Side-by-side comparison of the two widely studied GRPR radiotracers, radiolabeled NeoB and RM2, in a preclinical setting.

INTRODUCTION: NeoB and RM2 are the most investigated gastrin-releasing peptide receptor (GRPR)-targeting radiotracers in preclinical and clinical studies. Therefore, an extensive side-by-side comparison of the two radiotracers is valuable to demonstrate whether one has advantages over the other. Accordingly, this study aims to compare the in vitro and in vivo characteristics of radiolabeled NeoB and RM2 to guide future clinical studies. METHOD: The stability of the radiolabeled GRPR analogs was determined in phosphate buffered saline (PBS), and commercially available mouse and human serum. Target affinity was determined by incubating human prostate cancer PC-3 cells with [177Lu]Lu-NeoB or [177Lu]Lu-RM2, + / - increasing concentrations of unlabeled NeoB, RM2, or Tyr4-bombesin (BBN). To determine uptake and specificity cells were incubated with [177Lu]Lu-NeoB or [177Lu]Lu-RM2 + / - Tyr4-BBN. Moreover, in vivo studies were performed to determine biodistribution and pharmacokinetics. Finally, radiotracer binding to various GRPR-expressing human cancer tissues was investigated. RESULTS: Both radiotracers demonstrated high stability in PBS and human serum, but stability in mouse serum decreased substantially over time. Moreover, both radiotracers demonstrated high GRPR affinity and specificity, but a higher uptake of [177Lu]Lu-NeoB was observed in in vitro studies. In vivo, no difference in tumor uptake was seen. The most prominent difference in uptake in physiological organs was observed in the GRPR-expressing pancreas; [177Lu]Lu-RM2 had less pancreatic uptake and a shorter pancreatic half-life than [177Lu]Lu-NeoB. Furthermore, [177Lu]Lu-RM2 presented with a lower tumor-to-kidney ratio, while the tumor-to-blood ratio was lower for [177Lu]Lu-NeoB. The autoradiography studies revealed higher binding of radiolabeled NeoB to all human tumor tissues. CONCLUSION: Based on these findings, we conclude that the in vivo tumor-targeting capability of radiolabeled NeoB and RM2 is similar. Additional studies are needed to determine whether the differences observed in physiological organ uptakes, i.e., the pancreas, kidneys, and blood, result in relevant differences in organ absorbed doses when the radiotracers are applied for therapeutic purposes.

Imaging heterogeneity of peptide delivery and binding in solid tumors using SPECT imaging and MRI.

BACKGROUND: As model system, a solid-tumor patient-derived xenograft (PDX) model characterized by high peptide receptor expression and histological tissue homogeneity was used to study radiopeptide targeting. In this solid-tumor model, high tumor uptake of targeting peptides was expected. However, in vivo SPECT images showed substantial heterogeneous radioactivity accumulation despite homogenous receptor distribution in the tumor xenografts as assessed by in vitro autoradiography. We hypothesized that delivery of peptide to the tumor cells is dictated by adequate local tumor perfusion. To study this relationship, sequential SPECT/CT and MRI were performed to assess the role of vascular functionality in radiopeptide accumulation. METHODS: High-resolution SPECT and dynamic contrast-enhanced (DCE)-MRI were acquired in six mice bearing PC295 PDX tumors expressing the gastrin-releasing peptide (GRP) receptor. Two hours prior to SPECT imaging, animals received 25 MBq (111)In(DOTA-(ßAla)2-JMV594) (25 pmol). Images were acquired using multipinhole SPECT/CT. Directly after SPECT imaging, MR images were acquired on a 7.0-T dedicated animal scanner. DCE-MR images were quantified using semi-quantitative and quantitative models. The DCE-MR and SPECT images were spatially aligned to compute the correlations between radioactivity and DCE-MRI-derived parameters over the tumor. RESULTS: Whereas histology, in vitro autoradiography, and multiple-weighted MRI scans all showed homogenous tissue characteristics, both SPECT and DCE-MRI showed heterogeneous distribution patterns throughout the tumor. The average Spearman's correlation coefficient between SPECT and DCE-MRI ranged from 0.57 to 0.63 for the "exchange-related" DCE-MRI perfusion parameters. CONCLUSIONS: A positive correlation was shown between exchange-related DCE-MRI perfusion parameters and the amount of radioactivity accumulated as measured by SPECT, demonstrating that vascular function was an important aspect of radiopeptide distribution in solid tumors. The combined use of SPECT and MRI added crucial information on the perfusion efficiency versus radiopeptide uptake in solid tumors and showed that functional tumor characteristics varied locally even when the tissue appeared homogenous on current standard assessment techniques.

