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The reliable method for determination of identity and radiochemical purity (RCP) is of great importance in radiopharmaceutical development. This is especially relevant when more than one form of radiometal/ligand complex can be formed during radiolabelling, such as complexes of 99mTc or 188Re with meso-2,3-dimercaptosuccinic acid (DMSA), where depending on the pH, metal can occur either at +3 or +5 oxidation state. The aim of our study was to evaluate possibilities for optimization of chromatographic systems leading to specific and reliable analytical method for determination of the identity and RCP of DMSA complexes with 99mTc or 188Re.The commercial DMSA kits (POLATOM) were used for preparation of technetium-99m (III) and (V) complexes with DMSA. 99mTc(V)-DMSA complexes were prepared by addition of NaHCO3 to the kit vial prior to 99mTc-eluate to obtain pH ~8. 188Re(V)-DMSA was prepared either directly or using intermediate 188Re(III)-EDTA complex added to DMSA. RCP was evaluated by TLC using: ITLC-SG developed in methylethylketon, SG60 coated plates developed in: n-BuOH/H2O/CH3COOH and n-PrOH/H2O/CH3COOH systems, and in H2O. Comparative biodistribution studies were performed in normal Wistar rats.Using silica gel plates and n-PrOH, H2O and acetic acid in the developing solution, we observed that 99mTc/188Re(III)-DMSA and 99mTc/188Re(V)-DMSA complexes could be well separated from each other and from the impurities in the form of free pertechnetate/perrhenate. In vivo studies showed quite different biodistribution of 99mTc(III)- and 99mTc(V)-DMSA. The trivalent complex accumulated mainly in kidneys (40%ID), while 99mTc(V)-DMSA revealed high excretion with urine and relatively high concentration in osseous tissue (ca. 2 %ID/g). Accumulation of this complex in kidneys was very low (ca. 2.5 %ID). Biodistribution pattern of 188Re(V)-DMSA prepared directly was almost identical to that of 99mTc(V)-DMSA. Biodistribution results of the 188Re preparation obtained using 188Re(III)-EDTA intermediate indicated that the preparation contained the mixture of penta- and trivalent 188Re complexes. The quite high accumulation of radioactivity in kidneys (23 %ID) gave evidence of the presence of 188Re(III)-DMSA in this preparation, what was also confirmed by the results of TLC analysis performed using silica gel plate and n-propanol/water/acetic acid as developing system.Based on our study, we have made recommendation on the suitable methods for investigations of RCP of DMSA complexes, i.e.: SG60 plates developed in the mixture of n-propanol/water/acetic acid, which enable determination of the tri- and pentavalent DMSA complexes, as well as, the pertechnetate/perrhenate impurity, and developed in water for determination of the colloidal residue.

Myocardial perfusion studies are among the most often performed investigations in Nuclear Medicine. However, the development of radiopharmaceuticals for cardiology is an emerging discipline and several other radiotracers have been proven to be useful. Although the myocardial perfusion studies have a well established role in the management of cardiac disorders, still a number of radiopharmaceuticals are under development for a variety of specific cardiac indications and their eventual clinical role remains to be seen. The paper provides a short overview of currently used radiopharmaceuticals and potential molecular imaging radiotracers applicable in cardiology.

