Radioiodinated SB 207710 as a radioligand in vivo: imaging of brain 5-HT4 receptors with SPET

Single-photon emission tomography (SPET) and positron emission tomography (PET), when coupled to suitable radioligands, are uniquely powerful for investigating the status of neurotransmitter receptors in vivo. The serotonin subtype-4 (5-HT₄) receptor has discrete and very similar distributions in rodent and primate brain. This receptor population may play a role in normal cognition and memory and is perhaps perturbed in some neuropsychiatric disorders. SB 207710 [(1-butyl-4-piperidinylmethyl)-8-amino-7-iodo-1,4-benzodioxan-5-carboxylate] is a selective high-affinity antagonist at 5-HT₄ receptors. We explored radioiodinated SB 207710 as a possible radioligand for imaging 5-HT₄ receptors in vivo. Rats were injected intravenously with iodine-125 labelled SB 207710, euthanised at known times and dissected to establish radioactivity content in brain tissues. Radioactivity entered brain but cleared rapidly and to a high extent from blood and plasma. Between 45 and 75 min after injection, the ratios of radioactivity concentration in each of 12 selected brain tissues to that in receptor-poor cerebellum correlated with previous measures of 5-HT₄ receptor density distribution in vitro. The highest ratio was about 3.4 in striatum. SB 207710 was labelled with iodine-123 by an iododestannylation procedure. A cynomolgus monkey was injected intravenously with [¹²³I]SB 207710 and examined by SPET. Maximal whole brain uptake of radioactivity was 2.3% of the injected dose at 18 min after radioligand injection. Brain images acquired between 9 and 90 min showed high radioactivity uptake in 5-HT₄ receptor-rich regions, such as striatum, and low uptake in receptor-poor cerebellum. At 169 min the ratio of radioactivity concentration in striatum to that in cerebellum was 4.0. In a second SPET experiment, the cynomolgus monkey was pretreated with a selective 5-HT₄ receptor antagonist, SB 204070, at 20 min before [¹²³I]SB 207710 injection. Radioactivity in all brain regions was reduced almost to the level in cerebellum by 176 min after radioligand injection. These findings show that [¹²³I]SB 207710 is an effective radioligand for imaging brain 5-HT₄ receptors in vivo.For preliminary accounts of this work, see Pike VW et al., J Nucl Med 1998; 39 (Suppl):185; Eur J Nucl Med 1999; 26:991.     

Meniscectomy leads to an early increase in subchondral bone plate thickness in the rabbit knee

We evaluated morphological changes in the tibial bone after meniscectomy in a rabbit model. 15 rabbits subjected to a medial meniscectomy in the right knee and a sham-operation in the left. Histomorphometric parameters were evaluated in the subchondral bone plate and the underlying trabecular bone, 13, 25 and 40 weeks after surgery. 5 rabbits were used as unoperated controls. Meniscectomized knees had a thicker subchondral bone plate than sham-operated contralateral ones in 13 of the 15 rabbits (p = 0.01), but the trabecular bone showed no morphological differences. The meniscectomized knees of these rabbits developed mild osteoarthrosis, described elsewhere, which may have been partly due to a change in the mechanical properties of the thickened subchondral bone plate. Our findings suggest that the first bony response after meniscectomy occurs in the subchondral bone plate rather than in the trabecular bone.     

Effects of immediate switch from cyclosporine microemulsion to tacrolimus at first acute rejection in renal allograft recipients

BACKGROUND: A number of institutions have reported favorable results in renal transplant patients after conversion from cyclosporine (CsA) to tacrolimus at the time of acute rejection, but no prospective, controlled study has been performed to date. Here, we report the first randomized study comparing patients whose therapy was changed at a first episode of acute rejection to tacrolimus with those who were maintained on CsA microemulsion (ME). METHODS: This 3-month, prospective, open, multicenter, parallel-group study was conducted at 15 centers in seven European countries. In total, 119 renal graft recipients experiencing a first biopsy-proven acute rejection episode while receiving CsA-ME were randomized (1:1) to start tacrolimus-based therapy (n=61) or to continue CsA-ME-based therapy (n=58). RESULTS: Baseline characteristics were comparable for both groups. The initial rejection episode responded to steroid treatment in 93.4% (tacrolimus) and 63.8% (CsA-ME) (P=0.001), respectively. In patients at risk, the incidence of recurrent rejection events within 3 months was significantly lower with tacrolimus therapy (5/57, 8.8%) compared with CsA-ME therapy (15/44, 34.1%) (P=0.002). Patient and graft survival were similar in both study groups 3 months after randomization. The most frequently reported adverse events were increased serum creatinine (29.5% vs. 22.4%), hypertension (24.6% vs. 22.4%), and urinary tract infection (18.0% vs. 20.7%) for tacrolimus versus CsA-ME. Tremor was more common in tacrolimus treated-patients (17.4% vs. 2.1%, P=0.011). CONCLUSIONS: Early conversion to tacrolimus therapy benefited the resolution of acute rejection episodes and significantly reduced the risk of recurrent rejection compared with continuation of CsA-ME.     

