Synthesis and biodistribution study of a chlorotoxin derivative peptide labeled with 131-iodine for tumor therapy

Chlorotoxin is a 36-amino acid peptide found in the venom of the Leiurus quinquestriatus which blocks small-conductance chloride channels. Chlorotoxin binds preferentially to glioma cells that allow development of new methods for the treatment and diagnosis of several types of cancer. Thus chlorotoxin derivative was labeled with [131]I for further investigation. A chlorotoxin derivative was synthesized on a solid phase using a standard Fmoc strategy. Labeling with iodine-131 was performed through chloramine-T method and radiochemical analysis involved sephadex G-25 and HPLC methods. The stability of radiopeptide was checked in the presence of PBS and human serum at 37 °C up to 24 h. The biodistribution was studied in mice. The chemical purity of synthesized peptide as assessed by analytical RP-HPLC was 95%. Labeling of peptide resulted in a radiochemical yield of 80% with radiochemical purity of > 95% with specific activity of 0.740 GBq/ micro mol. Result of in vitro studies demonstrated acceptable stability of compound in human serum and PBS solution. Biodistribution data showed moderate blood clearance, with concentration of radioactivity in the kidneys, liver, intestine and stomach. Results indicates that the labeled Chlorotoxin derivative might be useful in determining tumor extent and also, tumor therapy of gliomas or possibily other cancers

Three different procedures in labeling of ubiquicidin with technetium 99m: a comparative study

UBI 29-41 [a derivative of antimicrobial peptide ubiquicidin] labelled with 99mTc is reported to discriminate between bacterial infections and sterile inflammatory processes. In this study, three lyophilized kit were performed, one of them based on the direct labelling with only SnCl[2] as reducing agent, and other two based on 6-hydrazinopyridine-3-carboxylic acid [HYNIC] and tricine as a coligands with or without ethylenediamine-N,N'-diacetic acid [EDDA]. Synthesis of UBI 29-41 was performed on solid phase using a standard Fmoc strategy. BOC-HYNIC was conjugated with peptide in solution. Three lyophilized kits were prepared as follows: kit 1:40 micro g UBI 29-41, 5 micro g SnCl[2], pH = 9; kit 2: 40 micro g UBI 29-41, 40 micro g SnCl[2], 20 mg tricine, pH = 5.2; kit 3: 40 micro g UBI 29-41, 40 micro g SnCl[2], 15 mg tricine, 5 mg EDDA, pH = 7. With addition of 99mTcO[4]-solution, kits were labeled under specific conditions, and the radiochemical purity was evaluated by ITLC and HPLC methods. Stability and protein binding in human serum followed by in vitro binding to bacteria were assessed. Biodistribution of radiopeptides in staphylococcus aureus infected rats muscles were studied using ex vivo counting and scintigraphy. Radiochemical analysis indicated rapid and high labeling yield [>95%] for the three kits. Binding to bacteria for kit 2 was to some extent higher than that was obtained for the two other kits. Specific accumulation in infected thigh muscles, as indicated by T/NT ratios was 3.29, 4.6 and 3.77 for kit 1, 2 and 3, respectively. The HYNIC-UBI 29-41 labeled in presence of tricine as coligands [kit 2] showed the most promising results for further in vivo evaluation

Synthesis, development and preclinical comparison of two new peptide based freeze-dried kit formulation 99mTc-EDDA-Tricine-HYNIC-TOC and 99mTc-EDDA-Trinice-HYNIC-TATE for somatostatin receptor positive tumor scintigraphy