Introduction: Interleukin-2 (IL-2) when radiolabelled with Tc-99m has been proved useful in imaging the side of lymphocytic infiltration in patients with autoimmune disorders and plays a significant role as a T-cell imaging agent. However, the labelling procedures used so far appeared to be rather complex and laborious. The aim of present study was to develop an efficient procedure of Tc-99m-labelling of recombinant human interleukin-2 (rhIL-2) via hydrazinonicotinamide (HYNIC) to develop a dry kit formulation.Methods: Various molar ratios of rhIL-2/HYNIC (from 1:2 to 1:12) were used at the conjugation step. The conjugates were purified on a PD-10 column to remove the excess of unbound HYNIC, as well as of any aggregates. The final peptide concentration was quantified by the BCA method, and the number of HYNIC molecules incorporated into a rhIL-2 molecule was determined based on the reaction with 2-sulfobenzaldehyde. The Tc-99m-labelling was optimized using various amounts of HYNIC rhIL-2, Tc-99m, SnCl2, tricine and nicotinic acid (NA). Quality control included GF-HPLC, ITLC, SDS-PAGE and biological assay. Biodistribution studies were performed in Swiss mice and Wistar rats.Results: Generally, the highest radiolabelling yields were achieved when the HYNIC rhIL-2 conjugates of ca. 2-4 HYNIC molecule substitution ratios were used. The optimal pH of the reaction medium was found to be in the range of 6.5 to 7.0. GF-HPLC analysis indicated that monomer and aggregates of Tc-99m-HYNIC rhIL-2 are formed during radiolabelling. At optimized conditions of wet radiolabel ling, the Tc-99m-HYNIC rhIL-2 monomer was obtained with radiochemical purity >99%, specific activity of ca. 4 GBq/mg rhIL-2 and overall yield lea. 65%. The two-vial freeze-dried kit was prepared: the first vial contained 30 mu g HYNIC rhIL-2, coligands, buffer and antioxidant; the second vial contained tricine and SnCl2. The monomer of Tc-99m-HYNIC rhIL-2 was obtained by gel chromatography on a PD-ID column. No differences between labelled and unlabelled IL2 in terms of biological activity were observed.Conclusions: Our study shows that rhIL-2 can be efficiently radiolabelled with Tc-99m via HYNIC, with tricine and NA as co-ligands using a two-vial freeze-dried kit. This enables the preparation of sterile and ready-to-use Tc-99m-HYNIC(tricine,NA)-rhIL-2 within 1 h. (C) 2010 Elsevier Inc. All rights reserved.

The alpha-fetoprotein derived growth inhibitory peptide (GIP) is a 34-amino acid peptide composed of three biologically active subfragments. GIP-34 and its three constituent segments have been synthesized, purified, and studied for biological activity. The GIP-34 and GIP-8 have been characterized as anticancer therapeutic peptides. An multicenter study was initiated to elucidate the means by which these peptide drugs could be targeted to tumor cells. The study first established which cancer types were specifically targeted by the GIP peptides in both in vitro and in vivo investigations. It was next demonstrated that radiolabeled peptide ((125)I GIP-34) is specifically localized to rodent breast tumors at 24 h post-injection. The radionuclide studies also provided evidence for a proposed cell surface receptor; this was confirmed in a further study using fluorescent-labeled GIP-nanobeads which localized at the plasma membrane of MCF-7 breast cancer cells. Finally, it was readily demonstrated that GIP conjugated to either fluorescein or doxorubicin (DOX) underwent tumor cell uptake; subsequently, DOX-GIP conjugates induced cytotoxic cell destruction indicating the utility of GIP segments as cancer therapeutic agents. Following a discussion of the preceding results, a candidate cell surface receptor family was proposed which correlated with previous published reports for a putative AFP/GIP receptor.