C2 monitoring in maintenance renal transplant recipients: is it worthwhile?

Presently, there is little knowledge regarding cyclosporine (CsA) concentration at 2 hr post-dose (C2) monitoring in maintenance patients. This study evaluates the actual C2 range in stable renal transplant recipients (who underwent transplantation >12 months ago). In addition, we investigated whether underexposure or overexposure to CsA (assessed by C2) affects graft function (as measured by serum [S]-creatinine). All renal transplant recipients in Norway receiving CsA were asked to participate; 1447 fulfilled the criteria. Valid C2 and CsA trough concentration (C0) measurements were performed in 1032 renal transplant recipients (71%) monitored by C0. Target C0 level was 75 to 125 mumol/L. CsA levels were measured using a Cloned Enzyme Donor Immunoassay method, and all analyses were performed in the same laboratory (overall mean [+/-standard deviation] CsA C0=112+/-31 mug/L, CsA C2=697+/-211 mug/L [range 81-1580 mug/L], CsA dose [mg/day]=208+/-61, CsA dose [mg/kg/day]=2.8+/-1.1, and S-creatinine=141+/-58 mumol/L). A univariate analysis of variance showed that patients with C2 levels between 700 and 800 mug/L (n=203, S-creatinine=136+/-49 mumol/L) had significantly lower S-creatinine levels compared with patients with C2 levels greater than 950 mug/L (n=94, S-creatinine=152+/-56 mumol/L) (P<0.02). The same was true for patients with C2 levels less than 450 mug/L (n=95, S-creatinine 141+/-72 mumol/L) (P<0.05) when compared with patients with C2 levels greater than 950 mug/L. There was no significant difference in S-creatinine between patients in the low and intermediate C2 group; 666 patients had C0 levels in the therapeutic range (75-125 mumol/L). A linear regression showed a significant relation between S-creatinine and C2 for these patients (P=0.03). The corresponding relation between S-creatinine and C0 was nonsignificant (P=0.3). Monitoring of C2 in maintenance patients is a valuable tool to detect overexposure to CsA. Until results from prospective studies are available, we recommend C0 in the therapeutic range and reduction in CsA in overexposed patients, aiming at a C2 value between 700 and 800 mug/L.     

Prognostic factors for the survival of surgically treated patients for non-small cell lung cancer

The survival and outcome rates of 284 patients who underwent surgical treatment for non-small cell lung cancer were assessed retrospectively. Resectability rate was 94.1%, hospital mortality 3.9% (n=11) and the mortality rates in patients who underwent pneumonectomy or lobectomy were 8.9% and 0.6%, respectively. The overall 5-year survival was 43.6%. Female gender, earlier stages of disease and a complete resection were strongly predictive for a long-term survival. Women in stage IA disease had a 5-year survival rate of 92.7%. The 5-year survival rate for patients in stages IIIA and N2 disease who underwent a complete resection was 21.9%, and 9% for those who did not undergo a complete resection. It is concluded that the best surgical results were observed in women who were operated on at an early stage of disease. A complete resection also contributed to a better outcome, even for patients in stage IIIA and N2 disease.     

Screening for gene mutations in a 500 kb neuroblastoma tumor suppressor candidate region in chromosome 1p; mutation and stage-specific expression in UBE4B/UFD2

Deletion of a part of the short arm of chromosome 1 is one of the most common chromosomal rearrangements observed in neuroblastoma (NBL) tumors and it is associated with a poor prognosis. No NBL tumor suppressor gene has yet been identified in the region. Our shortest region of overlap of deletions, ranging from marker D1S80 to D1S244, was shown to partly overlap a 500 kb region that was homozygously deleted in a NBL cell line. We have screened seven genes known to reside in or very close to this overlap consensus region, UBE4B/UFD2, KIF1B, DFFA, PGD, CORT, PEX14, and ICAT, for coding mutations in NBL tumor DNA. A few deviations from the reference sequences were identified; most interestingly being a splice site mutation that was detected in UBE4B/UFD2 in a stage 3 NBL with a fatal outcome. This mutation was neither present in the patients constitutional DNA nor in any of 192 control chromosomes analysed. Also, the expression of UBE4B/UFD2 was markedly diminished in the high-stage/poor-outcome tumors as compared to the low-stage/favorable-outcome tumors. Overall, the number of amino-acid changes in the genes of the region was low, which shows that mutations in these genes are rare events in NBL development. Given the data presented here, UBE4B/UFD2 stands out as the strongest candidate NBL tumor suppressor gene in the region at this stage.     

