Jong, M. de

Treatment of Gastroenteropancreatic Neuroendocrine Tumors with Peptide Receptor Radionuclide Therapy (Article)

2012-01-10T00:00:00Z

The primary treatment of gastroenteropancreatic neuroendocrine tumors (GEPNETs) is surgery with curative intent or debulking of the tumor mass. In case of metastatic disease, cytoreductive options are limited. A relatively new therapeutic modality, peptide receptor radionuclide therapy (PRRT) with radiolabeled somatostatin analogs, is currently available in a number of mostly European centers. Complete and partial responses obtained after treatment with [90Y-DOTA0,Tyr3]octreotide are in the same range as after treatment with [177Lu-DOTA0,Tyr3]octreotate (i.e. 10-30%). However, significant nephrotoxicity has been observed after treatment with [90Y-DOTA0,Tyr3]octreotide. Options to improve PRRT may include combinations of radioactive labeled somatostatin analogs, intra-arterial administration, and the use of radiosensitizing drugs combined with PRRT. Other therapeutic applications of PRRT may include additional therapy cycles in patients with progressive disease after benefit from initial therapy, PRRT in adjuvant or neoadjuvant setting, or PRRT combined with new targeted therapies, such as sunitinib or everolimus. Randomized clinical trials comparing PRRT with other treatment modalities, or comparing various radioactive labeled somatostatin analogs should be undertaken to determine the best treatment options and treatment sequelae for patients with GEPNETs. Copyright

Facilitating tumor functional assessment by spatially relating 3D tumor histology and In Vivo MRI: Image registration approach (Article)

2011-08-29T00:00:00Z

Background: Magnetic resonance imaging (MRI), together with histology, is widely used to diagnose and to monitor treatment in oncology. Spatial correspondence between these modalities provides information about the ability of MRI to characterize cancerous tissue. However, registration is complicated by deformations during pathological processing, and differences in scale and information content. Methodology/Principal Findings: This study proposes a methodology for establishing an accurate 3D relation between histological sections and high resolution in vivo MRI tumor data. The key features of the methodology are: 1) standardized acquisition and processing, 2) use of an intermediate ex vivo MRI, 3) use of a reference cutting plane, 4) dense histological sampling, 5) elastic registration, and 6) use of complete 3D data sets. Five rat pancreatic tumors imaged by T2*-w MRI were used to evaluate the proposed methodology. The registration accuracy was assessed by root mean squared (RMS) distances between manually annotated landmark points in both modalities. After elastic registration the average RMS distance decreased from 1.4 to 0.7 mm. The intermediate ex vivo MRI and the reference cutting plane shared by all three 3D images (in vivo MRI, ex vivo MRI, and 3D histology data) were found to be crucial for the accurate co-registration between the 3D histological data set and in vivo MRI. The MR intensity in necrotic regions, as manually annotated in 3D histology, was significantly different from other histologically confirmed regions (i.e., viable and hemorrhagic). However, the viable and the hemorrhagic regions showed a large overlap in T2*-w MRI signal intensity. Conclusions: The established 3D correspondence between tumor histology and in vivo MRI enables extraction of MRI characteristics for histologically confirmed regions. The proposed methodology allows the creation of a tumor database of spatially registered multi-spectral MR images and multi-stained 3D histology.

Comparative biodistribution of 12 111In-labelled gastrin/CCK2 receptor-targeting peptides (Article)

2011-08-01T00:00:00Z

Purpose Cholecystokinin 2 (CCK-2) receptor overexpression has been demonstrated in various tumours such as medullary thyroid carcinomas and small-cell lung cancers. Due to this high expression, CCK-2 receptors might be suitable targets for radionuclide imaging and/or radionuclide therapy. Several CCK-2 receptor-binding radiopeptides have been developed and some have been tested in patients. Here we aimed to compare the in vivo tumour targeting properties of 12111Inlabelled 1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetraacetic acid (DOTA)-conjugated gastrin/CCK2 receptor-binding peptides. Methods Two CCK8-based peptides and ten gastrin-based peptide analogues were tested. All peptides were conjugated with DOTA and labelled with111In. Biodistribution studies were performed in mice with subcutaneous CCK2/ gastrin receptor-expressing tumours and with receptornegative tumours contralaterally. Biodistribution was studied by counting dissected tissues at 1 and 4 h after injection. Results Both the CCK analogues displayed relatively low tumour uptake (approximately 2.5%ID/g) as compared to minigastrin analogues. Two linear minigastrin peptides (MG0 and sargastrin) displayed moderate tumour uptake at both 1 and 4 h after injection, but also very high kidney uptake (both higher than 48%ID/g). The linear MG11, lacking the penta-Glu sequence, showed lower tumour uptake and also low kidney uptake. Varying the N-terminal Glu residues in the minigastrin analogues led to improved tumour targeting properties, with PP-F11 displaying the optimal biodistribution. Besides the monomeric linear peptides, a cyclized peptide and a divalent peptide were tested. Conclusion Based on these studies, optimal peptides for peptide receptor radionuclide targeting of CCK2/gastrin receptor-expressing tumours were the linear minigastrin analogue with six D-Glu residues (PP-F11), the divalent analogue MGD5 and the cyclic peptide cyclo-MG1. These peptides combined high tumour uptake with low kidney retention, and may therefore be good candidates for future clinical studies.

Comparison of the binding and internalization properties of 12 DOTA-coupled and 111In-labelled CCK2/gastrin receptor binding peptides: A collaborative project under COST Action BM0607 (Article)

2011-08-01T00:00:00Z

Purpose Specific overexpression of cholecystokinin 2 (CCK2)/gastrin receptors has been demonstrated in several tumours of neuroendocrine origin. In some of these cancer types, such as medullary thyroid cancer (MTC), a sensitive diagnostic modality is still unavailable and therapeutic options for inoperable lesions are needed. Peptide receptor radionuclide therapy (PRRT) may be a viable therapeutic strategy in the management of these patients. Several CCK2R-targeted radiopharmaceuticals have been described in recent years. As part of the European Union COSTAction BM0607 we studied the in vitro and in vivo characteristics of 12 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated CCK2R binding peptides. In the presentstudy, we analysed binding and internalization characteristics. Stability, biodistribution and imaging studies have been performed in parallel by other centres involved in the project. Methods Determination of IC50values was performed using autoradiography, with DOTA-peptides displacing125I-CCK from receptors on tissue sections from human tumours. Saturation binding and internalization experiments were performed using111In-labelled peptides. The rat AR42J cell line and the human A431-CCK2R transfected cell line were utilized for in vitro experiments; dissociation constants (Kd) and apparent number of binding sites (Bmax) were determined. Internalization was determined in receptor-expressing cells by incubating with tracer amounts of peptide at 37 and 4°C for different times up to 120 min. Surface-bound peptide was then stripped either by acid wash or subsequent incubation with 1 μM unlabelled peptide at 4°C. Results All peptides showed high receptor affinity with IC50values ranging from 0.2 to 3.4 nM. Saturation experiments also showed high affinity with Kdvalues in the 10-9-10-8M range. Bmaxvalues estimated in A431- CCK2R cells ranged from 0.6 to 2.2×106per cell. All peptides showed high levels of internalization when incubated at 37°C. Conclusion All DOTA-conjugated peptides showed high receptor binding and internalization properties and appear suitable for further characterization, as described in other articles of this issue.

Preclinical animal research on therapy dosimetry with dual isotopes (Article)

2011-05-01T00:00:00Z

Preclinical research into radionuclide therapies based on radiation dosimetry will enable the use of any LET-equivalent radionuclide. Radiation dose and dose rate have significant influence on dose effects in the tumour depending on its radiation sensitivity, possibilities for repair of sublethal damage, and repopulation during or after the therapy. Models for radiation response of preclinical tumour models after peptide receptor radionuclide therapy based on the linear quadratic model are presented. The accuracy of the radiation dose is very important for observation of dose-effects. Uncertainties in the radiation dose estimation arise from incomplete assay of the kinetics, low accuracy in volume measurements and absorbed dose S-values for stylized models instead of the actual animal geometry. Normal dose uncertainties in the order of 20% might easily make the difference between seeing a dose-effect or missing it altogether. This is true for the theoretical case of a homogeneous tumour type behaving in vivo in the same way as its cells do in vitro. Heterogeneity of tumours induces variations in clonogenic cell density, radiation sensitivity, repopulation capacity and repair kinetics. The influence of these aspects are analysed within the linear quadratic model for tumour response to radionuclide therapy. Preclinical tumour models tend to be less heterogenic than the clinical conditions they should represent. The results of various preclinical radionuclide therapy experiments for peptide receptor radionuclide therapy are compared to the outcome of theoretical models and the influence of increased heterogeneity is analysed when the results of preclinical research is transferred to the clinic. When the radiation dose and radiobiology of the tumour response is known well enough it may be possible to leave the current phenomenological approach in preclinical radionuclide therapy and start basing these experiments on radiation dose. Then the use of a gamma ray-emitting radionuclides for a chemically comparable beta-particle-emitting paired isotope for therapy evaluation would be feasible.

Renal uptake of different radiolabelled peptides is mediated by megalin: SPECT and biodistribution studies in megalin-deficient mice (Article)

2011-04-01T00:00:00Z

Purpose: Radiolabelled peptides used for peptide receptor radionuclide therapy are excreted mainly via the kidneys and are partly reabsorbed and retained in the proximal tubular cells. The resulting high renal radiation dose can cause nephrotoxicity, limiting the maximum activity dose and the effectiveness of peptide receptor radionuclide therapy. The mechanisms of kidney reabsorption of these peptides are incompletely understood, but the scavenger receptor megalin has been shown to play a role in the reabsorption of111In-octreotide. In this study, the role of megalin in the renal reabsorption of various relevant radiolabelled peptides was investigated. Methods: Groups of kidney-specific megalin-deficient mice and wild-type mice were injected with111In-labelled somatostatin, exendin, neurotensin or minigastrin analogues. Single photon emission computed tomographic (SPECT) images of the kidneys were acquired and analysed quantitatively, or the animals were killed 3 h after injection and the activity concentration in the kidneys was measured. Results: Megalin-deficient mice showed significantly lower uptake of all studied radiolabelled peptides in the kidneys, ranging from 22% (111In-octreotide) to 65% (111In-exendin) of uptake in wild-type kidneys. Quantitative analysis of renal uptake by SPECT and ex vivo measurements showed a very good correlation. Conclusion: Megalin is involved in the renal reabsorption of radiolabelled octreotide, octreotate, exendin, neurotensin and minigastrin. This knowledge may help in the design of strategies to reduce this reabsorption and the resulting nephrotoxicity in peptide receptor radionuclide therapy, enabling more effective therapy. Small-animal SPECT is an accurate tool, allowing in vivo quantification of renal uptake and serial measurements in individual mice.