Improving radiopeptide pharmacokinetics by adjusting experimental conditions for bombesin receptor-targeted imaging of prostate cancer.

AIM: Prostate cancer (PC) is a major health problem. The Gastrin-Releasing Peptide Receptor (GRPR) offers a promising target for staging and monitoring of PC since it is overexpressed in PC and not in normal prostatic tissue. To improve receptor-mediated imaging we investigated the impact of various experimental conditions on pharmacokinetics using the Indium-111 labelled bombesin (BN) analogue AMBA. Besides frequently used androgen-resistant PC-3 also the clinically more relevant androgen sensitive VCaP celline was used as human PC xenograft in nude mice. METHODS: Non-purified [111In]AMBA was compared with HPLC-purified [111In]AMBA. Effect of specific activity was studied administering 0.1MBq [111In]AMBA supplemented with different amounts of AMBA (1-3000pmol). GRPR was saturated with Tyr4-BN 1 and 4h prior to injection of [111In]AMBA. RESULTS: GRPR-positive tissue showed a significant 2 to 3-fold increase in absolute uptake after HPLC-purification while keeping a stable tumor-to-pancreas ratio. Lowering specific activity resulted in decline in uptake to 43% in tumor, 49% in kidney and 92% in pancreas between 10 and 3000 pmol. Tumor-to-pancreas ratio improved six-fold from 0.1±0 after 10 pmol up to 0.6±0.2 after 3000 pmol (P<0.01). When saturating GRPR 4h prior to [111In]AMBA injection tumor-to-pancreas ratio improved from 0.10±0.3 to 0.22±0.2 (P<0.01) and tumor-to-kidney ratio increased from 0.92±0.16 to 3.45±0.5 (P<0.01). CONCLUSION: Besides specific peptide characteristics also the experimental conditions, such as HPLC-purification, variations in specific activity and saturation of the GRPR prior to [111In]AMBA administration essentially affect radiopeptide pharmacokinetics. Experimental conditions therefore need to be carefully selected in order to compose ideal standardised protocols for optimal targeting.

A transductionally retargeted adenoviral vector for virotherapy of Her2/neu-expressing prostate cancer.

The efficacy of adenovirus (Ad)-based gene therapy of solid tumors, such as prostate cancer, is limited. One of the many problems is that the virus infects many different cell types in the body, resulting in high toxicity, whereas the target cancer cells are often less prone to wild-type Ad infection. Our aim was to develop genetically de- and retargeted Ad vectors to reduce off-target effects and increase target infection for prostate cancer. We have previously reported an Ad5 vector specific for the cancer-associated receptor Her2/neu, created by inserting Her2/neu-reactive Affibody(®) molecules (ZH) into the HI loop of a coxsackievirus and adenovirus receptor binding-ablated fiber (Ad[ZH/1]). In addition to virus retargeting to Her2/neu, this virus was further modified from wild-type Ad by changing the RGD motif in the penton base to EGD and by substitution of the KKTK motif in the third shaft repeat to RKSK, resulting in the vector Ad[ZH/3]. The ZH-containing vectors could be produced to high titers and were specific for their target, resulting in efficient infection and killing of Her2/neu-positive androgen-dependent PC346C prostate cancer cells in vitro. Here we show that the oncolytic Ad[ZH/3] vector significantly prolonged survival time and reduced serum prostate-specific antigen levels in an orthotopic prostate tumor model in nude mice to the same extent as wild-type Ad5. Our results show that Her2/neu targeting using Ad-based vectors for prostate cancer is feasible and may serve as a basis for the development of gene therapy of human prostate cancer as well as other Her2/neu-expressing cancers.

