Significance of (111)In-DTPA chelate in renal radioactivity levels of (111)In-DTPA-conjugated peptides

Metabolic studies of (111)In-DTPA-labeled polypeptides and peptides showed that the radiolabeled (poly)peptides generated (111)In-DTPA-adducts of amino acid that possess long residence times in the lysosomal compartment of the tissues where (poly)peptides accumulated. However, a recent study suggested that metal-chelate-methionine (Met) might possess in vivo behaviors different from metal-chelate adducts of other amino acids. In this study, to elucidate whether some biological characteristics of Met may accelerate the renal elimination rate of (111)In-DTPA-adduct of Met into urine, (111)In-DTPA-Met(1)-octreotide was synthesized and the renal handling of (111)In-DTPA-Met was investigated using (111)In-DTPA-L-Phe(1)-octreotide (Phe represents phenylalanine), which was reported previously, as a reference. Both (111)In-DTPA-conjugated octreotide analogs were stable against 3-h incubation in murine serum at 37 degrees C. Both (111)In-DTPA-octreotide analogs also showed rapid clearance of the radioactivity from the blood and similar accumulation of the radioactivity in the kidney. No significant differences were observed in the renal radioactivity levels from 10 min to 24 h postinjection between the two. Metabolic studies indicated that (111)In-DTPA-Met(1)-octreotide and (111)In-DTPA-L-Phe(1)-octreotide generated (111)In-DTPA-adducts of Met and Phe, respectively, as the final radiometabolites at similar rates. These findings suggested that the long residence times of the radioactivity in tissues after administration of (111)In-DTPA-labeled peptides and polypeptides would be attributed to inherent characteristics of (111)In-DTPA chelate.     

Albumin-derived peptides efficiently reduce renal uptake of radiolabelled peptides

Purpose

In peptide-receptor radionuclide therapy (PRRT), the maximum activity dose that can safely be administered is limited by high renal uptake and retention of radiolabelled peptides. The kidney radiation dose can be reduced by coinfusion of agents that competitively inhibit the reabsorption of radiolabelled peptides, such as positively charged amino acids, Gelofusine, or trypsinised albumin. The aim of this study was to identify more specific and potent inhibitors of the kidney reabsorption of radiolabelled peptides, based on albumin.

Methods

Albumin was fragmented using cyanogen bromide and six albumin-derived peptides with different numbers of electric charges were selected and synthesised. The effect of albumin fragments (FRALB-C) and selected albumin-derived peptides on the internalisation of ¹¹¹In-albumin, ¹¹¹In-minigastrin, ¹¹¹In-exendin and ¹¹¹In-octreotide by megalin-expressing cells was assessed. In rats, the effect of Gelofusine and albumin-derived peptides on the renal uptake and biodistribution of ¹¹¹In-minigastrin, ¹¹¹In-exendin and ¹¹¹In-octreotide was determined.

Results

FRALB-C significantly reduced the uptake of all radiolabelled peptides in vitro. The albumin-derived peptides showed different potencies in reducing the uptake of ¹¹¹In-albumin, ¹¹¹In-exendin and ¹¹¹In-minigastrin in vitro. The most efficient albumin-derived peptide (peptide #6), was selected for in vivo testing. In rats, 5 mg of peptide #6 very efficiently inhibited the renal uptake of ¹¹¹In-minigastrin, by 88%. Uptake of ¹¹¹In-exendin and ¹¹¹In-octreotide was reduced by 26 and 33%, respectively.

Conclusions

The albumin-derived peptide #6 efficiently inhibited the renal reabsorption of ¹¹¹In-minigastrin, ¹¹¹In-exendin and ¹¹¹In-octreotide and is a promising candidate for kidney protection in PRRT.     

In vitro comparison of renal handling and uptake of two somatostatin receptor-specific peptides labeled with indium-111