Effect of interferon-a treatment on [68Ga-DOTA,Tyr3,Thre8] octreotide uptake in CA20948 tumors: A small-animal PET study (Article)

2011-04-01T00:00:00Z

In peptide receptor radionuclide therapy of neuroendocrine tumors, improvements have been made by increasing the affinity for receptors and by protecting critical organs (e.g., kidneys). However, tumor parameters involved in radiopeptide uptake are still under investigation. Interferon-a (IFNa) is used as biotherapy for neuroendocrine tumors. Several mechanisms of action are described, but the potential effect of IFNa on tumor uptake of labeled peptide has not been studied in vivo yet. Methods: Twenty-six male CA20948 tumor-bearing Lewis rats were imaged before and during IFNa treatment using quantitative small-animal PET with [68Ga-DOTA,Tyr3,Thre8]octreotide. Imaging was performed at days 0, 3, and 7. Animals were divided into 3 groups according to the treatment: Control (injected daily with saline), half (4 d of IFNa treatment from day 0 to day 3, then saline), and full (7 d of IFNa). A daily dose of IFNa (1.5 mIU) was administered subcutaneously. Quantitative PET results are expressed as percentage injected dose per cm3 and normalized to baseline (day 0) values. Tumor size was monitored by PET and caliper measurements. Results: Gross tumor uptake and tumor volumes increased in all groups over the 7-d period. On day 3, mean±SD ratios to day 0 were 1.2±0.2, 1.3±0.5, and 1.2±0.4, respectively, for control, half, and full groups. On day 7, respective values were 1.1±0.2, 1.3±0.6, and 1.5±0.4. At day 3, a comparison among groups showed no statistically significant difference. At day 7, the full group showed a significantly higher ratio in activity concentration than the control group (P = 0.021). A good correlation was found between tumor volumes assessed by small-animal PET and caliper measurements (R = 0.89, P<0.0001). Conclusion: As expected, over a period of 7 d, both tumor volumes and radiopeptide uptake increased in all animals. However, the activity concentration increased significantly more at day 7 in animals treated for 7 d with IFNα, compared with controls. This is the first, to our knowledge, in vivo indication that IFNa is able to increase tumor uptake of the labeled analog in a small-animal model of neuroendocrine tumors. The mechanisms underlying this effect (flow, vascular permeability, receptor upregulation) remain unknown and need to be further investigated. Copyright

Unfocused Extracorporeal Shock Waves Induce Anabolic Effects in Rat Bone (Article)

2011-01-05T00:00:00Z

Abstract. BACKGROUND: Extracorporeal shock waves are known to stimulate the differentiation of mesenchymal stem cells toward osteoprogenitors and induce the expression of osteogenic-related growth hormones. The aim of this study was to investigate if and how extracorporeal shock waves affected new bone formation, bone microarchitecture, and the mechanical properties of bone in a healthy rat model, in order to evaluate whether extracorporeal shock wave therapy might be a potential treatment for osteoporosis. METHODS: Thirteen rats received 1000 electrohydraulically generated unfocused extracorporeal shock waves to the right tibia. The contralateral, left tibia was not treated and served as a control. At two, seven, twenty-one, and forty-nine days after administration of the shock waves, in vivo single-photon-emission computed tomography (SPECT) scanning was performed to measure new bone formation on the basis of uptake of technetium-labeled methylene diphosphonate ((99m)Tc-MDP) (n = 6). Prior to and forty-nine days after the extracorporeal shock wave therapy, micro-computed tomography (micro-CT) scans were made to examine the architectural bone changes. In addition, mechanical testing, microcrack, and histological analyses were performed. RESULTS: Extracorporeal shock waves induced a strong increase in (99m)Tc-MDP uptake in the treated tibia compared with the uptake in the untreated, control tibia. Micro-CT analysis showed that extracorporeal shock waves stimulated increases in both trabecular and cortical volume, which resulted in higher bone stiffness compared with that of the control tibiae. Histological analysis showed intramedullary soft-tissue damage and de novo bone with active osteoblasts and osteoid in the bone marrow of the legs treated with extracorporeal shock waves. Microcrack analysis showed no differences between the treated and control legs. CONCLUSIONS: This study shows that a single treatment with extracorporeal shock waves induces anabolic effects in both cancellous and cortical bone, leading to improved biomechanical properties. Furthermore, treatment with extracorporeal shock waves results in transient damage to the bone marrow, which might be related to the anabolic effects. After further examination and optimization, unfocused extracorporeal shock waves might enable local treatment of skeletal sites susceptible to fracture.

Renal toxicity of radiolabeled peptides and antibody fragments: Mechanisms, impact on radionuclide therapy, and strategies for prevention (Article)

2010-07-01T00:00:00Z

Peptide-receptor radionuclide therapy (PRRT) with radiolabeled somatostatin analogs such as octreotide is an effective therapy against neuroendocrine tumors. Other radiolabeled peptides and antibody fragments are under investigation. Most of these compounds are cleared through the kidneys and reabsorbed and partially retained in the proximal tubules, causing dose-limiting nephrotoxicity. An overview of renal handling of radiolabeled peptides and resulting nephrotoxicity is presented, and strategies to reduce nephrotoxicity are discussed. Modification of size, charge, or structure of radiolabeled peptides can alter glomerular filtration and tubular reabsorption. Coinfusion of competitive inhibitors of reabsorption also interferes with the interaction of peptides with renal endocytic receptors; coinfusion of basic amino acids is currently used for kidney protection in clinical PRRT. Furthermore, nephrotoxicity may be reduced by dose fractionation, use of radioprotectors, or use of mitigating agents. Decreasing the risk of nephrotoxicity allows for administration of higher radiation doses, increasing the effectiveness of PRRT. COPYRIGHT

Androgen-regulated gastrin-releasing peptide receptor expression in androgen-dependent human prostate tumor xenografts (Article)

2010-06-15T00:00:00Z

Human prostate cancer (PC) overexpresses the gastrin-releasing peptide receptor (GRPR). Radiolabeled GRPR-targeting analogs of bombesin (BN) have successfully been introduced as potential tracers for visualization and treatment of GRPR-overexpressing tumors. A previous study showed GRPR-mediated binding of radiolabeled BN analogs in androgen-dependent but not in androgen-independent xenografts representing the more advanced stages of PC. We have further investigated the effect of androgen modulation on GRPR-expression in three androgen-dependent human PC-bearing xenografts: PC295, PC310 and PC82 using the androgen-independent PC3-model as a reference. Effects of androgen regulation on GRPR expression were initially studied on tumors obtained from our biorepository of xenograft tissues performing reverse transcriptase polymerase chain reaction (RT-PCR) and autoradiography (125I-universal-BN). A prospective biodistribution study (111In-MP2653) and subsequent autoradiography (125I-GRP and111In-MP2248) was than performed in castrated and testosterone resupplemented tumor-bearing mice. For all androgen-dependent xenografts, tumor uptake and binding decreased drastically after 7 days of castration. Resupplementation of testosterone to castrated animals restored GRPR expression extensively. Similar findings were concluded from the initial autoradiography and RT-PCR studies. Results from RT-PCR, for which human specific primers are used, indicate that variations in GRPR expression can be ascribed to mRNA downregulation and not to castration-induced reduction in the epithelial fraction of the xenograft tumor tissue. In conclusion, expression of human GRPR in androgendependent PC xenografts is reduced by androgen ablation and is reversed by restoring the hormonal status of the animals. This knowledge suggests that hormonal therapy may affect GRPR expression in PC tissue making GRPR-based imaging and therapy especially suitable for non-hormonally treated PC patients.

Nephrotoxicity in mice after repeated imaging using 111In- labeled peptides (Article)

2010-06-01T00:00:00Z

We determined the renal radiation dose of a series of111Inlabeled peptides using animal SPECT. Because the animals' health deteriorated, renal toxicity was assessed. Methods: Wild-type and megalin-deficient mice were imaged repeatedly at 3- to 6-wk intervals to quantify renal retention after injection of 40-50 MBq of111In- diethylenetriaminepentaacetic acid-labeled peptides (octreotide, exendin, octreotate, neurotensin, and minigastrin analogs), and the absorbed kidney radiation doses were estimated. Body weight, renal function parameters, and renal histology were determined at 16-20 wk after the first scan and compared with those in naive animals. Results: Because of high renal retention,111In-diethylenetriaminepentaacetic acid-exendin-4 scans resulted in a 70-Gy kidney radiation dose in wild-type mice. Megalin-deficient kidneys received 20-40 Gy. The other peptides resulted in much lower renal doses. Kidney function monitoring indicated renal damage in imaged animals. Conclusion: Micro-SPECT enables longitudinal studies in 1 animal. However, long-term nephrotoxic effects may be induced after high renal radiation doses, even with111In-labeled radiotracers. COPYRIGHT

Preclinical and Clinical Studies of Peptide Receptor Radionuclide Therapy (Article)

2010-05-01T00:00:00Z

In the 1980s, the111In-labeled somatostatin analog OctreoScan (Covidien, Hazelwood, MO) was developed for imaging of somatostatin receptor subtype 2 (sst2) overexpressing tumors. On the basis of this success, peptide receptor radionuclide therapy (PRRT) was developed using similar somatostatin analogs with different therapeutic radionuclides. Clinical application of PRRT demonstrated impressive results on tumor response, overall survival, and quality of life in patients with gastroenteropancreatic neuroendocrine tumors. The peptides 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), Tyr3-octreotate (DOTATATE) and DOTA, Tyr3-octreotide (DOTATOC) (brand name Onalta), predominantly targeting sst2, have been granted Orphan Drug status by the European Medicines Agency and the US Food and Drug Administration for application in PRRT. Besides somatostatin receptor-targeting peptides, multiple other radiopeptide analogs were developed targeting several other receptors overexpressed on various tumors. Some of these peptide analogs, including cholecystokinin, gastrin, gastrin-releasing peptide, arginine-glycine-aspartate (RGD)-peptides, and glucagon-like peptide 1 analogs appeared very promising in preclinical and clinical imaging and PRRT studies. Although the success of PRRT with radiolabeled somatostatin analogs has been established, there is still room for improvement. The therapeutic window of PRRT could be enlarged by the use of new and improved targeting compounds, of which new antagonists with excellent tumor to background ratios are very promising. Furthermore, locoregional administration, improved healthy tissue protection, and combination treatment can be applied to increase the effectiveness of PRRT. Combination treatment might include cocktails of different peptide analogs of different therapeutic radionuclides and of radiolabeled peptides with chemotherapeutic or radiosensitizing agents. This review summarizes results of PRRT and describes clinical and preclinical studies regarding PRRT optimizing strategies.

