate to high densities. Normal non-neoplastic control tissues from these organs lacked vascular GRP-receptors. In conclusion, tumoral vessels in all evaluated sites express GRP-receptors, suggesting a major biological function of GRP-receptors in neovasculature. Vascular GRP-receptor expression varies between the tumor types indicating tumor-specific mechanisms in their regulation. Urinary tract cancers express vascular GRP-receptors so abundantly, that they are promising candidates for vascular targeting applications.

Gastrin-releasing peptide receptors in the tumor vascular bed of various human cancers: high incidence in urinary tract cancers

e14575 Background: Tumoral Gastrin-releasing peptide (GRP) receptors are potential targets for diagnosis and therapy using radiolabeled or cytotoxic GRP analogs. GRP-receptor overexpression has been detected in cancer cells and, more recently, also in the vascular bed of selected tumors. More information on vascular GRP-receptors in cancer is required to asses their potential for vascular targeting applications. Methods: Frequent human cancers from the breast (n=134), lung (n=57), prostate (n=50), kidney (n=32), colon (n=46), urinary tract (n=26) and biliary tract (n=23) were analyzed using in vitro GRP-receptor autoradiography on tissue sections with the 125I-[Tyr4]-bombesin radioligand and/or the universal radioligand 125I-[D-Tyr6, ß-Ala11, Phe13, Nle14]-bombesin(6–14). GRP-receptor expressing tumoral vessels were evaluated in each tumor group for prevalence, quantity (vascular score) and GRP-receptor density. Results: Prevalence of vascular GRP-receptors is variable, ranging from 13% (prostate cancer) to 92% (urinary tract cancer). Different tumor-types within a given site may have divergent prevalence of vascular GRP-receptors (e.g. lung: small cell cancer: 0%; adenocarcinoma: 59%; squamous carcinoma: 83%). Also the vascular score varies widely, with highest score in urinary tract cancer (1.69), moderate scores in lung (0.91), colon (0.88), kidney (0.84) and biliary tract (0.69) cancers and low scores in breast (0.39) and prostate (0.14) cancers. Vascular GRP-receptors are expressed in the muscular vessel wall in moderate to high densities. Normal non- neoplastic control tissues from these organs lack vascular GRP-receptors. Conclusions: Tumoral vessels in all evaluated sites overexpress GRP-receptors, suggesting a major biological function of GRP-receptors in the tumor vascular bed. Vascular GRP-receptor expression varies between the tumor-types indicating tumor-specific mechanisms in their regulation. Urinary tract cancers express vascular GRP-receptors so abundantly, that they are promising candidates for vascular targeting applications. No significant financial relationships to disclose.

Overexpression of Gastrin-Releasing Peptide Receptors in Tumor-Associated Blood Vessels of Human Ovarian Neoplasms

Background: Peptide receptors, overexpressed in specific cancers, represent new diagnostic and therapeutic targets. In this study, receptors for the gastrin-releasing peptide (GRP), and other members of the bombesin-family of peptides, were evaluated in ovarian neoplasms. Methods: 75 primary, secondary and metastatic ovarian tumors were investigated for their bombesin-receptor subtype expression, incidence, localization and density using in vitro autoradiography on tissue sections with the universal radioligand 125I-[D-Tyr6, ß-Ala11, Phe13, Nle14]-bombesin(6-14) and the GRP-receptor subtype-preferring 125I-[Tyr4]-bombesin. Results: GRP-receptors were detected in 42/61 primary ovarian tumors; other bombesin-receptor subtypes (BB1, bb3) were rarely present (3/61). Two different tissue compartments expressed GRP-receptors: the tumoral vasculature was the predominant site of GRP-receptor expression (38/61), whereas neoplastic cells more rarely expressed GRP-receptors (14/61). GRP-receptor positive vessels were present in the various classes of ovarian tumors; generally, malignant tumors had a higher incidence of GRP-receptor positive vessels compared to their benign counterparts. The prevalence of such vessels was particularly high in ovarian carcinomas (16/19) and their metastases (5/5). The GRP-receptors were expressed in high density in the muscular vessel wall. Normal ovary (n=10) lacked GRP-receptors. Conclusions: The large amounts of GRP-receptors in ovarian tumor vessels suggest a role in tumoral vasculature and possibly angiogenesis. Further, these vessels might be targeted in vivo with bombesin analogs for diagnosis or for therapy.