Objective  Radiolabeled receptor-specific somatostatin analogs labeled with gamma- or beta-emitting radionuclides are useful for scintigraphic imaging and/or therapy of selected neuroendocrine tumors. However, significant renal uptake may result in radiotoxicological injury of the kidney and can limit clinical application of the agents. The aim of the study was to analyze renal handling, rate, and mechanism of renal accumulation of two somatostatin receptor-targeted peptides, [DOTA⁰, Tyr³, Thr⁸]-octreotide (DOTA-TATE) and [DOTA⁰, 1-Nal³]-octreotide (DOTA-NOC), labeled with indium-111 using in vitro methods. Methods  The perfused rat kidney and freshly isolated rat renal cells were used as experimental models. The perfusion was performed in a recirculation regimen at constant pressure with solution containing bovine albumin, erythrocytes, and a mixture of essential substrates. The renal cells were isolated from rat kidneys using two-phase collagenase perfusion. Accumulation studies were used to evaluate the renal uptake of the peptides and to compare their accumulation with that of passively or actively transported model drugs. The influence of selected inhibitors of receptor-mediated endocytosis and the inhibition of energy-dependent transport processes on the uptake were also investigated using isolated renal cells. Results  The renal clearance of ¹¹¹In-DOTA-NOC in the perfused rat kidney was significantly lower than that of ¹¹¹In-DOTA-TATE. Reverse situation was found in the case of renal retention. Pretreatment of the perfused kidney with maleate markedly decreased the renal retention. ¹¹¹In-DOTA-NOC was accumulated in the isolated renal cells at a higher rate than ¹¹¹In-DOTA-TATE (ratio 3: 1). The uptake of the radiopeptides in renal cells was higher than the uptake of not only the passively transported sucrose but also actively transported and accumulated methylglucose. The rank order of potency to inhibit the uptake by active endocytosis was approximately aprotinin > maleate > lysine. The uptake of the radiopeptides in the renal cells was temperature dependent. Conclusions  Both in vitro methods showed a higher renal accumulation of ¹¹¹In-DOTA-NOC in comparison with ¹¹¹In-DOTA-TATE. The renal uptake was partly decreased by inhibitors of receptor-mediated endocytosis and by a block of energy-dependent processes. A significant participation of active transport processes in renal accumulation of the studied peptides was confirmed.     

Reducing renal uptake of111In-DOTATOC: A comparison among various basic amino acids

PURPOSE: Several studies have reported significant renal toxicity after the use of a high dose of 90Y-DOTATOC. Thus, renal protection is necessary in treatments with 90Y-DOTA Tyr3-octreotide (DOTATOC). The infusion of certain positively charged amino acids has been shown to effectively reduce renal uptake of DOTATOC. In this study, we compared the effectiveness of three kinds of amino acids, D-lysine (lysine), L-arginine (arginine) and histidine, on renal protection in healthy rats and tried to determine which one was the most effective. METHODS: Twenty SD healthy male rats were divided into 4 groups: lysine, histidine, arginine, and control. The rats were injected with a dose of 400 mg/kg of amino acid or 2 ml of phosphate-buffered saline (PBS) (as control) intraperitoneally. All rats were sacrificed at 4 hrs after the injection of 1 MBq 111In-DOTATOC. Samples of the kidney were taken and weighed carefully. The counts of radioactivity were measured by a gamma counter and renal concentrations were calculated and expressed as percent injected dose per gram (% ID/g). RESULTS: The renal uptake of 111In-DOTATOC was significantly lower for all three kinds of amino acids when compared to the control group. The renal uptake of 111In-DOTATOC in the lysine group was significantly lower than those in the histidine and arginine groups. The renal uptake of 111In-DOTATOC in the histidine group was lower than that in the arginine group, but no statistical difference was noted. CONCLUSION: Among these three amino acids, lysine had the best reduction rate of renal uptake of DOTATOC. Histidine was more effective than arginine but no statistical difference was noted.     

Gelatin-based plasma expander effectively reduces renal uptake of 111In-octreotide in mice and rats.

111In-Diethylenetriaminepentaacetic acid-octreotide generally is used for the scintigraphic imaging of neuroendocrine and other somatostatin receptor-positive tumors. On the basis of the successful targeting of octreotide, radiolabeled somatostatin analogs, such as 90Y-(1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid [DOTA])0-Tyr3-octreotide and 177Lu-DOTA0-Tyr3-octreotate, were developed for peptide receptor radionuclide therapy. However, the maximum tolerated doses of these analogs are limited because of the high and persistent renal uptake that leads to relatively high radiation doses in the kidneys. Renal uptake can be reduced by coinfusion of basic amino acids or polypeptides. However, high doses of basic amino acids can induce severe side effects. It was reported that the infusion of gelatin-based plasma expanders resulted in increased low-molecular-weight proteinuria, suggesting that these plasma expanders interfere with the tubular reabsorption of peptides and proteins. In the present study, we analyzed the effects of several plasma expanders on the renal uptake of 111In-octreotide in rats and mice. METHODS: Wistar rats and BALB/c mice were injected with 0.5 or 0.1 mL of plasma expander, respectively. Thereafter, the animals received 111In-octreotide intravenously. Animals were killed at 20 h after the injection of the radiopharmaceutical. Organs were dissected, and the amount of radioactivity in the organs and tissues was measured. RESULTS: The administration of 20 mg of Gelofusine in rats or 4 mg in mice was as effective in reducing the renal uptake of 111In-octreotide as the administration of 80 or 20 mg of lysine in rats or mice, respectively, without reducing 111In-octreotide uptake in receptor-positive organs. Plasma expanders based on starch or dextran had no effect on the renal uptake of 111In-octreotide. CONCLUSION: The gelatin-based plasma expander Gelofusine significantly reduced the kidney uptake of 111In-octreotide as effectively as did lysine. Because Gelofusine is a well-known and generally used blood volume substitute that can be applied safely without the induction of toxicity, evaluation of this compound for its potential to reduce the kidney uptake of radiolabeled peptides in patients is warranted.     

