Martha Pedraza-López

In vitro and in vivo assessment of 99mTc-UBI specificity for bacteria.

Technetium-99m labeled ubiquicidin peptide 29-41 ((99m)Tc-UBI) is a cationic human antimicrobial peptide fragment that has been shown to bind bacteria in vitro and accumulates at sites of infection in experimental animals. To help determine if (99m)Tc-UBI is bound to the bacterial cell envelope by a simple nonspecific electrostatic interaction, a comparative study of the in vitro binding of (99m)Tc-UBI and two different (99m)Tc labeled cationic peptides ((99m)Tc-Tat-1-Scr and (99m)Tc-Tat-2-Scr) to bacteria and to two tumor cell line (LS174T and ACHN) was performed. The in vivo specificity of (99m)Tc-UBI for infection in mice was also evaluated using dual labels in the same animal and comparing the target/non-target ratio for (67)Ga-citrate and (99m)Tc-UBI at sites of induced infection and sterile inflammation. Under conditions of this study, the in vitro binding of (99m)Tc-UBI, (99m)Tc-Tat-1-Scr and (99m)Tc-Tat-2-Scr to S. aureus was 35, 78 and 87% respectively. While the binding of (99m)Tc-Tat-1-Scr and (99m)Tc-Tat-2-Scr was 37 and 33% to colon tumor cells (LS174T) and 39 and 41% to renal tumor cells (ACHN) respectively, the binding of (99m)Tc-UBI to both cell types was much lower at less than 4%. In vivo studies revealed that there is a significant difference (p < 0.05) in the radioactive accumulation of (99m)Tc-UBI between the sites of infection and inflammation compared to (67)Ga-citrate. Thus, (99m)Tc-UBI showed an average infection/inflammation ratio of 2.08 +/- 0.49 compared to 1.14 +/- 0.45 for (67)Ga-citrate. In conclusion, the in vitro and in vivo results provide evidence that a specific mechanism is responsible of the (99m)Tc-UBI bacterial intracellular accumulation.

Design, preparation, in vitro and in vivo evaluation of 99mTc-N2S2-Tat(49-57)-bombesin: A target-specific hybrid radiopharmaceutical

The gastrin-releasing peptide receptor (GRP-r) is over-expressed in various human tumors. Recently, (99m)Tc-EDDA/HYNIC-Lys(3)-bombesin ((99m)Tc-BN) was reported as a radiopharmaceutical with specific cell GRP-r binding and images in breast cancer patients demonstrated distinct radioactivity accumulation in malignant tissue. The HIV Tat-derived peptide has been used to deliver a large variety of cargoes into cells. Therefore, a new hybrid radiopharmaceutical of type (99m)Tc-N(2)S(2)-Tat(49-57)-Lys(3)-bombesin ((99m)Tc-Tat-BN) would increase cell uptake. The aim of this research was to prepare and assess in vitro and in vivo uptake kinetics in cancer cells of (99m)Tc-Tat-BN and to compare its cellular internalization with that of (99m)Tc-BN. Structures of N(2)S(2)-Tat-BN and Tc(O)N(2)S(2)-Tat-BN were calculated by an MM procedure. (99m)Tc-Tat-BN was synthesized and stability studies carried out by HPLC and ITLC-SG analyses in serum and cysteine solutions. In vitro internalization was tested using human prostate cancer PC-3 cells and breast carcinoma cell lines MDA-MB231 and MCF7. Biodistribution was determined in PC-3 tumor-bearing nude mice. Results showed a minimum energy of 271 kcal/mol for N(2)S(2)-Tat-BN and 300 kcal/mol for Tc(O)N(2)S(2)-Tat-BN. (99m)Tc-Tat-BN radiochemical purity was >90%. In vitro studies demonstrated stability in serum and cysteine solutions, specific cell receptor binding and internalization in three cell lines was significantly higher than that of (99m)Tc-BN (p<0.05). The tumor-to-muscle radioactivity ratio was 8.5 for (99m)Tc-Tat-BN and 7 for (99m)Tc-BN. Therefore, this hybrid is potentially useful in breast and prostate cancer imaging.

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.

Lys and Arg in UBI: a specific site for a stable Tc-99m complex?

The aim of this study was to help establish if ubiquicidin peptide 29-41 fragment (UBI) contains a specific site for 99mTc labeling by a new direct method under alkaline conditions. Since this peptide does not have cysteine residues, it is possible that neighboring arginine and lysine in the peptide amino acid sequence (Thr-Gly-Arg-Ala-Lys-Arg-Arg-Met-Gln-Tyr-Asn-Arg-Arg) could be a specific coordination site to form a stable 99mTc-UBI complex. Following direct labeling, the in vitro stability of 99mTc-UBI was compared to UBI radiolabeled by one indirect method using HYNIC/tricine and HYNIC/tricine/EDDA. Radiochemical purity of 99mTc-UBI averaged 97% compared to 88% for 99mTc-HYNIC-UBI/tricine and 98% for 99mTc-HYNIC-UBI/tricine/EDDA. Both 99mTc-HYNIC-UBI (tricine or EDDA) and 99mTc-UBI showed stability in human serum and solutions of cysteine. 99mTc-UBI radiochemical purity 24 h after dilution in 0.9% NaCl was greater than 90% at pH 9 and greater than 95% at pH 6.5. Under one set of experimental conditions, in vitro binding to bacteria of 99mTc-UBI was 35% and identical to that of 99mTc-HYNIC-UBI/tricine and 99mTc-HYNIC-UBI/tricine/EDDA at 32% and 31% respectively. The biodistribution of 99mTc-UBI in mice showed a rapid renal clearance. To help identify the site(s) of 99mTc binding following direct labeling, molecular mechanics and quantum-mechanical calculations were performed which showed that the amine groups of Arg(7) and Lys are the most probable site. The calculations show that these groups can form a square pyramid with two water molecules for the Tc cation (dxysp(3)). It will be necessary to isolate and characterize the 99Tc(V)(O)-UBI.(H2O)n complex to confirm these results.