Disconnecting the yin and yang relation of epidermal growth factor receptor (EGFR)-mediated delivery: a fully synthetic, EGFR-targeted gene transfer system avoiding receptor activation.

The epidermal growth factor receptor (EGFR) is upregulated within a high percentage of solid tumors and hence is an attractive target for tumor-targeted therapies including gene therapy. The natural EGFR ligand epidermal growth factor (EGF) has been used for this purpose, despite the risk of mitogenic effects due to EGFR activation. We have developed a fully synthetic, EGFR-targeted gene delivery system based on PEGylated linear polyethylenimine (LPEI), allowing evaluation of different EGFR-binding peptides in terms of transfection efficiency and EGFR activation. Peptide sequences directly derived from the human EGF molecule enhanced transfection efficiency with concomitant EGFR activation. Only the EGFR-binding peptide GE11, which has been identified by phage display technique, showed specific enhancement of transfection on EGFR-overexpressing tumor cells including glioblastoma and hepatoma, but without EGFR activation. EGFR targeting led to high levels of cell association of fluorescently labeled polyplexes after only 30 min of incubation. EGF pretreatment of cells induced enhanced cellular internalization of all polyplex types tested, pointing at generally enhanced macropinocytosis. EGF polyplexes diminished cell surface expression of EGFR for up to 4 hr, whereas GE11 polyplexes did not. In a clinically relevant orthotopic prostate cancer model, intratumorally injected GE11 polyplexes were superior in inducing transgene expression when compared with untargeted polyplexes.

Bone metabolism markers and bone mass in healthy pubertal boys and girls.

OBJECTIVE: During puberty, bone growth and mineralization as well as bone turnover increase dramatically. The relation between height velocity and bone turnover is already known, but there are few studies in which both bone metabolism markers and bone mass throughout puberty have been measured. DESIGN: Semi-longitudinal study. In 155 healthy boys (12.0 +/- 1.5 years; range 8.8-15.7 years) and 151 healthy girls (11.2 +/- 1.6 years; range 8.2-14.0 years) markers of bone formation and bone resorption were measured as well as sex steroids, IGF-1 and IGF-BP3, together with bone mineral content (BMC) and bone mineral density (BMD) of the lumbar spine, femur and total body during puberty. All bone measurements were repeated after 1 year. RESULTS: BMC and BMD increased throughout puberty in both sexes. Bone turnover markers increased significantly until maximum values were reached at stage G4 in boys and stage B3 in girls. Height velocity (HV) had a similar changing pattern. Sex steroids and IGF-1 increased and reached adult values at pubertal stage 4. The correlations between bone metabolism markers and BMC were highly significant in boys, while correlations between bone metabolism markers and the increase in BMC over 1 year were significant in both sexes, as was observed for the correlations with HV. CONCLUSIONS: Our data suggest that bone metabolism markers are good predictors of bone mass in boys and of bone mass increase in both sexes. In early puberty, sex steroids stimulate the pubertal growth spurt in conjunction with GH and IGF-1. The fast increase in height gives rise to an increase in bone turnover and bone mineral apposition. It is known that at the end of puberty high levels of oestradiol inhibit chondrocyte proliferation. This leads to a decline in height velocity and bone turnover. Bone mass still increases under the influence of sex steroids and IGF-1. The data in our study confirm previous reports that markers of bone turnover relate positively to height velocity.

Concurrent validity of a weight-bearing activity questionnaire in prepubertal and pubertal girls and boys.

OBJECTIVE: The purpose of this study was to evaluate the current validity of an interviewer-administered physical activity questionnaire against measurement of physical activity from vertical body accelerometer movements in prepubertal and pubertal children. METHODS: The Weight Bearing Activity Questionnaire for Kids (WBAQK) is an interviewer-administered questionnaire with a recall over 7 days and developed to assess weight-bearing activity in pre-pubertal and pubertal children. The Caltrac(TM) accelerometer was worn for 4-5 days (including 1 weekend day). Thirty-seven schoolgirls and 35 schoolboys participated, with a mean age of 11.2 0.3 years and 12.1 0.2 years, respectively. RESULTS: Weight-Bearing Score (WBS) and Metabolic Score (MS) derived from the WBAQK were significantly and positively related to the score of the Caltrac(TM). Weight-Bearing Score showed higher correlations in both boys (0.59) and girls (0.53) and slightly better compared to MS (0.54 and 0.35). The classification of boys and girls into high and low activity groups resulted also in a better agreement of WBS (71-72%) than of MS (60-67%) with Caltrac(TM). CONCLUSIONS: We conclude that the amount of weight-bearing activity can be estimated with the interviewer-administered WBAQK in boys and girls between 8 and 14 years of age.