Imaging of Experimental Rat Gliomas Using a Clinical MR Scanner

Background: Studies of brain tumor development in experimental animal models have to date mostly been based on post-mortem histological examinations. The use of magnetic resonance imaging (MRI) may provide a non-invasive technique for studying tumor growth and treatment effects in such animal models. However, most of these studies have been performed on purpose-dedicated small bore magnetic resonance (MR) systems, of high cost and limited availability. The purpose of this study was thus to obtain high-resolution images of experimental gliomas in the rat brain, using a clinical 1.5T MR scanner. Methods: Anesthesized rats bearing BT₄C brain tumors were positioned into a specially designed immobilizing device, and a small circular coil was positioned onto the skulls. Two T1 weighted series were acquired before and after subcutaneous contrast injections. A T2 weighted series was also obtained. The rats were then sacrified, the brains removed, and the histological tumor volumes were compared to the volumes obtained on MRI. Results: There were visible tumors in 10 of 13 animals scanned on MR. The rim of the tumors were visualized on T1 weighted series without contrast. On T1 images with contrast, the tumors were seen as high signal intensity areas. The T2 weighted images showed peritumoral edema. No necrosis or cystic parts of the tumors were detected. There was a consistency between the MR and the histology findings, showing a high degree of correlation between the two volume determination methods. Conclusions: High-resolution images of experimental rat gliomas can be obtained using a clinical MR scanner and a commercially available RF coil. This MRI technique may also be expanded to extraneural rat tumor models, for studies of tumor development and treatment.     

The synthetic retinoid RO 13-6307 induces neuroblastoma differentiation in vitro and inhibits neuroblastoma tumour growth in vivo

Retinoids modulate cell proliferation, differentiation and apoptosis in a variety of tumour cells including leukaemia and neuroblastoma, a childhood tumour of the sympathetic nervous system. 13-cis retinoic acid is in clinical use against minimal residual disease in neuroblastoma, where the effect seems to depend on dose, scheduling and tumour mass. Novel retinoids are searched for, to improve potency and lower toxicity. We investigated the effect of the synthetic retinoid Ro 13-6307 on neuroblastoma growth in vitro on SK-N-BE(2) and SH-SY5Y cells. Furthermore, effects on tumour growth and the toxicity profile were investigated in a rat xenograft model. Effects of Ro 13-6307 were compared to 13-cis RA (retinoic acid) in vitro and in vivo. Neuroblastoma cells treated with 1 microM Ro 13-6307 exhibited neuronal differentiation, decreased proliferation and accumulation of cells in G1 phase in at least the same magnitude as 5 microM 13-cis RA. No apoptosis was detected in vitro. Treatment of nude rats with neuroblastoma using Ro 13-6307, 0.12 mg p.o. daily, decreased neuroblastoma growth in vivo, in terms of tumour volume during treatment and tumour weight at sacrifice (p < 0.05). In contrast, Ro 13-6307, 0.08 mg p.o. daily, resulted in no significant reduction in tumour growth. All rats treated with Ro 13-6307 gained less weight than control rats, but they exhibited no other signs of toxicity. The toxicity profile of Ro 13-6307 was similar to what we found with 13-cis RA. Our preclinical results suggest that Ro 13-6307 may be a candidate retinoid for clinical oral therapy of neuroblastoma in children.     

Apoptosis and tumor remission in liver tumor xenografts by 4-phenylbutyrate

4-phenylbutyrate (triButyrate trade mark, PB) a derivative of the short-chain fatty acid, butyrate, possesses anti-tumor activity in vitro in different tumor cell lines. Unlike most cytostatic compounds, PB possesses low toxicity. In order to evaluate possible clinical use of PB in cancer therapy, hepatocarcinoma (Hep3B) and hepatoblastoma (HepT1) cell lines, as well as xenografts derived from those in nude rats, were treated with PB in different dose (1-100 mM) and time regimens. Treatment with 10 mM of PB for 24 h (or 5 mM for 48 h) was shown to significantly inhibit Hep3B cell growth in vitro. The HepT1 cell line was more sensitive to PB treatment: already 1 mM of PB for 24 h significantly inhibited the growth of the cells. PB also resulted in regression of xenografts derived from these cell lines in vivo, when administrated by mini-pump with an intratumor catheter, yielding 20 micro mol of PB per cm3 of tumor volume per day. TUNEL assay and caspase-3 activity measurements suggested apoptosis to be the cell death mechanism in both cell lines and xenografts. Increased histones H3 and H4 acetylation was shown in both cells and xenografts, and the inhibition of histone deacetylase is proposed as the main trigger for the anti-tumor action of PB. Concomitant induction of p21Waf1/Cip1 expression was detected by RNase protection assay and Western blotting. Reduction in expression of alpha-fetoprotein was found both in Hep3B cells and xenografts, suggesting also a differentiation effect by PB.