Kidney protection during peptide receptor radionuclide therapy with somatostatin analogues (Article)

2010-05-01T00:00:00Z

This review focuses on the present status of kidney protection during peptide receptor radionuclide therapy (PRRT) using radiolabelled somatostatin analogues. This treatment modality for somatostatin receptor-positive tumours is limited by renal reabsorption and retention of radiolabelled peptides resulting in dose-limiting high kidney radiation doses. Radiation nephropathy has been described in several patients. Studies on the mechanism and localization demonstrate that renal uptake of radiolabelled somatostatin analogues largely depends on the megalin/cubulin system in the proximal tubule cells. Thus methods are needed that interfere with this reabsorption pathway to achieve kidney protection. Such methods include coadministration of basic amino acids, the bovine gelatin-containing solution Gelofusine or albumin fragments. Amino acids are already commonly used in the clinical setting during PRRT. Other compounds that interfere with renal reabsorption capacity (maleic acid and colchicine) are not suitable for clinical use because of potential toxicity. The safe limit for the renal radiation dose during PRRT is not exactly known. Dosimetry studies applying the principle of the biological equivalent dose (correcting for the effect of dose fractionation) suggest that a dose of about 37 Gy is the threshold for development of kidney toxicity. This threshold is lower when risk factors for development of renal damage exist: age over 60 years, hypertension, diabetes mellitus and previous chemotherapy. A still experimental pathway for kidney protection is mitigation of radiation effects, possibly achievable by cotreatment with amifostine (Ethylol), a radiation protector, or with blockers of the renin-angiotensin-aldosterone system. Future perspectives on improving kidney protection during PRRT include combinations of agents to reduce renal retention of radiolabelled peptides, eventually together with mitigating medicines. Moreover, new somatostatin analogues with lower renal retention may be developed. Furthermore, knowledge on kidney protection from radiolabelled somatostatin analogues may be expanded to other peptides.

Of mice and humans: Are they the same? - Implications in cancer translational research (Article)

2010-04-01T00:00:00Z

Animal models have been instrumental in elucidating key biochemical and physiologic processes of cancer onset and propagation in a living organism. Most importantly, they have served as a surrogate for patients in the evaluation of novel diagnostic and therapeutic anticancer drugs, including radiopharmaceuticals. Experimental tumors raised in rodents constitute the major preclinical tool of new-agent screening before clinical testing. Such models for oncologic applications today include solid tumors raised in syngeneic fully immunocompetent hosts and human xenografts induced in immunodeficient mouse strains, and tumors spontaneously growing in genetically engineered mice represent the newest front-line experimental modality. The power of these models to predict clinical efficacy is a matter of dispute, as each model presents inherent strengths and weaknesses in faithfully mirroring the extremely complex process of human carcinogenesis. Differences in size and physiology, as well as variations in the homology of targets between mice and humans, may lead to translational limitations. Other factors affecting the predictive power of preclinical models may be animal handling during experimentation and suboptimal compilation and interpretation of preclinical data. However, animal models will remain a unique source of in vivo information and the irreplaceable link between in vitro studies and our patients. COPYRIGHT

Short-term dietary restriction and fasting precondition against ischemia reperfusion injury in mice (Article)

2010-02-01T00:00:00Z

Dietary restriction (DR) extends lifespan and increases resistance to multiple forms of stress, including ischemia reperfusion injury to the brain and heart in rodents. While maximal effects on lifespan require long-term restriction, the kinetics of onset of benefits against acute stress is not known. Here, we show that 2-4 weeks of 30% DR improved survival and kidney function following renal ischemia reperfusion injury in mice. Brief periods of water-only fasting were similarly effective at protecting against ischemic damage. Significant protection occurred within 1 day, persisted for several days beyond the fasting period and extended to another organ, the liver. Protection by both short-term DR and fasting correlated with improved insulin sensitivity, increased expression of markers of antioxidant defense and reduced expression of markers of inflammation and insulin/insulin-like growth factor-1 signaling. Unbiased transcriptional profiling of kidneys from mice subject to short-term DR or fasting revealed a significant enrichment of signature genes of long-term DR. These data demonstrate that brief periods of reduced food intake, including short-term daily restriction and fasting, can increase resistance to ischemia reperfusion injury in rodents and suggest a rapid onset of benefits of DR in mammals. © 2010 The Authors. Journal compilation

Albumin-derived peptides efficiently reduce renal uptake of radiolabelled peptides (Article)

2010-02-01T00:00:00Z

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 of111In-albumin,111In-minigastrin,111In-exendin and111In- octreotide by megalin-expressing cells was assessed. In rats, the effect of Gelofusine and albumin-derived peptides on the renal uptake and biodistribution of111In-minigastrin,111In-exendin and111In-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 of111In-albumin,111In-exendin and111In- 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 of111In-minigastrin, by 88%. Uptake of111In-exendin and111In-octreotide was reduced by 26 and 33%, respectively. Conclusions: The albumin-derived peptide #6 efficiently inhibited the renal reabsorption of111In-minigastrin,111In-exendin and111In-octreotide and is a promising candidate for kidney protection in PRRT.

Dose-response effect of Gelofusine on renal uptake and retention of radiolabelled octreotate in rats with CA20948 tumours (Article)

2009-12-01T00:00:00Z

Purpose: Peptide receptor radionuclide therapy using β-emitting radiolabelled somatostatin analogues like DOTA,Tyr3-octreotate shows beneficial results in patients suffering from somatostatin receptor overexpressing tumours. However, after high-dose therapy partial renal reabsorption of radiopeptides may lead to nephrotoxicity. Co-infusion of lysine/arginine lowers renal retention of these radiopeptides without affecting tumour uptake. Recently co-administration of Gelofusine has been described to have a comparable kidney-protecting effect in rats. In the present study optimal dosing of Gelofusine co-administration was studied in tumour-bearing rats. Methods: Doses of 40, 80, 120 or 160 mg/kg Gelofusine were co-injected with 15 μg DOTA,Tyr3-octreotate, labelled with 3 MBq111In for biodistribution (24 h post-injection, n=4 per group) and with 60 MBq111In for microSPECT imaging experiments at 3, 24 and 48 h post-injection. An additional group of rats received 80 mg/kg Gelofusine plus 400 mg/kg lysine co-injection. Biodistribution studies were performed both in older (475 g) and younger (300 g) rats, the latter bearing CA20948 tumours. Results: Co-injection of 40 mg/kg Gelofusine resulted in 40-50% reduction of renal uptake and retention of111In-DOTA,Tyr3-octreotate, whereas higher doses further increased the reduction to 50-60% in both groups of rats. Combining Gelofusine and lysine caused 70% reduction of renal uptake. The uptake of radiolabelled octreotate both in somatostatin receptor-expressing normal tissues and tumours was not affected by Gelofusine co-injection. Conclusion: In rats co-injection of 80 mg/kg Gelofusine resulted in maximum reduction of renal retention of111In-DOTA,Tyr3- octreotate, which was further improved when combined with lysine. Tumour uptake of radiolabelled octreotate was not affected, resulting in an increased tumour to kidney ratio.

Imaging of activated macrophages in experimental osteoarthritis using folate targeted animal SPECT/CT (Article)

2009-09-09T00:00:00Z

Objective. Evaluation of macrophage activation may provide essential information about aetiology and progression rate of osteoarthritis. Activated macrophages abundantly express the folate-receptor-beta (FR-β), which can be targeted using radioactive labelled folic acid. The purpose of this study was to investigate if macrophage activation can be monitored in small animal OA models using a folate radiotracer and to test if macrophage activation differs in different models of OA and subsequent different OA progression. Methods. Two rat models of OA were used: the monoiodoacetate (MIA) model, which is a fast progressing biochemical induced model and the anterior cruciate ligament transaction (ACLT) model that induces OA at a slower pace. Images were obtained using high resolution small animal SPECT/CT. Specificity of the technique was tested by eradicating macrophages using clodronate laden liposomes and blockade of the FR-β by cold folic acid. Results. The MIA model had a high initial activation with a peak after two weeks which disappeared after eight weeks. The ACLT model showed less activation but was still active 12 weeks after induction. The technique allowed monitoring of the disease process over time, in which late stage disease showed less macrophage activation than early onset stages especially in the fast progressing MIA model for OA. Conclusion. Macrophage activation in experimental OA could clearly be demonstrated and monitored by the folate radiotracer. The high resolution, high sensitivity and high specificity of the used technique allowed clear localisation of macrophage activity in a disease model, which is not known for abundant macrophage involvement.

