Preparation and preclinical evaluation of (66)Ga-DOTA-E(c(RGDfK))2 as a potential theranostic radiopharmaceutical.

INTRODUCTION: Integrin αvß3 plays an important role in angiogenesis and is over-expressed in tumoral endothelial cells and some other tumor cells. RGD (Arg-Gly-Asn) peptides labeled with (68)Ga (t1/2=68min) have showed good characteristics for imaging of αvß3 expression using positron emission tomography (PET). Gallium-66 has been proposed as a PET imaging alternative to (68)Ga and given the unique high energy of its emitted positrons (Emax 4.15MeV) it may also be useful for therapy. The aim of this research is to prepare [(66)Ga]DOTA-E-[c(RGDfK)]2 and evaluate in mice its potential as a new theranostic radiopharmaceutical. METHODS: High specific activity (66)Ga was produced via the (66)Zn(p,n) reaction, and the labelling method of DOTA-E-[c(RGDfK)]2 with (66)Ga was optimized. Radiochemical purity was determined by TLC, and in vitro stability and protein binding were determined. Serial microPET imaging and biodistribution studies were carried out in nude mice bearing C6 xenografts. Radiation absorbed dose estimates were based on the biodistribution studies, where tumor and organs of interest were collected at 0.5, 1, 3, 5 and 24h post-injection of [(66)Ga]DOTA-E-[c(RGDfK)]2. RESULTS: Our results have shown that [(66)Ga]DOTA-E-[c(RGDfK)]2 can be prepared with high radiochemical purity (>97%), specific activity (36-67GBq/µmol), in vitro stability, and moderate protein binding. MicroPET imaging up to 24 post-injection showed contrasting tumors reflecting αvß3-targeted tracer accumulation. Biodistribution studies and dosimetry estimations showed a stable tumor uptake, rapid blood clearance, and favorable tumor-to-tissue ratios. CONCLUSIONS: The peptide conjugated DOTA-E-[c(RGDfK)]2 labeled with (66)Ga may be attractive as a theranostic agent for tumors over-expressing αvß3 integrins.

Gold nanoparticles conjugated to [Tyr3]octreotide peptide.

A multifunctional system of gold nanoparticles (AuNP) capped by the [Tyr(3)]Octreotide (TOC) peptide was prepared and characterized by transmission electron microscopy (TEM) and UV-Vis, infrared and fluorescence spectroscopy. AuNP and AuNP-TOC fluorescence emission spectra were obtained both in solution and in murine AR42J-tumor tissues. Results suggest that AuNP were functionalized with TOC through interactions with the N-terminal amine of the phenylalanine, the amide groups and possibly with the indole group of the tryptophan residue. The fluorescence analyses in tissue revealed a recognition of the AuNP-TOC conjugate for the neuroendocrine tumor because of the lower energy position of the fluorescence resonance (692 nm) with respect to that of the AuNP in the same tumoral tissue (684 nm). The emission band observed in the near-infrared region (692 nm) opens the possibility for AuNP-TOC use in bioimaging.

[166Dy]Dy/166Ho hydroxide macroaggregates: an in vivo generator system for radiation synovectomy.

Radiation synovectomy is an effective treatment in patients suffering from inflammatory-rheumatoid and degenerative joint diseases. The aim of this work was to examine the feasibility of preparing dysprosium-166 (166Dy)/holmium-166(166Ho) hydroxide macroaggregates ([166Dy]Dy/166Ho-HM) as an in vivo generator for radiation synovectomy evaluating whether the stability of 166Dy-HM and 166Ho-HM complexes is maintained when the daughter 166Ho is formed. The Monte Carlo (MCNP4B) theoretical depth dose profile for the in vivo [166Dy]Dy/166Ho generator system in a joint model was calculated and compared with that produced by 90Y, 153Sm and 166Ho. 166Dy was obtained by neutron irradiation of enriched 164Dy2O3 in a Triga Mark III reactor. Macroaggregates were prepared by reaction of [166Dy]DyCl3 with 0.5 M NaOH in an ultrasonic bath. [166Dy]Dy/166Ho-HM was obtained with radiochemical purity >99.5% and with the majority of particles in the 2-5 microm range. In vitro studies demonstrated that the radio-macroaggregates are stable in saline solution and human serum without a significant change in the particle size over 14 d, suggesting that no translocation of the daughter nucleus occurs subsequent to beta- decay of 166Dy. Biological studies in normal rats demonstrated high retention in the knee joint even 7 d after [166Dy]Dy/166Ho-HM administration. The Monte Carlo (MCNP4B) theoretical depth dose profiles in a joint model, showed that the in vivo [166Dy]Dy/166Ho generator system would produce 25% and 50% less radiation dose to the articular cartilage and bone surface, respectively, than that produced by 90Y or pure 166Ho in a treatment with the same therapeutic dose to the synovium surface. Despite that 153Sm showed the best depth dose profile sparing doses to healthy tissues, the use of 166Dy could provide the advantage of being applied in patients that cannot be reached within a few hours from a nuclear reactor and to produce less radiation exposure to the medical personnel during the radiopharmaceutical administration.