Validation of transrectal ultrasonographic volumetry for orthotopic prostate tumours in mice.

Orthotopic human prostate tumour models in athymic nude mice are regarded as being most suitable for fundamental and pre-clinical research on prostate cancer. The anatomic localization of the tumour in the pelvis, however, provides little possibility for monitoring tumour growth or regression. To assess time-related changes in orthotopic tumour volume, we applied transrectal ultrasonography (TRUS) to the murine prostate. This technique has the advantages of allowing accurate monitoring of tumours during therapeutic manipulations and a reduction of animal use due to a reduction of sacrificing endpoints. To validate the TRUS method, the mouse prostate reconstitution model, RM-9, and the prostate-specific antigen (PSA) producing human prostate cancer xenograft PC-346 were used. Volumetric calliper measurements were performed with a 30 MHz ultrasound probe designed for intra-arterial use in humans. Tumour weight, determined at various time-points, was found to be closely related to actual tumour weight (R = 0.99) and, in the PC-346 model, to the level of PSA in the plasma. Furthermore, the interobserver variation for TRUS was low for tumours above 50 mg. Thus, TRUS for murine prostate tumours proves to be an accurate, reproducible and sensitive method.

Pubertal maturation characteristics and the rate of bone mass development longitudinally toward menarche.

To assess risks for osteoporosis and to compare bone mass in different groups of healthy children or children with diseases, it is important to have knowledge of their sexual maturation status during puberty. The aim of our study was to evaluate bone mass formation longitudinally in relation to pubertal maturation characteristics in healthy white girls. We investigated the bone mineral content (BMC) and the bone mineral density (BMD) at different skeletal sites in 151 girls with increasing pubertal stages in relation with their chronological age and with an early or late onset of puberty or menarche and with a slow or fast maturation. Bone mass was measured at the onset of puberty, during puberty, and at menarche. We conclude the following: (1) from midpuberty to menarche, the increase in bone mass formation is highest at all skeletal sites in white girls; (2) early mature girls at the onset of puberty have slightly but definitely lower bone masses at all skeletal sites and at all pubertal stages than late mature girls, whereas the average bone mass formation from the onset of puberty to menarche is similar in both groups; (3) girls with a slow rate of pubertal maturation have lower bone mass values 2 years after the onset of puberty, but at menarche bone mass is similar compared with fast maturers; and (4) it cannot be confirmed that there is an effect of menarcheal age on bone mass values at menarche.

Bone mineral density and markers of bone turnover in young adult survivors of childhood lymphoblastic leukaemia.