Bone marrow dosimetry in peptide receptor radionuclide therapy with [ 177Lu-DOTA0,Tyr3]octreotate (Article)

2009-07-01T00:00:00Z

Purpose: Adequate dosimetry is mandatory for effective and safe peptide receptor radionuclide therapy (PRRT). Besides the kidneys, the bone marrow is a potentially dose-limiting organ. The radiation dose to the bone marrow is usually calculated according to the MIRD scheme, where the accumulated activity in the bone marrow is calculated from the accumulated radioactivity of the radiopharmaceutical in the blood. This may underestimate the absorbed dose since stem cells express somatostatin receptors. We verified the blood-based method by comparing the activity in the blood with the radioactivity in bone marrow aspirates. Also, we evaluated the absorbed cross-dose from the source organs (liver, spleen, kidneys and blood), tumours and the so-called "remainder of the body" to the bone marrow. Methods: Bone marrow aspirates were drawn in 15 patients after treatment with [177Lu-DOTA0,Tyr3]octreotate. Radioactivity in the bone marrow was compared with radioactivity in the blood drawn simultaneously. The nucleated cell fraction was isolated from the bone marrow aspirate and radioactivity was measured. The absorbed dose to the bone marrow was calculated. The results were correlated to the change in platelet counts 6 weeks after treatment. Results: A strong linear correlation and high agreement between the measured radioactivities in the bone marrow aspirates and in the blood was found (r=0.914, p<0.001). No correlation between the calculated absorbed dose in the bone marrow and the change in platelets was found. There was a considerable contribution from other organs and the remainder of the body to the bone marrow absorbed dose. Conclusion: (1) After PRRT with [177Lu-DOTA0,Tyr3]octreotate, the radioactivity concentration in the bone marrow is identical to that in the blood; (2) There is no significant binding of the radiopharmaceutical to bone marrow precursor stem cells; (3) The contribution of the cross dose from source organs and tumours to the bone marrow dose is significant; and (4) There is considerable variation in bone marrow absorbed dose between patients. These findings imply that for individual dose optimization, individual calculation of the bone marrow absorbed dose is necessary.

Peptide-receptor radionuclide therapy for endocrine tumors (Article)

2009-07-01T00:00:00Z

Peptide-receptor radionuclide therapy (PRRT) with radiolabeled somatostatin analogs is a promising option for the treatment of somatostatin-receptor- positive endocrine tumors. Treatment with somatostatin analogs labeled with 111 In, 90 Y or 177 Lu can result in symptomatic improvement, although tumor remission is seldom achieved with 111 In-labeled analogs. In this Review, the findings of several studies on the use of PRRT for endocrine tumors are evaluated. Large variation in the antitumor effects of 90 Y-octreotide was reported between studies: an objective response (50% tumor regression) was achieved in 9-33% of patients. After treatment with 177 Lu-octreotate, an objective response was achieved in 29% of patients and a minor response (25-50% tumor regression) was achieved in 16% of patients; stable disease was present in 35% of patients. Treatment with 177 Lu-octreotate resulted in a survival benefit of several years and markedly improved quality of life. Serious, delayed adverse effects were rare after PRRT. Although randomized, clinical trials have not yet been performed, data on the use of PRRT compare favorably with those from other treatment approaches, such as chemotherapy. If these results can be replicated in large, controlled trials, PRRT might become the preferred option in patients with metastatic or inoperable gastroenteropancreatic neuroendocrine tumors.

Evaluation of a novel radiofolate in tumour-bearing mice: Promising prospects for folate-based radionuclide therapy (Article)

2009-06-01T00:00:00Z

Purpose: Folate-based radiopharmaceuticals have the potential to be used for imaging and therapy of tumours positive for the folate receptor (FR). We describe the in vitro and in vivo evaluation of a DOTA-folate conjugate. Methods: Radiolabelling of the DOTA-folate was carried out via standard procedures using111InCl3and177LuCl3, respectively. The distribution coefficient (log D) was determined in octanol/PBS (pH 7.4). Tissue distribution was investigated in nude mice bearing KB tumour xenografts at different time points after administration of111In-DOTA-folate (radiofolate 1) or177Lu-DOTA-folate (radiofolate 2) (1 MBq, 1 nmol per mouse). Pemetrexed (PMX, 400 μg) was injected 1 h prior to the radiofolate in order to reduce renal uptake. Images were acquired with a SPECT/CT camera 24 h after injection of the radiofolate (40-50 MBq, 3 nmol per mouse). Results: The hydrophilic character of the DOTA-folate was represented by a low log D value (radiofolate 1 -4.21±0.11). In vivo, maximal tumour uptake was found 4 h after injection (radiofolate 1 5.80±0.55% ID/g; radiofolate 2 7.51±1.25% ID/g). In FR-positive kidneys there was considerable accumulation of the radiofolates (radiofolate 1 55.88±3.91% ID/g; radiofolate 2 57.22±11.05% ID/g; 4 h after injection). However, renal uptake was reduced by preinjection of PMX (radiofolate 1 9.52±1.07% ID/g; radiofolate 2 13.43±0.54% ID/g; 4 h after injection) whereas the tumour uptake was retained (radiofolate 1 6.32±0.41% ID/g; radiofolate 2 8.99±0.43% ID/g; 4 h after injection). SPECT/CT images clearly confirmed favourable tissue distribution of the novel radiofolates and the positive effect of PMX. Conclusion: The preliminary requirements for the therapeutic use of the novel DOTA-folate are met by its favourable tissue distribution that can be ascribed to its hydrophilic properties and combined administration with PMX.

Peptide receptor imaging of prostate cancer with radiolabelled bombesin analogues (Article)

2009-06-01T00:00:00Z

Prostate Cancer (PC) is a type of cancer that is often diagnosed at very early stages due to improved detection among man in the Western world. Current imaging techniques are not optimal to determine extent of minimal early stage PC even though this is of great clinical importance. Human PC and high-grade PIN have shown high Gastrin-Releasing Peptide Receptor (GRPR) expression, while normal prostate tissue and BPH revealed to be predominantly GRPR-negative. Radiolabelled Gastrin-Releasing Peptide (GRP) or bombesin (BN) analogues targeting the GRPR can be used as non-invasive tools to diagnose, monitor and potentially treat PC. These BN analogues have already proven to be able to image PC in both tumour-bearing mice and clinical patients showing no important side effects. It's desirable that new peptides require fast-track standardised comparative testing in relevant PC models to select the best performing BN analogues for further evaluation in patients. Although knowledge about GRPR expression and development of new BN analogues can be extended, it is time to study performance of BN analogues for peptide receptor based imaging in patients validating results of PC imaging using histopathology as a golden standard.

Comparison of three radiolabelled peptide analogues for CCK-2 receptor scintigraphy in medullary thyroid carcinoma (Article)

2009-03-06T00:00:00Z

Purpose: Cholecystokinin 2 (CCK-2) receptor overexpression has been demonstrated in a high percentage of medullary thyroid carcinomas (MTC). Analogous to somatostatin receptors, CCK-2 receptors might be viable targets for radionuclide scintigraphy and/or radionuclide therapy. Several CCK-2 receptor-binding radiopeptides have been developed, and some have been carried through into clinical studies. However, these studies are mostly limited and difficult to compare. The aim of this study was to evaluate the diagnostic and therapeutic potential of three promising CCK-2 receptor-binding radiopeptides in patients with MTC. Methods: 111In-DOTA-(D)Asp-Tyr-Nle-Gly-Trp-Nle- Asp-Phe-NH2 (111In-DOTA-CCK), a CCK analogue, and the gastrin-based ligands 99mTc-N4-Gly-(D)Glu-(Glu) 5-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (99mTc- demogastrin 2) and 111In-DOTA-(D)Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe- NH2 (111In-DOTA-MG11) were each administered to the same group of six patients. Planar images made at 3-5, 7 and 24 h p.i. were used for comparison of tumour visualisation and renal uptake. Results: 99mTc-demogastrin 2 scintigraphy visualised all known lesions and new lesions in four of six patients. 111In-DOTA-CCK and 111In-DOTA-MG11 on the other hand missed several lesions; tumour uptake of these two radiopharmaceuticals was quite low. Comparison of retention of renal activity showed no major differences between the three radiopeptides. Conclusion: 99mTc-demogastrin 2 scintigraphy appeared most promising as a diagnostic tool in patients with MTC. Further studies are required to evaluate its value in patient management. Direct comparisons of the compounds studied strongly suggests that 111In-DOTA-CCK and 111In-DOTA-MG11 have less potential as imaging agents than 99mTc-demogastrin 2. These DOTA-linked compounds are considered unlikely to be useful for radionuclide therapy because of low tumour uptake.

Optimised labeling, preclinical and initial clinical aspects of CCK-2 receptor-targeting with 3 radiolabeled peptides (Article)

2008-11-01T00:00:00Z

Medullary thyroid carcinoma (MTC) expresses CCK-2 receptors.111In-labeled DOTA-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2(DOTA-MG11), DOTA-DAsp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2(DOTA-CCK), and99mTc-labeled N4-Gly-DGlu-(Glu)5-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2(99mTc-Demogastrin 2) are analogs developed for CCK-2 receptor-targeted scintigraphy. All 3 radiolabeled analogs were selected on the basis of their high CCK-2 receptor affinity and their good in vitro serum stability, with in vitro serum t1/2values of several hours. Radiolabeling of DOTA-peptides with111In requires a heating procedure, typically in the range of 80°-100°C up to 30 min. Following this procedure with DOTA-MG11 resulted in a >98 % incorporation of111In, however, with a radiochemical purity (RCP) of <50 %. The decrease in RCP was found to be due to oxidation of the methionine residue in the molecule. Moreover, this oxidized compound lost its CCK-2 receptor affinity. Therefore, conditions during radiolabeling were optimised: labeling of DOTA-MG11 and DOTA-CCK with111In involved 5 min heating at 80°C and led to an incorporation of111In of >98 %. In addition, all analogs were radiolabeled in the presence of quenchers to prevent radiolysis and oxidation resulting in a RCP of >90 %. All 3 radiolabeled analogs were i.v. administered to 6 MTC patients: radioactivity cleared rapidly by the kidneys, with no significant differences in the excretion pattern of the 3 radiotracers. All 3 radiolabeled analogs exhibited a low in vivo stability in patients, as revealed during analysis of blood samples, with the respective t1/2found in the order of minutes. In patient blood, the rank of radiopeptide in vivo stability was:99mTc-Demogastrin 2 (t1/210-15 min)>111In-DOTA-CCK (t1/2≈5-10 min)>111In-DOTA-MG11 (t1/2<5 min).