Labeling of biotin with [166Dy]Dy/166Ho as a stable in vivo generator system.

The aim of this work was to synthesize [166Dy]Dy/166Ho-DTPA-Biotin to evaluate its potential as a new radiopharmaceutical for targeted radiotherapy. Dysprosium-166 (166Dy) was obtained by neutron irradiation of enriched 164Dy(2)O(3) in a Triga Mark III reactor. The labeling was carried out in aqueous media at pH 8.0 by addition of [166Dy]DyCl(3) to diethylenetriaminepentaacetic-alpha,omega-bis(biocytinamide) (DTPA-Biotin). Radiochemical purity was determined by high-performance liquid chromatography (HPLC) and TLC. The biological integrity of labeled biotin was studied evaluating its avidity for avidin in an agarose column and by size-exclusion HPLC analysis of the radiolabeled DTPA-Biotin with and without the addition of avidin. Stability studies against dilution were carried out by diluting the radiocomplex solution with saline solution and with human serum at 37 degrees C for 24 h. The [166Dy]Dy/166Ho-labeled biotin was obtained with a 99.1+/-0.6% radiochemical purity. In vitro studies demonstrated that [166Dy]Dy/166Ho-DTPA-Biotin is stable after dilution in saline and in human serum and no translocation of the daughter nucleus occurs subsequent to beta(-) decay of 166Dy that could produce release of 166Ho(3+). Avidity of labeled biotin for avidin was not affected by the labeling procedure. Biodistribution studies in normal mice showed that the [166Dy]Dy/166Ho-DTPA-Biotin has a high renal clearance. In conclusion, the radiolabeled biotin prepared in this investigation has adequate properties to work as a stable in vivo generator system for targeted radiotherapy.

99mTc-glucarate for detection of isoproterenol-induced myocardial infarction in rats.

Infarct-avid radiopharmaceuticals are necessary for rapid and timely diagnosis of acute myocardial infarction. The animal model used to produce infarction implies artery ligation but chemical induction can be easily obtained with isoproterenol. A new infarct-avid radiopharmaceutical based on glucaric acid was prepared in the hospital radiopharmacy of the INCMNSZ. 99mTc-glucarate was easy to prepare, stable for 96 h and was used to study its biodistribution in rats with isoproterenol-induced acute myocardial infarction. Histological studies demonstrated that the rats developed an infarct 18 h after isoproterenol administration. The rat biodistribution studies showed a rapid blood clearance via the kidneys. Thirty minutes after 99mTc-glucarate administration the standardised heart uptake value S(h)UV was 4.7 in infarcted rat heart which is six times more than in normal rats. ROIs drawn over the gamma camera images showed a ratio of 4.4. The high image quality suggests that high contrast images can be obtained in humans and the 96 h stability makes it an ideal agent to detect, in patients, early cardiac infarction.

[Radiopharmacokinetic and gammagraphic studies for calculating personalized dosimetry]. / Estudios radiofarmacocinéticos y gammagráficos para cálculos de dosimetría personalizada.

In nuclear medicine radiation absorbed doses are important in the patient's risk/benefit evaluation and are estimated by means of biological and complex mathematical models. The biological model includes radiopharmacokinetic data obtained through blood and urine samples taken at given intervals. A useful mathematical model is the MIRD model and with the value for the time of residence tau the MIRDOSE3 computer program uses several anatomic models and calculates radiation absorbed dose for 25 organs. At the Radiopharmacy Unit of the Nuclear Medicine Department at INCMNSZ two new bone seeking radiopharmaceuticals, 99mTc-ABP and 188Re-ABP, have been designed, characterized and animal-tested. Radiopharmaceutical parameters and sequential scanning were obtained for diagnostic 99mTc-ABP in 10 normal subjects and the aim was to use % 24 hour urine elimination and % bone uptake to calculate radiation absorbed dose and extrapolate the values to 188Re-ABP as the basis for a therapeutic treatment. 99mTc-ABP was eliminated in women's urine 63.2 +/- 7.3%/activity and 70 +/- 11%/activity in men. In women 36.8 +/- 7.3% of the radiopharmaceutical remains on the bone surface and in men 30 +/- 11%. ROIs were drawn on the images and the time-integrated renal cpm/pixel/ROI gave a residence time tau = 0.52 h. Cumulative bone activity A calculated with A = 1.443 (T1/2) A0 was 2358 +/- 469 MBq h for women and 1923 +/- 707 MBq h for men. Residence time tau was 3.19 +/- 0.63 h in women and 2.6 +/- 0.95 h in men. Radiation absorbed dose for the whole body was 0.0020 +/- 0.0004 mGy/MBq for women and 0.0013 +/- 0.0005 mGy/MBq for men. For women's bone marrow it was 0.0063 +/- 0.0013 mGy/MBq and for men 0.0041 +/- 0.0015 mGy/MBq. 188Re-ABP behaves as 99mTc-ABP therefore, the effective dose given by 188Re, a beta emitter, would be for women 0.0936 mSv/MBq and for men 0.0608 mSv/MBq. These characteristics and the radionuclidic characteristics of 188Re indicate that 188Re-ABP might be a good bone metastases pain palliation radiopharmaceutical.