OBJECTIVE: In order to determine if a serious disease like childhood acute lymphoblastic leukaemia (ALL) and the treatment necessary to cure the patients has long term effects on bone mass, we assessed bone mineral density (BMD) and several parameters involved in bone formation in a group of young adult survivors of ALL. DESIGN AND PATIENTS: Fourteen male and ten female survivors, treated for ALL in childhood, were cross-sectionally studied, at a mean age of 25.1 years (range 20.1-34.9). All patients, except for two, had received cranial irradiation as part of their treatment (mean radiation dose 2460 cGy). MEASUREMENTS: Height and weight were measured. Bone mineral density (BMD) was assessed using dual energy X-ray absorptiometry in the lumbar spine, femoral neck, femoral trochanter and at 1/3 distal and ultradistal in the radius. Early morning serum levels of LH, FSH, oestradiol or testosterone, IGF-1 and IGF-BP3 were determined as well as several specific markers of bone turnover. RESULTS: Mean height, expressed as standard deviation score (SDS) was -1.12, significantly reduced. BMD in the lumbar spine, femoral neck and at 1/3 distal and ultradistal in the radius, was significantly lower compared to the reference population (P < 0.05). No correlation was found between the BMD values and the cumulative dose of administered cytotoxic drugs, the age at diagnosis of ALL or the duration of follow-up. Mean IGF-1 and IGF-BP3 SDS-scores were -1.24 and -0.78 respectively, significantly reduced. GH stimulation tests performed in a subgroup of 9 patients showed an insufficient peak GH response in at least one test in all tested patients. The values of LH, FSH oestradiol or testosterone were within the normal adult range. Serum markers of bone formation and bone resorption were in the normal range, indicating that bone turnover was normal at the time of the study. CONCLUSIONS: Bone development in patients cured of acute lymphoblastic leukaemia is disturbed, resulting in a significantly reduced bone mineral density. Impaired growth hormone activity, as a long term effect of cranial irradiation, may be one of the underlying causes as well as the illness itself and the administered cytotoxic drugs. Since a reduced bone mineral density predispose patients to osteoporosis, intervention in order to improve bone mass should be considered.

Changes in body composition during 12 months after discontinuation of growth hormone therapy in young adults with growth hormone deficiency from childhood.

Little is known about the velocity of changes in body composition after discontinuation of GH therapy at final height. The aim of this study was to describe the changes of fat distribution in male and female GH deficient young adults during the first year after discontinuation of GH therapy. Ten Dutch GH deficient young adults who had reached final height were retested and confirmed to be still GH deficient. These preliminary results demonstrate that the greatest gain in subcutaneous fat, measured by skinfold thickness, was observed in the first 3 months after GH withdrawal. Intra-abdominal fat was measured by CT scan at 0 and 12 months study time. The mean gain in intra-abdominal fat after 12 months was dramatically high (48%). We conclude that the subcutaneous and intra-abdominal fat mass increased dramatically in young GH deficient Dutch adults with GH deficiency, after discontinuation of therapy, especially in the first three months. This indicates that GH therapy should be restarted as soon as possible, after reconfirming the diagnosis of GH deficiency in adults. This will reduce the risk for these patients of diseases associated with overweight, such as cardiovascular diseases.

Clinical utility of markers of bone turnover in children and adolescents.

We review the past years' literature on biochemical markers of bone turnover. A general introduction to markers of bone formation and bone resorption is followed by reference values of bone turnover in neonates, infants, and prepubertal and pubertal children. We describe intervention with calcium and physical activity in healthy children and bone turnover in patients. The predictive value of a single measurement of bone markers in individuals is poor, due to the large biologic intraperson variation for bone markers in general. Serious osteoporosis can be diagnosed by the combined results of measurement of several bone formation and resorption markers.

Volumetric measurements of bone mineral density of the lumbar spine: comparison of three geometrical approximations using dual-energy X-ray absorptiometry (DXA)

Measurements of bone mineral density using dual-energy X-ray absorptiometry (DXA) gives area values (g cm-2) rather than true volumetric values (g cm-3). To calculate the vertebral volume using planar postero-anterior and lateral DXA values, several different geometrical approximations were used: cubic, cylindrical with a circular cross-section and cylindrical with an elliptical cross-section. The aim of this study was to compare these geometrical approximations with each other and with a reference standard, defined as the volume found on a computed tomographic (CT) scan. L2 and L3 were evaluated in a phantom study. Volume approximations by the cube or cylinder with circular cross-section geometry showed more than a 50% overestimation (range 54-74%). However, the elliptical cylinder approach showed very good agreement: 2.1% and 1.2% for L2 and L3, respectively, when compared to the CT volumes. In addition, we performed four patient studies with both CT and DXA to evaluate the elliptical cylinder estimate in a clinical setting. For L2 and L3, the mean relative difference was less than 2%. We conclude that the elliptical cylinder approach results in the most accurate bone volume estimates in both the phantom and patients.

Genetic diversity of the biofilm covering Montacuta ferruginosa (Mollusca, bivalvia) as evaluated by denaturing gradient gel electrophoresis analysis and cloning of PCR-amplified gene fragments coding for 16S rRNA.