Expression of the SST receptor 2 in uveal melanoma is not a prognostic marker (Article)

2008-08-07T00:00:00Z

Introduction: Uveal melanoma (UM) cells and neurohormone-producing cells both originate from the neural crest. Somatostatin receptors subtype 2 (SSTR2) are over-expressed in several tumors, often from neuroendocrine origin, and synthetic antagonists like octreotide and octreotate are being used as diagnostic or therapeutic agents. We investigated the SSTR2 expression in UM, and determined whether this expression was related to prognosis of the disease. Materials and methods: UM cell lines and fresh primary UM samples were tested for SSTR2 expression by autoradiography (AR) using 125I-Tyr3-octreotate. Furthermore, UM cell lines were analyzed for SSTR2 mRNA expression with quantitative real-time RT-PCR. Results: Using AR, cell-surface SSTR2 expression was demonstrated in two UM metastatic cell lines, but no expression was detected in three cell lines derived from primary UM. However, all primary and metastatic UM cell lines showed mRNA expression levels for SSTR2 using quantitative real-time RT-PCR. Only three of 14 primary UM demonstrated moderate SSTR2 expression, and this expression was not significantly associated with tumor-free survival or any tested prognostic factor. Conclusions: Based on the rare and low expression of SSTR2 found in primary UM specimens and in UM cell lines, we conclude that SSTR2 is not widely expressed in UM. Furthermore, SSTR2 expression was not associated with tumor-free survival and prognostic factors. Therefore SSTR2 is not suited as prognostic marker or therapeutic target in UM.

Pemetrexed improves tumor selectivity of 111In-DTPA-folate in mice with folate receptor-positive ovarian cancer (Article)

2008-04-01T00:00:00Z

Folate-based radiopharmaceuticals can be used as imaging agents and for potential radiotherapy of folate receptor (FR)-positive malignant tissue (e.g., ovarian carcinomas). However, substantial FR expression in the kidneys results in undesired renal retention of radioactivity. Recently, we found that the preinjection of an antifolate significantly improved tumor selectivity of organometallic99mTc-radiofolates in mice. The aim of this study was to corroborate the effect of pemetrexed with the clinically tested111In-DTPA-folate (DTPA is diethylenetriaminepentaacetic acid) in a human ovarian cancer xenografted mouse model. Methods: In vivo studies were performed in female athymic nude mice bearing subcutaneous FR-positive ovarian tumors (IGROV-1 and SKOV-3) or metastases (after intraperitoneal SKOV-3 cell inoculation). Biodistribution studies were performed 1, 4, and 24 h after administration of111In-DTPA-folate (0.7 MBq/mouse, 0.35 μg) with or without preinjection of pemetrexed (PMX, 400 mg) 1 h before the radiofolate. Images were acquired with a high-resolution, high-sensitivity SPECT/CT camera, 4 and 24 h after injection of the radiotracer (30-50 MBq/ mouse, 4.5-10 μg). Results: In biodistribution studies the tumor uptake of111In-DTPA- folate (IGROV-1: 9.79 ± 3.21 %ID/g [percentage injected dose per gram]; SKOV-3: 7.57 ± 0.61 %ID/g, 4 h after injection) was high and retained over the time of investigation. However, considerable retention of radioactivity was found in kidneys (85-105 %ID/g, 4 h after injection), resulting in unfavorably low tumor-to-kidney ratios (∼0.10). Preinjection of PMX resulted in a significant reduction of renal uptake (20%-30% of control values, P < 0.03) at all time points after injection of111In-DTPA-folate, whereas the tumor uptake was retained. Thus, the tumor-to-kidney ratio was significantly increased to ∼0.50. SPECT/CT images confirmed the superior tumor-to-background ratio in mice injected with PMX. These findings were particularly evident in mice with SKOV-3 metastases that could be visualized only when111In-DTPA-folate was administered in combination with PMX. Conclusion: The application of PMX resulted in a significant reduction of undesired radioactivity accumulation in kidneys, whereas the tumor uptake remained unaffected. These observations suggest a general validity of the reducing effect of PMX on the uptake of radiofolates in kidneys. Our findings will lead the way toward the development of folate-based radiotherapy. Copyright

Update: Improvement strategies for peptide receptor scintigraphy and radionuclide therapy (Article)

2008-04-01T00:00:00Z

Somatostatin receptor-targeting peptides are widely used for the imaging and therapy of neuroendocrine tumors. Peptide-receptor radionuclide therapy (PRRT) in neuroendocrine tumor patients with radiolabeled somatostatin analogs has resulted in symptomatic improvement, prolonged survival, and enhanced quality of life. The side-effects of PRRT are few and mostly mild, certainly when using kidney protective agents. If a more widespread use of PRRT is possible, such therapy might become the therapy of first choice in patients with metastasized or inoperable neuroendocrine gastroenteropancreatic tumors. Yet, much profit can be gained from improving the receptor-targeting strategies available and developing new strategies. This review presents an overview of several options to optimize receptor-targeted imaging and radionuclide therapy. These include the optimization of peptide analogs, increasing the number of receptors on the tumor site, and combining PRRT with other treatment strategies. The development of new peptide analogs with increased receptor-binding affinity and improved stability might lead to a higher accumulation of radioactivity inside tumor cells. Analogs of somatostatin have been widely studied. However, much profit can be gained in improving peptide analogs targeting other tumor-related receptors, including gastrin-releasing peptide (GRP) receptors, neurotensin (NT) receptors, cholecystokinin (CCK) receptors, and glucagon-like peptide-1 (GLP-1) receptors. Several peptide analogs targeting these receptors are well on their way to clinical utilization. The literature shows that it is possible to increase the receptor density on tumor cells by using different methods, which results in higher binding and internalization rates and thus a higher contrast during peptide-receptor scintigraphy. In PRRT treatment, this would enable the administration of higher therapeutic doses to tumors, which might lead to a higher cure rate in patients. Combinations of radionuclide therapy with other treatment modalities, such as chemotherapy or pretreatment with radiosensitizers, might increase the impact of the treatment. Further, the administration of higher dosages of radioactivity to the patient, enabled by combinations of PRRT with strategies reducing the radiation dose to healthy organs, will improve the outcome of tumor treatment. Also, targeting one or several tumor-specific receptors by using combinations of therapeutic agents, as well as by reducing nontarget uptake of radioactivity, will enlarge the therapeutic window of PRRT. Clinical studies will provide more insight in the effects of combining treatment strategies in cancer patients.

SPECT study of folate receptor-positive malignant and normal tissues in mice using a novel 99mTc-radiofolate (Article)

2008-02-01T00:00:00Z

The folate receptor (FR) is overexpressed on epithelial cancers (FR-α) and on activated, but not resting, macrophages (FR-β) involved in a variety of inflammatory and autoimmune diseases. Therefore, folate-based radiopharmaceuticals have the potential to be used as imaging agents of FR-positive tumor and inflammatory cells. In this study SPECT/CT of FR-positive malignant and normal tissues and organs in mice was performed using an improved organometallic99mTc-radiofolate. Methods: The99mTc radiolabeling of the histidine-folate was performed using the tricarbonyl technique described earlier for preparation of other organometallic radiofolates. Nude male mice with FR-positive KB tumor xenografts were used. Biodistribution studies were performed 1, 4, and 24 h after injection of the99mTc-Hisfolate (1.5 MBq/mouse). Images were acquired with a dedicated small-animal SPECT/CT camera 24 h after injection of the radiofolate (500 MBq/mouse). Ex vivo autoradiography was performed on tumors and FR-positive normal tissues. Adjacent sections were used for in vitro autoradiography of FRs after decay of the injected radioactivity. Results: The SPECT/CT studies revealed accumulation of the radiotracer in FR-positive KB tumor xenografts and kidneys as reported previously. At the same time, specific uptake of the radiofolate in normal tissues - that is, salivary glands and choroid plexus - could be visualized with SPECT. FR-specific accumulation in these tissues and organs was confirmed by coinjection of excess folic acid, which resulted in a complete blockade of radiofolate uptake. In addition, ex vivo and in vitro autoradiography of these organs and tissues confirmed FR expression and displayed radioactivity distribution patterns almost identical to those found on SPECT images. In biodistribution studies we found a high tumor uptake (4.29 ± 0.67 %ID/g [percentage of the injected dose per gram of tissue], 4 h after injection) that was almost completely retained over time (3.51 ± 0.37 %ID/g, 24 h after injection). Conclusion: The novel99mTc- histidine-folate showed improved in vivo characteristics compared with other organometallic radiofolates that allowed imaging of FR-positive malignant (KB tumor xenografts) and kidneys. For the first time, to our knowledge, specific tracer uptake in salivary glands and the choroid plexus could be visualized using a high-resolution animal SPECT/CT camera. Copyright

Targeting of a CCK2 receptor splice variant with 111In-labelled cholecystokinin-8 (CCK8) and 111In-labelled minigastrin (Article)

2008-02-01T00:00:00Z

Purpose: Radiolabelled cholecystokinin (CCK) and gastrin-derived peptides potentially can be used for peptide receptor radionuclide therapy (PRRT). Recently, a splice variant version of the CCK2R has been identified, designated CCK2i4svR. Constitutive expression of this receptor has been demonstrated in human colorectal cancer and in pancreatic cancer, but not in normal tissue. So far, it has never been shown whether radiolabelled peptides can target the CCK2i4svR in vivo. In this paper, we investigated the potential of sulfated111In-labelled DOTA-CCK8 (sCCK8), a pan-CCKR-binding peptide, and [111In]DOTA-minigastrin (MG0), a CCK2R selective peptide, for the targeting of the CCK2i4svR. Materials and methods: The receptor binding affinity of [111In]DOTA-sCCK8 and [111In]DOTA-MG0 for the CCK2R and CCK2i4svR was determined using stably transfected HEK293 cell lines, expressing either CCK2R or CCK2i4svR. Tumour targeting was studied in HEK293-CCK2i4svR tumour-bearing athymic mice. Results: [111In]DOTA- sCCK8 as well as [111In]DOTA-MG0 specifically bound both CCK2R and CCK2i4svR with affinities in the low nanomolar range. In vivo experiments revealed that accumulation of both peptides in CCK2i4svR-positive tumours was similar (3.21±0.77 and 3.01±0.67%ID/g, sCCK8 and MG0, respectively, 24 h p.i.). Kidney retention of [111In]DOTA-MG0 (32.4±7.5%ID/g, 24 h p.i.) was markedly higher than that of [111In]DOTA-sCCK8 (2.75±0.31%ID/g, 24 h p.i.). Conclusion: We demonstrated that the CCK2i4svR is a potential target for PRRT using a radiolabelled sulfated CCK8 peptide. As this receptor is expressed on colorectal and pancreatic tumours, but not in normal tissue, these tumours are potentially new targets for PRRT with CCK8 and gastrin analogs.

