Studies on batch formulation of a freeze dried kit for the preparation of 99mTc-HYNIC-TATE for imaging neuroendocrine tumors.

AIM: To formulate freeze dried cold kits for preparation of 99mTc-HYNIC-TATE suitable for use at hospital radiopharmacy and establish clinical utility of 99mTc-HYNIC-TATE prepared using kits for detection of neuroendocrine tumors (NETs). METHODS: Standardization of reagent concentrations for formulation of freeze dried kits of HYNIC-TATE was carried out. Consistency in formulation was tested by six batch preparation. Quality control tests were carried out to establish compliance of specifications of purity and safety criteria for both kits and 99mTc-HYNIC-TATE formulated using kits. Clinical utility of 99mTc-HYNIC-TATE prepared using kits was demonstrated in patients with histopathologically confirmed well-differentiated NETs. RESULTS: Pharmaceutical grade HYNIC-TATE kits compliant with all the quality control criteria were formulated and successfully radiolabeled with 99mTc. Radiopharmaceutical was successfully utilized for detection of NETs in patients and comparison with uptake of 99mTc-HYNIC-TOC and 177Lu-DOTA-TATE was made. CONCLUSION: The formulated kits are robust and provide consistently high radiolabeling yields (> 95%) with 99mTc in short time periods requiring no additional purification. Initial clinical trials demonstrate the utility of 99mTc-HYNIC-TATE using formulated kits.

Formulation and purification of therapeutic dose of 90Y-labeled peptides: Some interesting radiochemistry aspects.

Yttrium-90 obtained from most of the 90Sr/90Y generators contains 90Sr impurity above permissible limit for human administration. A protocol has been optimized for formulation of therapeutic dose of 90Y-DOTA-Tyr3-octreotate (90Y-DOTA-TATE) and removal of 90Sr impurity from it. The radiochemical purity of 90Y-DOTA-TATE was found to be >98% and it met the requirements for clinical use. The radiopharmaceutical was used in preliminary clinical investigation in patients with neuroendocrine tumors. This promising strategy would aid toward widespread clinical utilization of 90Sr/90Y generators.

PK study of octreotide based on LC-MS/MS combining protein precipitation and impurity extraction technique.

AIM: To establish a robust methodology for quantitative analysis of therapeutic peptide in biological samples. MATERIALS & METHODS: Octreotide was chosen as a model therapeutic peptide, and oxidized-octreotide was synthesized as internal standard. Protein precipitation combining liquid-liquid extraction technique was adopted to enhance the recovery and reduce the endogenous interferences effectively. A LC-MS/MS method for the quantification of octreotide in plasma has been optimized and validated according to FDA guidelines. RESULTS: Linearity, selectivity, accuracy, precision, stability, matrix effect and recovery were within bioanalytical method validation acceptance criteria as FDA guidelines. The methodology was then successfully applied into the studies for octreotide. CONCLUSION: This robust methodology would be useful for the PK studies for octreotide and other therapeutic peptides.

Radiolabeling of DOTATOC with the long-lived positron emitter 44Sc.

The positron-emitting radionuclide (44)Sc with a half-life of 3.97 h and a ß(+) branching of 94.3% is of potential interest for clinical PET. As so far it is available from a (44)Ti/(44)Sc generator in Mainz, where long-lived (44)Ti decays to no-carrier-added (nca) (44)Sc. The (44)Sc is a trivalent metal cation and should be suitable for complexation with many well established bifunctional chelators conjugated to peptides or other molecular targeting vectors. Thus, the aim of this work was to investigate the potential of (44)Sc for labeling of DOTA-conjugated peptides. DOTA-D-Phe(1)-Tyr(3)-octreotide (DOTATOC) was used as a model molecule to study and optimize labeling procedure. Reaction parameters such as buffer conditions, concentration of peptide, pH range, reaction temperature and time were optimized. Addition of 21 nmol of DOTATOC to (44)Sc in ammonium acetate buffer pH 4.0 provided labeling yields >98% within 25 min of heating in an oil-bath at 95°C. This time can be reduced to 3 min only by applying microwave supported heating. (44)Sc-DOTATOC was found to be stable in 0.9% NaCl, PBS pH 7.4, fetal calf and human serums, and also in the presence of competing metal cations (Fe(3+), Ca(2+), Cu(2+), Mg(2+)), as well as other ligand competitors, like EDTA and DTPA, even after almost 25 h incubation at 37°C. Present study shows that nca (44)Sc forms stable complexes with the macrocyclic ligand DOTA and that (44)Sc-DOTATOC and analog targeting vectors may be synthesized for further preclinical and clinical investigations.