Uptake of (188)Re-beta-naphthyl-peptide in cervical carcinoma tumours in athymic mice.

Radiolabelled somatostatin analogues have been used in diagnostic and therapeutic nuclear medicine to treat cancerous tumours. Lanreotide, a cyclic octapeptide, beta-naphthyl-peptide, with antiproliferative action on human small cell lung carcinoma was (188)Re labelled and characterised, and its biodistribution was studied in mice. Molecular modelling indicates that the lipophilic radiopharmaceutical might be an oxo-rhenium (V) penta-coordinated complex. The implanted human cervical tumour of epidermoid origin was positive for cytokeratins and Vimentin. Uptake of (188)Re-labelled peptide in the implanted tumour in athymic mice was 6.2+/-2.9% and was rapidly cleared via the hepatobiliary system. (188)Re-beta-naphthyl-peptide might be a potential therapeutic agent.

Uptake of the 188Re(V)-DMSA complex by cervical carcinoma cells in nude mice: pharmacokinetics and dosimetry.

The uptake of the rhenium-188 (188Re(V)-DMSA) complex of dimercaptosuccinic acid by cervical carcinoma cells in nude mice was evaluated. The pharmacokinetics and dosimetry calculations in normal rats were also evaluated. The images obtained in mice did not show significant accumulation in metabolic organs and the biodistribution studies showed that 3.52 +/- 0.76% of the injected activity per gram (n = 4) was taken up by the tumor. This percentage produces a cumulated activity of 35.63 +/- 8.40 MBq h and an equivalent dose per injected activity of 260 +/- 8.91 mSv/MBq. Pharmacokinetics and dosimetry of the 1887e(V)-DMSA complex indicate that this radiopharmaceutical could be evaluated in patients with soft tissue tumors, since the risk of radiation damage to the kidney or red bone marrow could not be an obstacle for its application in therapeutic nuclear medicine.

Labelling of Re-ABP with 188Re for bone pain palliation.

Etidronate and medronate have been labelled with technetium-99m (99mTc-HEDP, 99mTc-MDP) for bone scanning and, with rhenium-188 (188Re-HEDP) to palliate the pain resulting from bone metastases. The objective of this study was to label alendronate, ABP, a new bisphosphonate, with SnF2-reduced-188Re. The reagents for the 5 mg ABP kit were SnF2, KReO4 and gentisic acid at acid pH. The chemical, spectroscopic and microscopic characteristics, quality control, rat bone uptake of [188Re]Re-ABP and similarities with 99mTc-ABP are presented. We conclude that this is a promising new radiopharmaceutical for bone metastases pain palliation.

Assessment of radiation-induced DNA damage caused by the incorporation of 99mTc-radiopharmaceuticals in murine lymphocytes using single cell gel electrophoresis.

The DNA damage induced by the 99mTc-radiopharmaceuticals incorporation to the cell was determined by the single-cell gel electrophoresis in murine lymphocytes in vitro. The 99mTc-hexamethyl-propylene amine oxime (99mTc-HMPAO) and 99mTc-2, 5-dihydroxybenzoic acid (99mTc-gentisic acid) induced nearly 100% of cells with breaks and/or alkali labile sites, which is explained by the action of the Auger electrons produced by the decay of the 99mTc. These results agree with the doses of 1.6 and 1.0 Gy estimated by subcellular dosimetry for 99mTc-HMPAO that is incorporated in the cytoplasm, and the 99mTc-gentisic acid, which remains bonded to the cell membrane, respectively. The results imply that Auger electrons are able to cause important DNA damage, when the radionuclide is incorporated in the range of a few microns from the nuclei.

Labeling peptides with rhenium-188.

A direct labeling technique via EHDP for the preparation of 188Re-somatostatin analogue peptide beta-(2-naphthyl)-D-Ala-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-amide complex was developed. The influence of reaction conditions such as pH, temperature, weak ligand concentration and stannous chloride concentration were investigated. Methods of analysis were also established permitting identification of radiochemical impurities which may be present in the radiopharmaceutical solution. Results showed that under the procedure reported herein 188Re-peptide complex can be prepared with a radiochemical purity of 90% and a specific activity up to 1.8 GBq mg-1 without radiolytic degradation of the product.