Targeting malignant gliomas with a glial fibrillary acidic protein (GFAP)-selective oncolytic adenovirus (Article)

2007-12-01T00:00:00Z

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein abundantly expressed in malignant gliomas. We have constructed a novel oncolytic adenovirus, Ad5-gfa2(B)3-E1, for treatment of these tumors. In this construct, the E1 region is under control of the tissue-specific GFAP promoter (gfa2) with three additional copies of the glial specific 'B' enhancer. Infection of a GFAP-positive cell line with Ad5-gfa2(B)3-E1 resulted in E1A and E1B expression at 75% and 30% of the levels obtained after wtAd5 infection. Q-PCR showed that Ad5-gfa2(B)3-E1 replicated 4.5 times more efficiently in the GFAP-positive than in the GFAP-negative cell lines. Cell viability assays showed efficient elimination of GFAP-positive cells by Ad5-gfa2(B)3-E1, in some cell lines as efficiently as wtAd5, while the elimination was attenuated in GFAP-negative cell lines. When tested in human tumor xenografts in nude mice, Ad5-gfa2(B)3-E1 effectively suppressed the growth of GFAP-positive SNB-19 glial tumors but not of GFAP-negative A549 lung tumors. In Ad5-gfa2(B)3-E1, the E3 region was deleted to create space for future insertion of heterologous therapeutic genes. Experiments with d17001, an E3-deleted variant of wtAd5, confirmed that the specificity of Ad5-gfa2(B)3-E1 replication was based on the promoter driving E1 and not on the E3 deletion. Strategies to further improve the efficacy of Ad5-gfa2(B)3-E1 for the treatment of malignant gliomas include the insertion of therapeutic genes in E3 or retargeting to receptors that are more abundantly expressed on primary glioma cells than CAR. Copyright

Somatostatin receptor subtype 2-mediated uptake of radiolabelled somatostatin analogues in the human kidney (Article)

2007-11-01T00:00:00Z

Purpose: Renal irradiation is a dose-limiting factor in peptide receptor radionuclide therapy using radiolabelled somatostatin analogues. This irradiation is mainly caused by reabsorption of radiolabelled peptides in the proximal tubule. In the human kidney, somatostatin receptors are expressed in the vasa recta, tubuli and glomeruli. It is not clear to what extent these receptors contribute to the total kidney radioactivity uptake. Methods: Retrospectively, [111In-DTPA0]octreotide scans of ten selected patients with carcinoids (well-differentiated gastrointestinal endocrine tumour) with liver metastases were evaluated. For each patient, two scans were obtained: one scan was performed without (control) and one during treatment with unlabelled octreotide. Kidney, tumour, spleen and liver uptake was measured in both scans. Results: The interval between the two scans per patient varied from 50 to 397 days. Octreotide treatment substantially lowered kidney [111In-DTPA0]octreotide uptake in eight out of ten patients. Kidney uptake in all patients was reduced to 82%±15% of control, (p<0.01). A correlation between kidney uptake and spleen uptake was found (r=0.67, p<0.05). Serum creatinine was unchanged. Surprisingly, tumour and liver [111In-DTPA0]octreotide uptake was not significantly influenced by unlabelled octreotide therapy, but spleen uptake was significantly lowered by treatment (30.6% of control, p<0.002). Conclusion: We conclude that the somatostatin receptor plays a role in the total renal uptake of radiolabelled somatostatin analogues. The long interval between scans might explain the finding that tumour and liver metastasis uptake of [111In-DTPA0]octreotide was unchanged. Further studies are needed to confirm and eludicate the results of this study.

Peptide Receptor Radionuclide Therapy with radiolabelled somatostatin analogues in patients with somatostatin receptor positive tumours (Article)

2007-08-01T00:00:00Z

Peptide Receptor Radionuclide Therapy (PRRT) with radiolabelled somatostatin analogues is a promising treatment option for patients with inoperable or metastasised neuroendocrine tumours. Symptomatic improvement may occur with all of the various 111In, 90Y, or 177Lu-labelled somatostatin analogues that have been used. Since tumour size reduction was seldom achieved with 111Indium labelled somatostatin analogues, radiolabelled somatostatin analogues with beta-emitting isotopes like 90Y and 177Lu were developed. Reported anti-tumour effects of [90Y-DOTA0,Tyr3]octreotide vary considerably between various studies: Tumour regression of 50% or more was achieved in 9 to 33% (mean 22%). With [177Lu-DOTA0,Tyr3]octreotate treatments, tumour regression of 50% or more was achieved in 28% of patients and tumour regression of 25 to 50% in 19% of patients, stable disease was demonstrated in 35% and progressive disease in 18%. Predictive factors for tumour remission were high tumour uptake on somatostatin receptor scintigraphy and limited amount of liver metastases. The side-effects of PRRT are few and mostly mild, certainly when using renal protective agents: Serious side-effects like myelodysplastic syndrome or renal failure are rare. The median duration of the therapy response for [90Y-DOTA0,Tyr3]octreotide and [177Lu-DOTA0,Tyr3]octreotate is 30 months and more than 36 months respectively. Lastly, quality of life improves significantly after treatment with [177Lu-DOTA0,Tyr3]octreotate. These data compare favourably with the limited number of alternative treatment approaches, like chemotherapy. If more widespread use of PRRT is possible, such therapy might become the therapy of first choice in patients with metastasised or inoperable gastroenteropancreatic neuroendocrine tumours. Also the role in somatostatin receptor expressing non-GEP tumours, like metastasised paraganglioma/ pheochromocytoma and non-radioiodine-avid differentiated thyroid carcinoma might become more important.

Renal uptake and retention of radiolabeled somatostatin, bombesin, neurotensin, minigastrin and CCK analogues: species and gender differences (Article)

2007-08-01T00:00:00Z

Introduction: During therapy with radiolabeled peptides, the kidney is most often the critical organ. Newly developed peptides are evaluated preclinically in different animal models before their application in humans. In this study, the renal retention of several radiolabeled peptides was compared in male and female rats and mice. Methods: After intravenous injection of radiolabeled peptides [somatostatin, cholecystokinin (CCK), minigastrin, bombesin and neurotensin analogues], renal uptake was determined in both male and female Lewis rats and C57Bl mice. In addition, ex vivo autoradiography of renal sections was performed to localize accumulated radioactivity. Results: An equal distribution pattern of renal radioactivity was found for all peptides: high accumulation in the cortex, lower accumulation in the outer medulla and no radioactivity in the inner medulla of the kidneys. In both male rats and mice, an increasing renal uptake was found: [111In-DTPA]CCK8<[111In-DTPA-Pro1,Tyr4]bombesin≈[111In-DTPA]neurotensin<[111In-DTPA]octreotide≪[111In-DTPA]MG0. Renal uptake of [111In-DTPA]octreotide in rats showed no gender difference, and renal radioactivity was about constant over time. In mice, however, renal uptake in females was significantly higher than that in males and decreased rapidly over time in both genders. Moreover, renal radioactivity in female mice injected with [111In-DTPA]octreotide showed a different localization pattern. Conclusions: Regarding the renal uptake of different radiolabeled peptides, both species showed the same ranking order. Similar to findings in patients, rats showed comparable and constant renal retention of radioactivity in both genders, in contrast to mice. Therefore, rats appear to be the more favorable species for the study of the renal retention of radioactivity.

Dose-dependent effects of (anti)folate preinjection on 99mTc-radiofolate uptake in tumors and kidneys (Article)

2007-08-01T00:00:00Z

Introduction: The folate receptor (FR) is frequently overexpressed in tumors and can be targeted with folate-based (radio)pharmaceuticals. However, significant accumulation of radiofolates in FR-positive kidneys represents a drawback. We have shown that preadministration of the antifolate pemetrexed (PMX) significantly improved the tumor-to-kidney ratio of radiofolates in mice. The aim of this study was to investigate the dose dependence of these effects and whether the same results could be achieved with folic acid (FA) or 5-methyl-tetrahydrofolate (5-Me-THF). Methods: Biodistribution was assessed 4 h postinjection of the organometallic99mTc-picolylamine monoacetic acid folate in nude mice bearing FR-positive KB tumor xenografts. PMX (50-400 μg/mouse) was injected 1 h previous to radioactivity. The effects of FA and 5-Me-THF (0.5-50 μg/mouse) were investigated likewise. Tissues and organs were collected and counted for radioactivity and the values tabulated as percentage of injected dose per gram tissue (% ID/g). Results: PMX administration reduced renal retention (<1.6% ID/g vs. control: >10% ID/g), while the tumor uptake (average 1.35%±0.40% ID/g vs. control: 1.79%±0.49% ID/g) was only slightly affected independent of the PMX dose. Replacement of PMX by FA or 5-Me-THF (50 μg/mouse) resulted in a significant renal blockade (<0.1% ID/g) but at the same time in an undesired reduction of tumor uptake (<0.2% ID/g). Conclusions: Selective reduction of radiofolate uptake in kidneys under retention of high tumor accumulation could be achieved in combination with PMX over a broad dose range but not with FA or 5-Me-THF.

Novel 111In-labelled bombesin analogues for molecular imaging of prostate tumours (Article)

2007-08-01T00:00:00Z

Purpose: It has been shown that some primary human tumours and their metastases, including prostate and breast tumours, overexpress gastrin-releasing peptide (GRP) receptors. Bombesin (BN) is a neuropeptide with a high affinity for these GRP receptors. We demonstrated successful scintigraphic visualisation of BN receptor-positive tumours in preclinical studies using the radiolabelled BN analogue [111In-DTPA-Pro1,Tyr4]BN. However, the receptor affinity as well as the serum stability of this analogue leave room for improvement. Therefore new111In-labelled BN analogues were synthesised and evaluated in vitro and in vivo. Methods and results: The receptor affinity of the new BN analogues was tested on human GRP receptor-expressing prostate tumour xenografts and rat colon sections. Analogues with high receptor affinity (low nM range) were selected for further evaluation. Incubation in vitro of GRP receptor-expressing rat CA20948 and human PC3 tumour cells with the111In-labelled analogues resulted in rapid receptor-mediated uptake and internalisation. The BN analogue with the best receptor affinity and in vitro internalisation characteristics, Cmp 3 ([111In-DTPA-ACMpip5,Tha6,βAla11,Tha13,Nle14]BN(5-14)), was tested in vivo in biodistribution studies using rats bearing GRP receptor-expressing CA20948 tumours, and nude mice bearing human PC3 xenografts. Injection of111In-labelled Cmp 3 in these animals showed high, receptor-mediated uptake in receptor-positive organs and tumours which could be visualised using planar gamma camera and microSPECT/CT imaging. Conclusion: With their enhanced receptor affinity and their rapid receptor-mediated internalisation in vitro and in vivo, the new BN analogues, and especially Cmp 3, are promising candidates for use in diagnostic molecular imaging and targeted radionuclide therapy of GRP receptor-expressing cancers.