Introduction The aim of the study was to compare in vitro and in vivo a novel DOTA-chelated bombesin (BN) analog of the amino acid sequence, QRLGNQWAVGHLM-CONH 2 (BN[2–14]NH 2 ), labeled with 90 Y and 177 Lu, for its potential use in targeted radiotherapy of tumors expressing gastrin releasing peptide (GRP) receptors. The same amino acid sequence, but with different chelator, referred as BN1.1 ( Gly - Gly - Cys - Aca - QRLGNQWAVGHLM - CONH 2 ), has already been studied and reported; however, the DOTA-chelated one, suitable for labeling with M +3 type radiometals, was not yet described. Methods The conditions for labeling of DOTA-BN[2–14]NH 2 with noncarrier added 90 Y and with 177 Lu [specific activity (SA), 15 Ci/mg Lu] were investigated and optimized to provide 90 Y-DOTA-BN[2–14]NH 2 and 177 Lu-DOTA-BN[2–14]NH 2 of high SA. The stability of the radiolabeled compounds in human serum was evaluated over a period of 24 h. The human prostate cancer cell line PC-3, known to express GRP receptors, was used for in vitro evaluation of radiolabeled peptide affinity to GRP receptors and for assessment of cytotoxicity of both nonlabeled and radiolabeled peptide. Biodistribution accompanied by receptor blocking was studied in normal Swiss mice. Results 90 Y-DOTA-BN[2–14]NH 2 and 177 Lu-DOTA-BN[2–14]NH 2 were obtained with radiochemical yield >98% and high SA (67.3 GBq 90 Y/μmol and 33.6 GBq 177 Lu/μmol, respectively). They were stable when incubated in human serum for up to 24 h. The binding affinities of DOTA-BN[2–14]NH 2 and both nat Y- and nat Lu-labeled analogs to GRP receptors were high (IC 50 =1.78, 1.99, and 1.34 nM, respectively), especially for the nat Lu-DOTA-BN[2–14]NH 2 complex. The cytotoxicity study of DOTA-BN[2–14]NH 2 to PC-3 cells revealed an IC 50 =6300 nM after 72 h of exposition, while the labeled derivatives showed no significant cytotoxic effect. The internalization rate to PC-3 cells was more rapid for 177 Lu-labeled peptide (84.87%) than for the 90 Y-labeled one (80.79%), while the efflux rate was slower for 177 Lu-DOTA-BN[2–14]NH 2 (46.8% vs. 61.74%). The biodistribution study of both derivatives in normal mice revealed a specific binding to GRP receptor-positive tissues, which could be blocked by coinjection of cold peptide. The effect of receptor blockage in vivo was also more pronounced for the 177 Lu-labeled peptide than that for the 90 Y-labeled (81% vs. 42%, respectively). Conclusions Our studies demonstrated that DOTA-BN[2–14]NH 2 can be labeled with 90 Y (NCA) and 177 Lu (CA) with high radiochemical yields. The in vitro and in vivo comparison between 90 Y-DOTA-BN[2–14]NH 2 and 177 Lu-DOTA-BN[2–14]NH 2 indicated that the change of radiometal in the complex from Y to Lu influence the binding affinity to the GRP receptors with preference to the 177 Lu-labeled derivative.

Purpose Antitumor activity of the dichloroplatinum(II)–histamine complexes labeled with I-125 or I-131 was investigated in a transplantable murine adenocarcinoma (MA) model. Methods The tumor model was obtained in C3H/W female mice after subcutaneous inoculation of the tumor cells derived from the mice bearing a mammary tumor of spontaneous origin. Antitumor activities of the platinum-histamine complexes were investigated in three independent experiments, which differed in applied doses of preparations (PtCl 2 Hist, PtCl 2 [ 125 I]Hist, PtCl 2 [ 131 I]Hist, PtCl 2 Hist/PtCl 2 [ 125 I]Hist and PtCl 2 Hist/PtCl 2 [ 131 I]Hist), treatment schedules as well as stages of the disease progress in the animals used. Experiment 1 included long-term, multidose treatment with low single doses (treatment duration 31–32 days; 8–10 doses of ca. 0.25∙MTD Pt each). Experiment 2 included short-term, multidose treatment with higher single doses (4×ca. 0.5∙MTD Pt up to Day 13 of the treatment). Experiment 3 included long-term concomitant multidose treatment with higher single doses (9×0.9–0.4∙MTD Pt up to Day 33). Results The long-term treatment with the platinum-histamine preparations revealed inhibiting activity on the tumor growth and size in comparison to control groups. The most intensive and significant antitumor effects were observed for the radioactive complexes. The tumor growth delay factors (GDFs) observed in Experiment 1 were 0.4, 0.7, and 1.2 for PtCl 2 Hist, PtCl 2 Hist/PtCl 2 [ 131 I]Hist, and PtCl 2 Hist/PtCl 2 [ 125 I]Hist, respectively. Significant ( P .05 ) prolongations of median survivals (MS) were found in Experiment 2 following the treatment with higher single doses of PtCl 2 Hist and PtCl 2 His/PtCl 2 [ 125 I]Hist (Ratio MS tr /MS con ca. 1.4). A slightly less potent activity was observed for PtCl 2 Hist/PtCl 2 [ 131 I]Hist, and no survival improvement was found for the groups treated mostly with the radiation (PtCl 2 [ 125 I]Hist and PtCl 2 [ 131 I]Hist). The intensive and long-term concomitant scheduling of the radioactive platinum–histamine complexes labeled with I-125 and I-131 (Experiment 3) resulted in a significant inhibition of the tumor growth (GDF=1.9) and survival prolongation of the tumor-bearing mice (MS tr /MS con =1.5, P =.023). The treatment-related toxicity was mild. Conclusion An enhancement of the antitumor activity due to the multidose concomitant treatment with a combination of cytotoxic/cytostatic dichloroplatinum(II)–histamine and the attached iodine radionuclides was shown in the murine model of experimental neoplasm.