An electro-amalgamation approach to isolate no-carrier-added 177Lu from neutron irradiated Yb for biomedical applications.

INTRODUCTION: A novel two-step separation process for the production of no-carrier-added (NCA) (177)Lu from neutron irradiated Yb target through an electrochemical pathway employing mercury-pool cathode has been developed. METHODS: A two-cycle electrolysis procedure was adopted for separation of (177)Lu from (177)Lu/Yb mixture in lithium citrate medium. The influence of different experimental parameters on the separation process was investigated and optimized for the quantitative deposition of Yb in presence of (177)Lu. The first electrolysis was performed for 50 min in the (177)Lu/Yb feed solution at pH 6 applying a potential of 8 V using platinum electrode as anode and mercury as the cathode. The second electrolysis was performed under the same conditions using fresh electrodes. The radionuclidic and chemical purity of (177)Lu was determined by using gamma ray spectrometry and atomic absorption spectrometry. The suitability of (177)Lu for biomedical applications was ascertained by labeling 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid D-Phe(1)-Tyr(3)-octreotate(DOTA-TATE) with (177)Lu. RESULTS: This process could provide NCA (177)Lu with >99.99% radionuclidic purity and an overall separation yield of ∼99% was achieved within 3-4 h. The Hg content in the product was determined to be <1 ppm. Radiolabeling yield of >98% was obtained with DOTA-TATE under the optimized reaction conditions. CONCLUSIONS: An efficient strategy for the separation of NCA (177)Lu, suitable for biomedical applications, has been developed.

Separation of positional isomers of mono-poly(ethylene glycol)-modified octreotides by reversed-phase high-performance liquid chromatography.

The purpose of this study was to develop a reversed-phase high-performance liquid chromatographic method (RP-HPLC) for separating each positional isomer from low- to high-molecular-weight mono-PEGylated octreotides prepared by polyethylene glycol (PEG) derivatives with various molecular weights (2, 5, or 20kDa). In the gradient elution using acetonitrile and 10mM phosphate buffer at pH 7.0 on a Phenomenex Gemini C-18 column (250mmx4.6mm id, 5microm), each positional isomer of the mono-PEGylated octreotides was completely resolved with good resolution (PEG-2K: 7.6, PEG-5K: 6.6, and PEG-20K: 3.1). The optimal RP-HPLC condition also resolved the degradation products of mono-PEG-octreotide isomers in thermal stability studies at 55 degrees C and enzymatic stability studies with trypsin. In conclusion, the developed RP-HPLC method will be valuable for studying the effect of PEGylation site and the attached PEG size on the physicochemical and pharmacological properties of PEGylated octreotides.

PEGylation of octreotide: I. Separation of positional isomers and stability against acylation by poly(D,L-lactide-co-glycolide).

PURPOSE: To investigate the mechanism by which polyethylene glycol (PEG) conjugation (PEGylation) prevents the acylation of octreotide by poly(d,l-lactide-co-glycolide) (PLGA). METHODS: Octreotide was chemically modified by reaction with succinimidyl propionate-monomethoxy PEG. Each PEGylated octreotide species with different PEG number and modified position was separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with endoproteinase Lys-C digestion. Acylation of octreotide and PEGylated octreotides was observed with hydrophobic and hydrophilic PLGA. RESULTS: Two mono- and one di-PEGylated octreotides were separated by RP-HPLC. MALDI-TOF MS of the PEGylated products after Lys-C digestion at different pH revealed that the two mono-PEGylated octreotides were modified at the N-terminus and Lys(5) residue, respectively. The interaction of octreotide with PLGA involved an initial adsorption followed by acylation and the subsequent release of octreotide and acylated octreotide. The initial adsorption of octreotide was dependent on the acidity of PLGA. PEGylation of octreotide significantly inhibited the initial adsorption and acylation by PLGA. In particular, the acylation could be completely prevented by mono-PEGylation at the N-terminus of octreotide. CONCLUSIONS: This study shows that the N-terminus of octreotide is the preferred PEGylation site to prevent acylation in degrading PLGA microspheres. The mono-N-terminally PEGylated octreotide may possibly serve as a new source for somatostatin microsphere formulation.