Five stabilized 111in-labeled neurotensin analogs in nude mice bearing HT29 tumors (Article)

2007-06-01T00:00:00Z

Neurotensin (NT) receptors are overexpressed in different human tumors, such as human ductal pancreatic adenocarcinoma. New stable neurotensin analogs with high receptor affinity have been synthesized by replacing arginine residues with lysine and arginine derivatives. The aim of this study was to explore the biodistribution, tumor uptake, kidney localization, and stability characteristics of these new analogs in order to develop new diagnostic tools for exocrine pancreatic cancer. Four111In-labeled DTPA-chelated NT analogs and one111In-labeled DOTA-chelated NT analog were evaluated in NMRI nude mice bearing NT receptor-positive HT29 tumors. Experiments with a coinjection of unlabeled NT or lysine were performed to investigate receptor-mediated uptake and kidney protection, respectively. In addition, the in vivo serum stability of the most promising analog was analyzed. In the biodistribution study in mice, at 4 hours postinjection, a low percentage of the injected dose per gram (%ID/g) of tissue for all compounds was found in NT receptor-negative organs, such as the blood, spleen, pancreas, liver, muscle, and femur. A high uptake was found in the colon, intestine, kidneys, and in implanted HT29 tumors. The coinjection of excess unlabeled neurotensin significantly reduced tumor uptake, showing tumor uptake to be receptor-mediated. To a lesser extent, this was also observed for the colon, but not for other tissues. We concluded that DTPA-(Pip)Gly-Pro-(PipAm)Gly-Arg-Pro- Tyr-tBuGly-Leu-OH and the DOTA-linked counterpart have the most favorable biodistribution properties regarding tumor uptake.

Amifostine protects rat kidneys during peptide receptor radionuclide therapy with [177Lu-DOTA0,Tyr3]octreotate (Article)

2007-05-01T00:00:00Z

Purpose: In peptide receptor radionuclide therapy (PRRT) using radiolabelled somatostatin analogues, the kidneys are the major dose-limiting organs, because of tubular reabsorption and retention of radioactivity. Preventing renal uptake or toxicity will allow for higher tumour radiation doses. We tested the cytoprotective drug amifostine, which selectively protects healthy tissue during chemo- and radiotherapy, for its renoprotective capacities after PRRT with high-dose [177Lu-DOTA0,Tyr3]octreotate. Methods: Male Lewis rats were injected with 278 or 555 MBq [177Lu-DOTA0,Tyr3]octreotate to create renal damage and were followed up for 130 days. For renoprotection, rats received either amifostine or co-injection with lysine. Kidneys, blood and urine were collected for toxicity measurements. At 130 days after PRRT, a single-photon emission computed tomography (SPECT) scan was performed to quantify tubular uptake of99mTc-dimercaptosuccinic acid (DMSA), a measure of tubular function. Results: Treatment with 555 MBq [177Lu-DOTA0,Tyr3]octreotate resulted in body weight loss, elevated creatinine and proteinuria. Amifostine and lysine treatment significantly prevented this rise in creatinine and the level of proteinuria, but did not improve the histological damage. In contrast, after 278 MBq [177Lu-DOTA0,Tyr3]octreotate, creatinine values were slightly, but not significantly, elevated compared with the control rats. Proteinuria and histological damage were different from controls and were significantly improved by amifostine treatment. Quantification of99mTc-DMSA SPECT scintigrams at 130 days after [177Lu- DOTA0,Tyr3]octreotate therapy correlated well with 1/creatinine (r2=0.772, p<0.001). Conclusion: Amifostine and lysine effectively decreased functional renal damage caused by high-dose [177Lu-DOTA0,Tyr3]octreotate. Besides lysine, amifostine might be used in clinical PRRT as well as to maximise anti-tumour efficacy.

Indication for different mechanisms of kidney uptake of radiolabeled peptides (Article)

2007-04-01T00:00:00Z

Nephrotoxicity due to renal reabsorption of radiolabeled peptides limits the tumor dose in peptide receptor radiotherapy (PRRT). Therefore, we evaluated the ability of several agents to inhibit the renal accumulation of different radiopeptides. Methods: Male Wistar rats (4 per group) were injected intravenously with 1 MBq of111In-labeled octreotide (OCT), minigastrin (MG), bombesin (BOM), or exendin (EX), together with a potential inhibitor of renal uptake (lysine [Lys], poly-glutamic acid [PGA], and Gelofusine [GF], a gelatin-based plasma expander) or phosphate-buffered saline as a control. Organ uptake at 20 h after injection was determined as the percentage of injected activity per gram (%IA/g). Lys, PGA, and GF were also combined to determine whether an additive effect could be obtained. The localization of the peptides in the kidneys was investigated by autoradiography using a phosphor imager. Results: OCT accumulation in the kidney was inhibited by Lys and GF (40.7%-45.1%), whereas PGA was ineffective. On the other hand, renal uptake of BOM, MG,and EX was inhibited by PGA and GF (15.4%-85.4%), whereas Lys was ineffective. The combination of GF and Lys showed additive effects in inhibiting OCT uptake, whereas PGA and GF had additive effects for the inhibition of EX uptake. The amount of kidney uptake correlated with the number of charged amino acids. All radiopeptides were localized in the renal cortex, as indicated by autoradiography. Conclusion: Inhibition of renal accumulation of the radiopeptides tested could be achieved by either Lys or PGA but not by both at the same time, suggesting 2 different uptake mechanisms. The differences in renal accumulation of radiopeptides may be related to the number of charges of a molecule. GF is the only compound that inhibited renal accumulation of all radiopeptides tested. Additional experiments are needed to further elucidate these findings and to optimize inhibition of renal accumulation of radiopeptides to reduce the kidney dose in PRRT. Copyright

De schoonheid van het verval (Inaugural Lecture)

2007-03-23T00:00:00Z

In de tijd dat hoogleraren zich nog een eigen chauffeur konden veroorloven, lang geleden dus, was er een hoogleraar die regelmatig lezingen hield. Op een avond was hij weer op weg naar een lezing en plotseling stelde zijn chauffeur in de auto een vraag. ‘Professor’, zei hij, ‘Iedere keer als u een lezing houdt, zit ik in de zaal te luisteren. Ik heb uw verhaal nu zo vaak gehoord dat ik denk dat ik die lezing ook wel eens kan geven. Wat zou u ervan vinden als we vanavond de rollen eens zouden omdraaien?’. De professor was wel in voor een geintje en toen zij op de plaats van bestemming aankwamen, stelde de chauffeur zich voor als de professor en de echte professor ging achter in de zaal zitten. De chauffeur begon zijn verhaal en de professor moest toegeven dat de chauffeur het minstens zo goed deed als hijzelf en - als hij heel eerlijk was - misschien zelfs nog wel wat beter. Maar, dacht hij, straks komen de vragen en dan gaat het vast niet meer zo goed. Na afl oop van de lezing waren er inderdaad vragen, maar de eerste vragen waren al vaker bij vorige lezingen gesteld, zodat de chauffeur daar prima raad mee wist. Toen kwam er echter een vraag waarvan ook de professor vond dat die zeker niet eenvoudig was. De chauffeur trok wat wit weg en er verschenen zweetdruppeltjes op zijn voorhoofd, maar plotseling klaarde zijn gezicht op en hij antwoordde: ‘Dit is een heel lastige vraag, althans op het eerste gehoor, maar hij is eigenlijk heel eenvoudig, zo eenvoudig dat zelfs mijn chauffeur die daar achter in de zaal zit hem kan beantwoorden’. Mijnheer de Rector Magnifi cus, Mijnheer de Decaan, Zeer gewaardeerde toehoorders, Inleiding

Peptide receptor radionuclide therapy (Article)

2007-03-01T00:00:00Z

Peptide receptor radionuclide therapy with radiolabelled somatostatin analogues is an emerging and convincing treatment modality for patients with unresectable, somatostatin-receptor-positive neuroendocrine tumours. Using radiolabelled somatostatin analogues for imaging became the gold standard for staging of neuroendocrine tumours. The somatostatin receptor is strongly over-expressed in most tumours, resulting in high tumour-to-background ratios. Consequently, the next step was to try to treat these patients by increasing the radioactivity of the administered radiolabelled somatostatin analogue in an attempt to bring about tumour cure. Many patients have been treated successfully with this approach, roughly 25% of them achieving objective tumour shrinkage >50%. Serious side-effects have been rare. This article reviews the effectiveness and safety of the different radiolabelled somatostatin analogues used. Furthermore, clinical issues - including indication and timing of therapy - are discussed. Finally, important directions for future research are mentioned to illustrate new strategies for increasing therapy efficacy.

Long-term toxicity of [177Lu-DOTA0,Tyr 3]octreotate in rats (Article)

2007-02-01T00:00:00Z

Purpose and methods: Studies on peptide receptor radionuclide therapy (PRRT) using radiolabelled somatostatin analogues have shown promising results with regard to tumour control. The efficacy of PRRT is limited by uptake and retention in the proximal tubules of the kidney, which might lead to radiation nephropathy. We investigated the long-term renal toxicity after different doses of [177Lu-DOTA0,Tyr3]octreotate and the effects of dose fractionation and lysine co-injection in two tumour-bearing rat models. Results: Significant renal toxicity was detected beyond 100 days after start of treatment as shown by elevated serum creatinine and proteinuria. Microscopically, tubules were strongly dilated with flat epithelium, containing protein cylinders. Creatinine levels rose significantly after 555 MBq [177Lu-DOTA0,Tyr3]octreotate, but were significantly lower after 278 MBq (single injection) or two weekly doses of 278 MBq. Renal damage scores were maximal after 555 MBq and significantly lower in the 278 and 2x278 MBq groups. Three doses of 185 MBq [177Lu-DOTA0,Tyr3]octreotate with intervals of a day, a week or a month significantly influenced serum creatinine (469±18, 134±70 and 65±15 μmol/l, respectively; p<0.001). Renal histological damage scores were not significantly influenced by dose fractionation. Lysine co-administration with three weekly treatments of 185 MBq significantly lowered serum creatinine and proteinuria. Conclusion: Injection of high doses of [177Lu-DOTA0,Tyr3]octreotate resulted in severe renal damage in rats as indicated by proteinuria, elevated serum creatinine and histological damage. This damage was dose dependent and became overt between 100 and 200 days after treatment. Dose fractionation had significant beneficial effects on kidney function. Also, lysine co-injection successfully prevented functional damage.