99mTc-N2S2-Tat (49-57)-bombesin internalized in nuclei of prostate and breast cancer cells: kinetics, dosimetry and effect on cellular proliferation.

BackgroundThe gastrin-releasing peptide receptor (GRP-r) is overexpressed in prostate and breast cancers. technetium-99m-bombesin (99mTc-BN) has been reported as a radiopharmaceutical with specific cell GRP-r binding. The HIV Tat (49-57)-derived peptide has been used to deliver a large variety of molecules to cell nuclei. A new hybrid radiopharmaceutical of type 99mTc-N2S2-Tat(49-57)-Lys3-BN (99mTc-Tat-BN) internalized in cancer cell nuclei could act as an effective system of targeted radiotherapy using Auger and internal conversion electron emissions near DNA. AimThe aim of this study was to assess the in-vitro nucleus internalization kinetics of 99mTc-Tat-BN in GRP r-positive cancer cells and to evaluate the subcellular-level radiation-absorbed dose associated with the observed effect on cancer cell DNA proliferation. Methods 99mTc-Tat-BN in-vitro internalization kinetics were evaluated in human prostate cancer PC-3 cells and breast carcinoma cell lines MCF7 and MDA-MB231. Nuclei from cells were isolated using a nuclear extraction kit. Total disintegration in each subcellular compartment was calculated by the integration of experimental time–activity kinetic curves. Nucleus internalization was corroborated by confocal microscopy images using immunofluorescently labelled Tat–BN. The PENELOPE code was used to simulate and calculate the absorbed dose by the contribution of Auger and internal conversion electrons in the cytoplasm and nucleus using geometric models built from immunofluorescent cell images. A cell proliferation kit was used to evaluate DNA concentration after cancer cell incubation with 99mTc-Tat-BN. ResultsThe results showed that 59.7, 61.2 and 41.5% of total disintegration per unit of 99mTc-Tat-BN activity (1 Bq) bound to the cell occurred in the nucleus of PC-3, MCF7 and MDA-MB231, respectively. The 99mTc-Tat-BN absorbed doses delivered to nuclei were 0.142 mGy/decay (PC-3), 0.434 mGy/decay (MCF7) and 0.276 mGy/decay (MDA-MB231). 99mTc-Tat-BN produced a significant decrease in PC-3 (52.98%), MCF7 (45.71%) and MDA-MB231 (35.80%) cellular proliferation with respect to untreated cells. ConclusionThe hybrid radiopharmaceutical could be potentially useful as a therapeutic agent for prostate and breast cancers.

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.

Specificity of 99mTc-UBI for detecting infection foci in patients with fever in study.

Fever of unknown origin (FUO) represents a challenge to clinical medicine, and bacterial cultures have been considered the ‘gold standard’ in discriminating between fevers resulting from bacterial infection and sterile inflammations. In nuclear medicine, a synthetic radiolabeled antimicrobial peptide (99mTc-UBI) is used to image the molecular localization of infectious microorganisms. ObjectiveThe aim of this research was to determine the absolute and relative frequencies of 99mTc-UBI, by molecular imaging, to detect infection foci in patients with fever in study or FUO. MethodsImages (207) from 196 patients with FUO acquired with 99mTc-UBI and a Siemens gammacamera were read by two nuclear medicine physicians and classified as positive or negative for infection foci. The diagnostic value was corroborated with our gold standard, which comprises bacterial cultures of biopsies, blood and urine, plus laboratory studies, morphological images (radiographs, nuclear magnetic resonance, computed tomography) and the clinical history of each patient. The absolute and relative frequencies of 99mTc-UBI were calculated from the molecular images versus the gold standard. ResultsThe specificity of 99mTc-UBI for localizing infection foci and for discarding sterile inflammation was 95.35%, the sensitivity was 97.52%, the positive predictive value was 96.72%, the negative predictive value was 96.47%, the accuracy was 96.62%, and the observed agreement between the bacterial culture and the molecular image was 96.62% (P=0.0001). ConclusionConsidering that the absolute and relative frequencies are very high, we propose that, in the future, 99mTc-UBI molecular imaging could be the gold standard to detect infection sites and to discard sterile inflammation.

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.

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

INTRODUCTION Integrin αvβ3 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 αvβ3 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 αvβ3-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 αvβ3 integrins.

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.