Increased mammary uptake on 111In pentetreotide scintigraphy

The normal scintigraphic features of 111In pentetreotide include uptake in the pituitary, thyroid, liver, spleen, kidneys, urinary bladder, gallbladder, and bowel. In premenopausal women, faint activity can be seen in the breasts. We present 2 cases of symmetrical, markedly increased mammary uptake. Our hypothesis to explain this pattern of uptake is an upregulation of the somatostatin type 2 receptor caused by high blood levels of estradiol during pregnancy. Estradiol levels during the menstrual cycle could therefore explain the variable degree of uptake commonly seen in premenopausal women.     

Submandibular uptake on indium-111 leukocyte scans in children

Report is made of false-positive localization of In-111 leukocytes in the submandibular gland area in children. Possible mechanisms are discussed, but this may represent a normal finding in children.     

Significance of nodal uptake on indium 111 labeled leukocyte scans

A retrospective study of 132 indium 111 labeled leukocyte scans on 129 patients was undertaken to determine the incidence and significance of activity in the neck, axilla, and groin suggestive of regional lymph nodes. Possible nodal uptake was observed in 35 out of 108 (32%) adults and 11 out of 21 (52%) children. Activity was characterized as symmetric or asymmetric. Based on medical record review, positive scans were divided into two groups: with or without clinical correlation. Nodal visualization was common in children and showed a high degree of clinical correlation. In adults, asymmetric uptake in the axilla and groin was frequently associated with disease. Cervical activity, however, often remained unexplained and may represent false-positive uptake in mandibular marrow or submandibular glands. In summary, nodal uptake may represent a significant finding, reflecting known disease or may signal subclinical disease.     

Indium-111 octreotide uptake in the surgical scar

Indium-111 octreotide uptake has been reported in various somatostatin receptor positive tumors, granulomas and autoimmune diseases in which activated leucocytes may play a role, subcutaneous cavernous hemangioma and angiofibroma. We present Indium-111 octreotide uptake in a surgical abdominal scar tissue 1.5 and 6 months after surgery in a patient who had been treated for recurrent carcinoid tumor in the rectosigmoid junction. Indium-111 octreotide uptake in a surgical scar may be related to the binding to somatostatin receptors in the activated lymphocytes and fibroblasts that is previously reported.     

抗菌肽的分子设计研究进展

抗菌肽是一类具有抗菌、抗病毒、抗肿瘤等生物活性的小分子多肽,与传统抗生素相比,不易产生耐药性;但因其毒性强、稳定性差、合成成本高等原因,抗菌肽还不能广泛应用于临床。为此,药物学家们对其进行了结构改造,获得了高活性、低毒性的抗菌肽。该文综述了抗菌肽分子设计的研究进展及发展方向。     

Indium-111 pentetreotide lung uptake in infectious lung disease

Bilateral diffuse lung uptake of In-111 pentetreotide (OCT) was observed during a whole-body scan performed in a 68-year-old woman with Cushing's syndrome and suspected ectopic adrenocorticotropic hormone secretion. A few days later, she was found to have bilateral bacterial pneumonia (of mixed anaerobic origin). Cushing's syndrome was finally proved to be of pituitary origin. The OCT lung uptake in pneumonia probably resulted from tracer binding by somatostatin receptors on the inflammatory leukocytes. Although the rapid wash-out from experimentally induced abscesses does not make OCT a suitable tracer for detecting acute infections, the images and data here reported suggest that infectious lung disease should be excluded before diagnosing lung involvement by neuroendocrine tumors.     

Oral versus intravenous administration of lysine: equal effectiveness in reduction of renal uptake of [111In-DTPA]octreotide.

During tumor therapy with radiolabeled somatostatin analogs, the kidneys are dose limiting. Renal uptake in patients can effectively be reduced by a 4- to 10-h intravenous infusion of a lysine/arginine solution, thereby increasing the maximum radiation dose to the tumor without renal side effects. Oral administration of amino acids could facilitate this labor-intensive procedure. Therefore, the effects of oral versus intravenous administration of D-lysine were compared in rats injected with [111In-diethylenetriaminepentaacetic acid (DTPA)]octreotide. METHODS: Rats were intravenously injected with 3 MBq/0.5 microg [111In-DTPA]octreotide and also received D-lysine intravenously or orally in various concentrations and following various time schedules. Twenty-four hours after injection, a biodistribution study and renal ex vivo autoradiography were performed. RESULTS: Renal uptake was reduced significantly-up to 40%-in all lysine-treated groups, without affecting the uptake in other organs. CONCLUSION: Renal uptake of this radiolabeled peptide can be reduced up to 40% both by oral and by intravenous administration of lysine in rats.     