Somatostatin receptor [sstr] scintigraphy with [111In diethylenetriaminepentaacetic acid]-octreotide [[111In-DTPA]-OC] has became a routine diagnostic procedure in oncology. However, it suffers from some drawbacks concerning the limited availability, suboptimal imaging properties and elevated radiation burden of 111In. In this study synthesis, conjugation and preclinical evaluation of two new freeze-dried kit formulation based on somatostatin analogues, Tyr3-Octreotide [TOC] and Tyr3-octreotate [TATE], designed for the labeling with 99mTc are described. After cleavage from the resin and preparation of the cyclized peptides, these compounds were conjugated with 6-hydrazinopyridine-3-carboxylic acid [HYNIC] in solution. Radiolabeling of HYNIC peptide conjugates was performed at high specific activity using one-step kits formulation based on tricine and ethylenediamine-N,N? -diacetic acid [EDDA] as co-ligands. Both, 6-hydrazinopyridine-3-carboxylic acid0-Tyr3-Octreotide [HYNIC-TOC] and 6-hydrazinopyridine-3-carboxylic acid0-Tyr3-Octreotate [HYNIC-TATE], showed a specific and high rate of internalization after 4 h in AR4-2J rat pancreatic tumor cells, [11.2 +/- 0.8 and 18.1 +/- 1.2 respectively]. Biodistribution studies in AR4-2J tumor-bearing rats showed rapid clearance of both analogues from all sstr-negative tissues except the kidneys. The specific uptake in tumor and sstr-positive tissues especially pituitary, pancreas and adrenals were observed. After 4 h the adrenals to pancreas uptake ratio for HYNIC-TOC was higher than that of HYNIC-TATE. Although both compounds had high kidney and low liver excretion, for HYNIC-TATE, it was lower. The results suggest these two new peptide based freeze-dried kits might be of great promise for clinical application in imaging of somatostatin receptor-positive tumors

[Evaluation and comparison of different quality control methods in radiochemical purity determination of new peptide kits 99mTc-EDDA-Tricine-HYNIC-Tyr3-Octreotide and 99mTc-EDDA-Tricine-HYNIC-Tyr3-octreotate for imaging somatostatin receptor positive tumors]

Using labeled peptides enjoys high importance in nuclear medicine. Somatostatin analogs labeled with different radionuclide are vastly investigated for diagnosis or treatment of somatostatin receptor positive tumors. Labeling somatostatin analogs with 99mTc as alternative of 111In needs doing different quality control method for determination of labeling yield and complex stability for clinic applications. For determining radiochemical purity, three different methods were compared and evaluated including HPLC, TLC and Sep-Pak. All the three methods were suitable for determining of radiochemical purity in peptide kits, although using HPLC was more effective rather than the others. HPLC was the best method for determination of radiochemical purity of the peptide kits. TLC or Sep-Pak method should only be employed if HPLC is not available in nuclear medicine clinics, and after training and gaining sufficient operational experience. In this research, a new monoclonal antibody against colon cancer cells was prepared and antigen concentration in different cells determined by a radioimmunoassay method using iodine [I-125] labeled protein G. 125I-labeled protein G percent binding to white blood cell, HT29, LS180 and MCF7 cell lines were 7.1%, 91.2%, 75.8% and 40.2%, respectively. Regarding importance of monoclonal antibody applications, it is necessary to find an efficient method for their evaluation in cancer therapy. In this method, a radioactive agent with no count restriction was used. Also by this method, amount of the antigen can be easily quantified

Preparation, formulation and quality control of one step Kit - 99mTc -EDDA/HyNic - Tyr - Octreotide as a peptide radiopharmaceutical for imaging somatostatin receptor positive tumors

The high expression of somatostatin receptors in many tumours, have made receptor scintigraphy with 111In-DTPA-Octreotide a widly used procedure in nuclear medicine. Despite its clinical success, some limitation and drawbacks of radiolabelling with 111In remain, especially those concerned with the cost, availability and physical decay properties of this radionuclide. 99mTc-EDDA/HYNIC-Tyr3-TOC was studied as a new agent with the potential to replace Octreoscan in somatostatin receptor scintigraphy. This hydrazinonicotinic acid derivatized somatostatin complex contains ethylenediamine N,N diacetic acid [EDDA] as a co-ligand resulting in a high in vitro and in vivo stability. High labeling yields [>90%] were achieved at high specific activities. Charactrization via HPLC, biodistribution and receptor binding of the resulting complex are described. The formulation developed enables rapid and simple labeling of 99mTc-EDDA/HYNIC-TOC in a manner suitable for clinical setting