The shell of the bivalve Montacuta ferruginosa, a symbiont living in the burrow of an echinoid, is covered with a rust-colored biofilm. This biofilm includes different morphotypes of bacteria that are encrusted with a mineral rich in ferric ion and phosphate. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Also, the possible roles of the microorganisms in the processes of mineral deposition within the biofilm, as well as their impact on the biology of the bivalve, were assessed by phenotypic inference. The genetic diversity was determined by denaturing gradient gel electrophoresis (DGGE) analysis of short (193-bp) 16S ribosomal DNA PCR products obtained with primers specific for the domain Bacteria. This analysis revealed a diverse consortium; 11 to 25 sequence types were detected depending on the method of DNA extraction used. Individual biofilms analyzed by using the same DNA extraction protocol did not produce identical DGGE profiles. However, different biofilms shared common bands, suggesting that similar bacteria can be found in different biofilms. The phylogenetic affiliations of the sequence types were determined by cloning and sequencing the 16S rRNA genes. Close relatives of the genera Pseudoalteromonas, Colwellia, and Oceanospirillum (members of the gamma-Proteobacteria lineage), as well as Flexibacter maritimus (a member of the Cytophaga-Flavobacter-Bacteroides lineage), were found in the biofilms. We inferred from the results that some of the biofilm bacteria could play a role in the mineral formation processes.

Symbiotic relationship of thiothrix spp. with An echinoderm

Immunoassay procedures were used to investigate the symbiotic relationship of Thiothrix spp. in the intestinal cecum of the spatangoid species Echinocardium cordatum. Thiothrix spp. were identified in nodule samples from E. cordatum digestive tubes based on microscopic examination, enzyme-linked immunosorbent assay, and indirect immunofluorescence. Thiothrix spp. protein made up as much as 84% of the total protein content of the nodules. This is the first identification of Thiothrix spp. internally symbiotic with marine invertebrates.

Androstenedione, dehydroepiandrosterone sulfate, and estradiol levels throughout female puberty: relation to height velocity.

Sex steroids are important contributors to the pubertal growth spurt. Both androgens and estrogens have been related to this moment of rapid growth, but the role of estrogens is thought to be the most important one. Since exogenous estrogens are not capable to induce an appropriate growth spurt in girls, there might be an additional contributing factor involved. In a recent pilot study of 32 healthy pubertal girls, we found that the peak height velocity (HV) is preceded by relatively high levels of dehydroepiandrosterone sulfate and androstenedione (delta4A) as compared with the end-pubertal level. In the present study we evaluated HV in relation to dehydroepiandrosterone sulfate and delta4A levels in 149 healthy girls of various Tanner stages. HV was correlated with delta4A and estradiol levels in Tanner stages I-III. These results suggest that, like estrogens, delta4A might be an important stimulator of the female growth spurt.

Immunoadsorption assay for bone alkaline phosphatase compared with wheat germ agglutinin precipitation assay in serum from (pre)pubertal girls.

The performance characteristics of two bone alkaline phosphatase (ALP; EC 3.1.3.1) assays, a wheat germ agglutinin (WGA) precipitation assay and a new immunoadsorption assay (IAA), were compared. The within- and between-run imprecision of the IAA (3.6-4.2% and 3.6-7.7%) was comparable with that of the WGA assay. The mean cross-reactivity with liver ALP appeared to be 4% in the WGA assay and 11% in the IAA. The reference ranges in a group of 155 healthy Caucasian (pre)pubertal schoolgirls were: 149-401 U/L (total ALP, 30 degrees C), 105-349 U/L (bone ALP, 30 degrees C, WGA assay), and 58-205 U/L (bone ALP, 25 degrees C, IAA). Comparison of the WGA assay (x) with the IAA (y) demonstrated a correlation coefficient of 0.95 [Deming regression equation: y = (0.56 +/- 0.01)x + (2.0 +/- 1.5); Sy[symbol: see text]x = 5.3 U/L]. Correlation studies of the WGA assay and the IAA results with total ALP demonstrated r = 0.98 and 0.96, respectively.