The aim of the study was to examine in vitro and in vivo binding of radiolabelled analogues of P149 peptide by experimental mammary adenocarcinoma with the intention of potential application for diagnosis and internal radiotherapy of tumours.The 36-amino acid peptide (P149-QY) of 90% homology to 447-480 peptide fragment of hAFP was synthesised and radiolabelled with iodine-125. The biodistribution of P149-Q[125I]-Y was studied in experimental mammary tumours. For in vitro experiments, extract from mouse mammary tumours were prepared and incubated with radioiodinated P149-QY peptide in the presence of a cross-linking reagent.The gel electrophoresis analysis (SDS-PAGE) showed that radioiodinated P149-QY peptide formed a complex with adenocarcinoma proteins of about 30 kDa. The biodistribution of P149-Q[125I]-Y studied in experimental mammary tumours revealed a higher pharmacokinetic rate in comparison with the whole radioiodinated AFP molecule. A moderate uptake of P149-Q[125I]-Y in the tumour tissue was observed (3.2% ID/g at 30-min p.i.v). However, a faster radioactivity clearance from blood and normal tissues resulted in an increase in the tumour/muscle (T/M) ratio, i.e. from 2.3 to 3.4 after 30 mins and 24 h p.i.v, respectively.The present study shows that radioiodinated P149-QY peptide reveals some positive features as the AFP receptor radioligand, however, some additional structural modifications of the initial peptide molecule are necessary for full retention of the ligand-receptor interaction of its radiolabelled forms.

Recently, a new PtCl2-histamine complex, and its radioactive analogues labelled with I-131 and I-125 have been synthesised and investigated both in vitro and in vivo. In this preliminary report the survival rate of radioactive platinum-[125I] histamine therapy in tumour-bearing mice is demonstrated.A murine model of transplantable colon adenocarcinoma (C38) in C57BL/6 mice (15 days postimplantation) was used for the experiment. Three groups of animals were treated every 2-3 days with five intraperitoneal injections of the following preparations: PtCl2Hist (total dose of Pt--125 micromol/kg), PtCl2[125I]Hist (total dose of I-125--4.2 MBq; Pt--13 micromol/kg), and "Active/Cold"--PtCl2[125I]Hist/PtCl2Hist (I-125--4.2 MBq; Pt--125 micromol/kg). A solution of 15% dimethylformamide in saline was applied to the control group. A survival analysis with the Kaplan-Meier estimation of survival curves and a statistical comparison by a log-rank test was applied to evaluate the anticancer activity of the tested preparations.Treatment of the animals with platinum-histamine preparations resulted in a significant prolongation of survivals, especially if the radioactive complex with carrier-added PtCl2Hist (p0.005) was applied. The highest, almost a 60% prolongation of survival was observed in the Active/Cold group (MStr/MScon ratio = 1.58, 95% CI 1.22-1.93). For this group there was the lowest risk of death (hazard ratio HR = 0.29), whereas HR = 0.45 and 0.47 were found in the animals treated with unattended PtCl2Hist and 125I-labelled complex, respectively.The significant enhancement of in vivo anti-cancer activity by a concomitant combination of the therapeutic factors, i.e. cytotoxic/cytostatic activity of the platinum(II)-histamine and the Auger electrons effects generated by the attached I-125 radionuclide, was found on the murine model of transplantable colon adenocarcinoma.