Increased uptake of 111In-octreotide in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by an uncontrolled accumulation and activation of lung fibroblasts. A modulation of fibroblast activation has been observed in various systems with octreotide, a synthetic somatostatin analog with strong affinity for the somatostatin receptor subtype 2 (sst2). One aim of our study was to evaluate the expression of somatostatin receptors in the lungs of patients with IPF. A second aim was to evaluate the relationship between 111In-octreotide uptake and the effect of pulmonary fibrosis as assessed by lung function tests and parameters and by radiologic findings. METHODS: We investigated 11 patients with IPF, 6 patients with pulmonary fibrosis associated with systemic sclerosis (SSc), and 19 patients with disease not of the lung (control patients). The expression of somatostatin receptors was evaluated in vivo using 111In-octreotide scintigraphy. We evaluated the relationship between 111In-octreotide uptake and the activity of pulmonary fibrosis as assessed by lung function tests, bronchoalveolar lavage (BAL) cellularity, and high-resolution CT (HRCT) of the chest. Planar images and thoracic SPECT (24 h) were performed after injection of 222 MBq of 111In-octreotide. Lung uptake was quantified using the lung-to-background ratio (L/B). In addition, the expression of sst2 was evaluated in vitro, in frozen lung-tissue samples using autoradiography, and in human cultures of lung fibroblasts using a ligand-binding assay. RESULTS: Compared with lung uptake in control patients (median L/B, 1.25; range, 1.14-1.49), lung uptake was increased in all 11 IPF patients (median L/B, 2.63; range, 1.59-3.13; P < 0.001) and in 4 of 6 SSc patients (median L/B, 1.68; range, 1.42-2.16). The L/B was lower in SSc patients than in IPF patients (P = 0.011). Increased uptake correlated with the alteration of lung function (carbon monoxide diffusing capacity [rho = -0.655; P = 0.038], diffusing capacity for carbon monoxide and alveolar volume ratio [rho = -0.627; P = 0.047], vital capacity [rho = -0.609; P = 0.054], and total lung capacity [rho = -0.598; P = 0.058]) and with the intensity of alveolitis (total BAL cellularity [rho = 0.756; P = 0.045], neutrophil counts [rho = 0.738; P = 0.05]), and HRCT fibrosis score (rho = 0.673; P = 0.007). Autoradiography suggested that vascular structures were a prominent binding site. Lung fibroblasts expressed somatostatin receptors in vitro as measured by binding assay. CONCLUSION: Our preliminary results identified an increased expression of sst2 in (mainly idiopathic) pulmonary fibrosis. Lung uptake correlates with the alteration of lung function and with the intensity of alveolitis.     

Development of 111In-labeled tumor-associated antigen peptides for monitoring dendritic-cell-based vaccination

Dendritic cells (DC) are professional antigen-presenting cells capable of inducing potent immune responses. In our ongoing clinical trials, human leukocyte antigen (HLA)-A2.1+ melanoma patients are vaccinated with mature DC, presenting tumor-derived peptides in major histocompatibility complexes (MHC) to naive T cells. Previously, we have shown that both intradermally and intranodally injected (111)In-labeled mature DC migrate to draining lymph nodes. However, little is known about the fate of the MHC-peptide complex after injection of these peptide-loaded DC. The aim of the present study was to develop radiolabeled, tumor-derived peptides to monitor their binding to MHC Class I. METHODS: The HLA-A2.1 binding peptide gp100:154-162mod (gp100:154m) was conjugated with diethylenetriamine pentaacetic acid (DTPA) either at the N-terminus (alpha-DTPA-gp100:154m) or at the epsilon amino group of the Lys(154) residue (epsilon-DTPA-gp100:154m) and labeled with (111)In. RESULTS: The maximum specific activity for both peptides was 13 GBq/micromol. The IC50 of the alpha-[(111)In]DTPA-gp100:154m peptide was >75 microM. The IC50 of the (111)In-labeled epsilon-DTPA-gp100:154m was 3 microM, similar to the unconjugated peptide. MHC binding studies showed specific binding of the epsilon-[(111)In]DTPA-gp100:154m peptide to the JY cells at 4 degrees C. Interestingly, no specific binding was observed for the alpha-[(111)In]DTPA-gp100:154m peptide. In contrast to the alpha-[(111)In]DTPA-gp100:154m peptide, the epsilon-[(111)In]DTPA-gp100:154m peptide was recognized by cytotoxic T cells. CONCLUSION: When DTPA was conjugated to the epsilon NH2 group of the Lys(154) residue, MHC binding of the peptide was preserved and could still be recognized by cytotoxic T cells. These studies allow the noninvasive determination of the behavior of MHC-peptide complexes on DC in vivo.