Anaphylactoid reaction from amifostine (Article)

2007-01-01T00:00:00Z

Plasma membrane transport of thyroid hormones and its role in thyroid hormone metabolism and bioavailability (Article)

2001-01-01T00:00:00Z

Although it was originally believed that thyroid hormones enter target cells by passive diffusion, it is now clear that cellular uptake is effected by carrier-mediated processes. Two stereospecific binding sites for each T4 and T3 have been detected in cell membranes and on intact cells from humans and other species. The apparent Michaelis-Menten values of the high-affinity, low-capacity binding sites for T4 and T3 are in the nanomolar range, whereas the apparent Michaelis- Menten values of the low-affinity, high-capacity binding sites are usually in the lower micromolar range. Cellular uptake of T4 and T3 by the high-affinity sites is energy, temperature, and often Na+ dependent and represents the translocation of thyroid hormone over the plasma membrane. Uptake by the low-affinity sites is not dependent on energy, temperature, and Na+ and represents binding of thyroid hormone to proteins associated with the plasma membrane. In rat erythrocytes and hepatocytes, T3 plasma membrane carriers have been tentatively identified as proteins with apparent molecular masses of 52 and 55 kDa. In different cells, such as rat erythrocytes, pituitary cells, astrocytes, and mouse neuroblastoma cells, uptake of T4 and T3 appears to be mediated largely by system L or T amino acid transporters. Efflux of T3 from different cell types is saturable, but saturable efflux of T4 has not yet been demonstrated. Saturable uptake of T4 and T3 in the brain occurs both via the blood-brain barrier and the choroid plexus-cerebrospinal fluid barrier. Thyroid hormone uptake in the intact rat and human liver is ATP dependent and rate limiting for subsequent iodothyronine metabolism. In starvation and nonthyroidal illness in man, T4 uptake in the liver is decreased, resulting in lowered plasma T3 production. Inhibition of liver T4 uptake in these conditions is explained by liver ATP depletion and increased concentrations of circulating inhibitors, such as 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, indoxyl sulfate, nonesterified fatty acids, and bilirubin. Recently, several organic anion transporters and L type amino acid transporters have been shown to facilitate plasma membrane transport of thyroid hormone. Future research should be directed to elucidate which of these and possible other transporters are of physiological significance, and how they are regulated at the molecular level.

Changes in renal tri-iodothyronine and thyroxine handling during fasting (Article)

2000-01-01T00:00:00Z

OBJECTIVE: Liver handling of thyroid hormones (TH) has been known to alter significantly during fasting. This study investigates whether renal handling of TH is also changed during fasting. METHODS: We measured urinary excretion rates and clearances of free tri-iodothyronine (T(3)) and free thyroxine (T(4)) in healthy subjects prior to and on the third day of fasting. RESULTS: During fasting, both mean T(3) and T(4) urinary excretion decreased significantly to a mean value of 42% of control. Also, total and free (F) serum T(3) concentrations declined significantly, but serum T(4) did not change. Both FT(3) and FT(4) clearance decreased significantly during fasting (62% and 42% of control). The fasting-induced decrease in uric acid clearance correlated well with the decrease in FT(3) clearance (r=0.94; P<0.001). Serum concentrations of non-esterified fatty acids (NEFA) were significantly elevated during fasting. CONCLUSIONS: The findings cannot be fully explained by the fasting-induced decrease in serum T(3), and are in accordance with inhibition of uptake of T(3) and T(4) at the basolateral membrane of the tubular cell. This inhibition may be caused by a decreased energy state of the tubular cell and by other factors such as ketoacidosis and/or increased NEFA concentrations during fasting.

Uptake of 3,3',5,5'-tetraiodothyroacetic acid and 3,3',5'-triiodothyronine in cultured rat anterior pituitary cells and their effects on thyrotropin secretion (Article)

1995-01-01T00:00:00Z

We compared the uptake, metabolism, and biological effects of tetraiodothyroacetic acid (Tetrac) and rT3 in anterior pituitary cells with those of T4 and T3. Cells were isolated from adult male Wistar rats and cultured for 3 days in medium with 10% fetal calf serum. Uptake was measured at 37 C in medium with 0.1% BSA for [125I]Tetrac (200,000 cpm; 240 pM) and [125I]T4 (100,000 cpm; 175 pM) or with 0.5% BSA for [125I]rT3 (100,000 cpm; 250 pM) and [125I]T3 (50,000 cpm; 50 pM). The free fraction of Tetrac was 1% that of T4 (in medium with 0.1 and with 0.5% BSA), and the free fraction of rT3 was half that of T3. Uptake of the four tracers increased sharply up to 1 h of incubation and then leveled off. Expressed as femtomoles per pM free hormone, uptake at equilibrium was 1.16 +/- 0.16 (n = 6) for Tetrac, 0.15 +/- 0.01 (n = 6) for T4, 0.023 +/- 0.003 (n = 6) for rT3, and 0.21 +/- 0.02 (n = 6) for T3. Cell-associated radioactivity after incubation for 24 h with [125I]Tetrac was represented for 15% by [125I]Triac; after incubation with [125I]T4 for 15-20% by [125I]T3, after incubation with [125I]rT3 for 6% by [125I]3,3'-T2, while [125I]T3 was still for 98% [125I]T3. Exposure of cells for 2 h to 100 nM TRH stimulated TSH release by 90-135%. Tetrac was effective in reducing this response at a free concentration of 0.05 pM, but rT3 was effective only at a free concentration of 16 nM. A free Tetrac concentration of 5 pM was equally effective as 50 pM free T4 in reducing the TSH response to TRH. In human serum, Tetrac was exclusively bound to T4-binding prealbumin. The free Tetrac fraction was 0.001% in control subjects and rose 2- to 12-fold in patients with nonthyroidal illness. As uptake of [125I]Tetrac in the pituitary was higher than that of T4 and T3, and it was more potent than T4 in reducing TSH release, Tetrac may be of potential significance for the regulation of TSH secretion in vivo.

Uptake of triiodothyroacetic acid and its effect on thyrotropin secretion in cultured anterior pituitary cells (Article)

1994-01-01T00:00:00Z

The uptake of [125I]triiodothyroacetic acid ([125I]Triac) in anterior pituitary cells was investigated and compared with that of [125I]T3. Furthermore, the effects of Triac, T3, and T4 on TSH release were compared. Cells isolated from adult male Wistar rats were cultured for 3 days in medium with 10% fetal calf serum. Uptake was measured at 37 C with [125I]Triac (100,000 cpm; 120 pM) or [125I]T3 (50,000 cpm; 50 pM) in medium with 0.5% BSA. In this medium, the ratio of the free fractions of Triac, T3, and T4 was 1:8:1. Exposure of cells to 100 nM TRH for 2 h stimulated TSH release by 80-110% (P < 0.001). Comparing total hormone levels (1 nM to 1 microM), Triac and T3 were equally effective in reducing this response, and both were 10-fold more effective than T4. The time course (15 min to 4 h) of [125I]Triac uptake was similar to that of [125I]T3, showing equilibrium after 1 h. Unlabeled Triac (1 microM) reduced the uptake of [125I]Triac and [125I]T3 at all time intervals. Expressed per pM free hormone, the cellular and nuclear uptake of [125I]Triac were twice those of [125I]T3. The 15-min uptake of [125I]Triac was reduced by incubation with 10 nM unlabeled Triac (35%; P < 0.001). Maximum inhibition (56%; P < 0.001) was found with 10 microM Triac. A similar effect was seen with 10 microM T3, T4, or 3,3',5,5'-tetraiodothyroacetic acid. Preincubation (30 min) and incubation (15 min) with 10 microM oligomycin reduced the cellular ATP content by 51% (P < 0.001), [125I]T3 uptake by 77% (P < 0.001), and [125I]Triac uptake by only 25% (P < 0.001). The temperature dependence of [125I]Triac and [125I]T3 uptake was the same. Preincubation and incubation with 10 microM monensin (reduces the Na+ gradient) or 10 microM monodansylcadaverine (inhibits receptor-mediated endocytosis) reduced 15-min [125I] Triac uptake by 15% (P < 0.005) and 19% (P < 0.005), respectively. The data show that 1) Triac, on the basis of the free hormone concentration, is more potent than T3 or T4 in suppressing TSH secretion; and 2) the rapid uptake of [125I]Triac by the anterior pituitary occurs by a carrier-mediated mechanism that is only partially dependent on ATP or the Na+ gradient.

Thyroid hormone transport into liver cells: its (patho)physiological significance (Doctoral Thesis)

1993-12-09T00:00:00Z

In the experiments described in this thesis transmembrane transport of thyroid hormones into liver cells is investigated, in particular the regulatory role of this transport process in the bioavailability of thyroid hormones in (patho )fysiological conditions. This first chapter is an introduction to the studies described in Chapters 3-9. It opens with a short description of different aspects regarding thyroid hormones, i.e. synthesis, transport in blood to the different organs, transport into cells, nuclear binding and the different routes of intracellular metabolism. Only the main issues will be discussed here. The isolated rat liver perfusion system was used to study the transport process in most of our studies. Therefore, this perfusion system is also described in this chapter and compared to isolated rat hepatocytes in primary culture, in which many studies on thyroid hormone transport have been performed. Furthermore, a twocompartment model, which describes thyroid hormone kinetics in the liver perfusion system, is dealt with here. At the end of the chapter the scope of the thesis and a short overview of the questions that were addressed are presented