The platinum-iodohistamine complex with in vitro cytostatic activity toward colon and mammary cancer cells has been synthesised recently in our laboratory. The pharmacokinetics of radioactive complex analogues, labelled with I-131 and I-125, has been examined in murine model of spontaneous mammary adenocarcinoma. The present work is devoted to the examination of the potential use of liposomes as a carrier system for the radioactive platinum-[*I]iodohistamine complex in vivo.Encapsulations of the Pt- [(125)I]iodohistamine were studied using a different molar ratio of the complex and liposomes with positive surface charge, as well as various incubation procedures. Biodistribution of the initial and the liposomal form of the complex were studied in C3H tumour-bearing mice with spontaneously developed and transplantable (16C) mammary adenocarcinoma.Comparative biodistribution studies in C3H/16C mice and in mice with spontaneously developed mammary tumour have shown that in the former model pharmacokinetics of the Pt-[125I]iodohistamine complex is more predictable and more similar to that observed for cisplatin. Therefore, the transplantable tumour model is more advantageous for the complex and its liposomal form evaluation. In C3H/16C mice, significant differences in the biodistribution between the radioactive platinum complex and its liposomal form were observed. The concentration of the activity in blood after 2 h p.i.v. was two times lower for the encapsulated complex, and the uptake of the radioactivity by liver, spleen, and lungs was twice as high as that obtained for the free Pt-[(125)I]iodohistamine preparation. The radioactivity in tumour was almost constant for liposomal platinum complex (ca. 2% ID/g), although it was two times lower compared to the initial platinum complex.The results of the present study indicate that platinum-[*I]iodohistamine can be efficiently incorporated into cationic liposomes (c. 40%). However, the uptake of the encapsulated complex by the liver and spleen macrophages demands further modification of the lipid membrane.

A novel class of water-soluble Pt(IV) complexes with histamine (Hist) and radioiodinated histamine ([ 125 I/ 131 I]Hist) has been synthesised with the goal of potential application for concomitant anticancer radio-chemotherapy of solid tumours. The prepared complex of 1:2 metal:ligand stoichiometry ([Pt(IV)(Hist) 2 (OH) 2 ]Cl 2 ) was characterised by microanalysis, mass spectrometry, and chromatographic methods. Cytotoxic/cytostatic activities of the complex were examined by flow cytometry method using the MCF-7 cells line. A slightly lower cytotoxicity of the Pt(IV) complex comparing to cisplatin was found (IC 50 59 and 48 μM, respectively). Both cisplatin and the histamine complex show a cytostatic activity by blocking MCF-7 cells in S-phase of cell cycle. Biodistribution studies in normal rats revealed the highest accumulation of the 131 I-labelled complex in liver and kidneys (41.3% and 12.4% ID after 24 h post-intravenous injection (p.i.v.)). The similar pharmacokinetics was observed in tumour-bearing C3H/W mice, however, a lower accumulation in liver was observed following an intraperitoneal comparing to an intravenous administration. A concentration of the complex in tumour increased with time post-intraperitoneal injection (1.2 and 2.5%ID/g after 2 and 24 h (p.i.), respectively). An increasing tumour/muscle ratio was also observed (2.2 and 4.5 after 2 and 24 h p.i., respectively), and that suggests a penetration of the complex into the tumour cells, and a permanent binding with some cellular components, probably with the DNA.

Morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) and its analogs modified at position 3: [D-Phe(3)]morphiceptin, [D-ClPhe(3)]morphiceptin and [D-Cl(2)Phe(3)]morphiceptin were synthesized and labeled with [(125)I] or [(131)I]. Their binding to membranes isolated from experimental adenocarcinoma was examined in vitro with the use of a cross-linking assay followed by the Western blot technique. The radioactive complex had molecular weight of about 65 kDa and was detectable by anti-mu-opioid receptor polyclonal antibody. Expression of the mu-opioid receptor in mouse mammary adenocarcinoma was confirmed by reverse transcriptase-polymerase chain reaction. The binding studies showed the highest affinity and capacity for [D-Phe(3)]morphiceptin (K(d) 0.39 and B(max) 1112) and [D-ClPhe(3)]morphiceptin (K(d) 1.8 and B(max) 220). Morphiceptin and its D-Cl(2)Phe analog had significantly lower B(max) values (131 and 83, respectively). Biodistribution experiments in tumor-bearing C3H/Bi mice with the use of the (131)I-labeled peptides confirmed the results of our in vitro studies. The highest accumulation of radioactive peptides in the tumor tissue was also found for peptides with D-Phe and D-ClPhe.

Purpose Identification of vulnerable plaques remains crucial for better cardiovascular risk assessment. At least 20% of inflammatory cells within unstable (vulnerable) plaques comprise T lymphocytes, which contain receptors for interleukin-2 (IL-2); those receptors can be identified by scintigraphy with radiolabelled IL-2.The aim of this study was to identify the “inflamed” (vulnerable) plaques by scintigraphy using IL-2 labelled with 99mTc in the selected, high cardiovascular risk group of end-stage renal disease (ESRD) patients. Methods A total of 28 patients (18 men, 10 women, aged 55.2 ± 9.6 years, 17 on peritoneal dialysis, 11 on haemodialysis) underwent common carotid artery (CCA) scintigraphy with the use of 99mTc-hydrazinonicotinamide (HYNIC)-IL-2. In all cases, ultrasound examination of the CCA was performed and levels of selected proinflammatory factors, atherogenic markers and calcium-phosphate balance parameters were measured. Finally, the target to non-target (T/nT) ratio of IL-2 uptake in atherosclerotic plaques with intima-media thickness (IMT), classic cardiovascular risk factors and concentrations of the measured factors were compared. Results Increased 99mTc-HYNIC-IL-2 uptake in atherosclerotic plaques in 38/41 (91%) cases was detected. The median T/nT ratio of focal 99mTc-HYNIC-IL-2 uptake in atherosclerotic plaques was 2.35 (range 1.23–3.63). The mean IMT value on the side of plaques assessed by scintigraphy was 0.79 ± 0.18 mm (median 0.8, range 0.5–1.275). Correlations between T/nT ratio and homocysteine (R = 0.22, p = 0.037), apolipoprotein B (apoB) (R = 0.31, p = 0.008), apoB to apoA-I ratio (R = 0.29, p = 0.012) and triglyceride concentration (R = 0.26, p = 0.021) were detected. A lower T/nT ratio in patients with better parameters of nutritional status (haemoglobin, albumin, adiponectin) in comparison with patients with worse nutritional parameters (3.20 ± 0.5 vs 2.16 ± 0.68, p = 0.025) was revealed as well as a difference between values of T/nT ratio in groups of patients with values of apoB, soluble CD40 ligand and asymmetric dimethylarginine above and below median (3.18 ± 0.52 vs 2.16 ± 0.68, p = 0.031). No statistically significant association was found between T/nT ratio and mean value of either IMT or classic cardiovascular risk factors. Conclusion Scintigraphy with the use of 99mTc-HYNIC-IL-2 can be a tool for inflamed atherosclerotic (vulnerable) plaque visualization within CCA in ESRD patients. Quantitative results of carotid artery scintigraphy with 99mTc-HYNIC-IL-2 correlate with serum concentration of selected cardiovascular risk markers.