Intravenous administration of recombinant human IL-10 suppresses the development of anti-thy 1-induced glomerulosclerosis in rats.

AIM: Interleukin-10 (IL-10) is a cytokine with potent antifibrotic and anti-inflammatory properties. However, IL-10 has a very short plasma half-life in vivo. This prompted the question whether a short intravenous treatment might have prolonged effects on more chronic processes like sclerosis. METHODS: Glomerulosclerosis was induced by anti-Thymocyte 1 (Anti-Thy 1) antibody. Four days after induction, an intravenous injection of recombinant human IL-10 (rhIL-10) was given for 3 consecutive days. Untreated rats received vehicle only (phosphate-buffered saline). Parameters of inflammation and fibrosis were assessed at protein and mRNA levels. Untreated rats showed renal histopathological changes as compared to normal rats. RESULTS: Glomerular matrix expansion and inflammatory cell influx was observed and an increase in glomerular-inducible nitric oxide synthetase and α-smooth muscle actin (α-SMA) were found on the protein level, factors that were clearly attenuated by IL-10 treatment. In particular, the decrease of matrix metalloproteinase-13 levels between days 4 and 7 was completely prevented by IL-10. In contrast, IL-10 did not significantly reduce mRNA levels for procollagen α1(1), α-SMA, and transforming growth factor 1. CONCLUSION: A short-term treatment with rhIL-10 after induction of Anti-Thy 1 antibody nephritic rats attenuated intraglomerular inflammation, and at the protein level also influenced the parameters reflecting matrix deposition and degradation. Despite in fact that IL-10 was shown to be effective in the inhibition of matrix deposition, it had no beneficial effect on proteinuria. LAY ABSTRACT: Interleukin-10 is a cytokine with potent antifibrotic and anti-inflammatory properties. Its short plasma half-life raises the question whether a short intravenous treatment might have prolonged effects on chronic disease like sclerosis. To confirm this, recombinant human interleukin-10 was used to treat glomerulosclerosis in rats. The disease was induced by Anti-Thy 1 antibody. Four days after induction, an intravenous injection of IL-10 was given for 3 consecutive days. Untreated rats received vehicle only (phosphate-buffered saline). Parameters of inflammation and fibrosis were assessed at protein and mRNA levels. In this study, untreated rats showed renal histopathological changes as compared to normal rats. Glomerular matrix expansion and inflammatory cell influx was observed, and increases in glomerular nitric oxide synthetase and α-smooth muscle actin α-SMA were found on the protein level. In contrast, treated rats clearly showed reduction of all these parameters. In particular, the decrease of anti-matrix metalloproteinase-13 (MMP-13) levels between days 4 and 7 was completely prevented by IL-10. However, IL-10 did not significantly reduce mRNA levels for procollagen α1(1), α-SMA, and TGFß-1. Based on these results, it can be concluded that a short-term treatment with rhIL-10 after induction of Anti-Thy 1 antibody in nephritic rats attenuated intraglomerular inflammation, and at the protein level also influenced the parameters reflecting matrix deposition and degradation. Despite in fact that IL-10 was shown to be effective in the inhibition of matrix deposition, it had no beneficial effect on proteinuria.

Pharmacokinetics and whole body distribution of elastase derived angiostatin (K1-3) in rats.

In the current study, we determined short-term pharmacokinetics and whole body distribution of elastase derived angiostatin [angiostatin(k1-3)] in rats after i.v. injection of radiolabelled protein. Since in gamma-camera studies, no tumor specific angiostatin(k1-3) accumulation was observed, general pharmacokinetics were studied in tumor free rats. By one-compartment model fitting of the data, Km 7.3 +/- 1.7 microg x ml(-1), Vmax 0.94 +/- 0.19 microg x min(-1), V, 10.9 +/- 2.5 ml and intrinsic clearance (Vmax/Km) 0.128 ml x min(-1) were calculated. Of the injected dose (I.D.) of angiostatin(k1-3), 12.1 +/- 2.1% per gram tissue was present in the kidneys 10 min after injection. Accumulation of angiostatin(k1-3) was detectable in spleen, liver, lungs and heart 10 min after injection. Sixty minutes after injection, kidney associated angiostatin(k1-3) had decreased, whereas in stomach and small intestines a small increase was seen. Immunohistochemical analysis demonstrated specific staining of interstitial cells of the kidney, liver Kupffer cells and endothelium of larger blood vessels of the lungs. Renal clearance of angiostatin(k1-3) and/or fragments is a major route of elimination, whereas lack of accumulation of radioactivity in the faeces indicates little hepatic elimination or hepatic elimination followed by enterohepatic cycling of the protein's degradation products. Instant blood coagulation at the site of vascular activation and the occurrence of respiratory problems upon administration of higher doses of angiostatin(k1-3) warrants further investigation of the protein's potential side effects. The data presented can be applied to study the relation between angiostatin(k1-3) treatment regimens, blood concentration levels, anti-tumor activity and harmful effects.

Validation of S-1'-[18F]fluorocarazolol for in vivo imaging and quantification of cerebral beta-adrenoceptors.

S-1'-[18F]fluorocarazolol (S-(-)-4-(2-hydroxy-3-(1'-[18F]fluoroisopropyl)-aminopropoxy)carba zole, a non-subtype-selective beta-adrenoceptor antagonist) has been investigated for in vivo studies of beta-adrenoceptors. Previous results indicated that uptake of this radioligand in heart and lung can be inhibited by beta-adrenoceptor agonists and antagonists. In the present study, blocking, displacement and saturation experiments were performed in rats, in combination with metabolite analysis to investigate the suitability of this radioligand for in vivo positron emission tomography (PET) imaging and quantification of beta-adrenoceptors in the brain. The results demonstrate that, (i) the uptake of S-1'-[18F]fluorocarazolol reflects specific binding to beta-adrenoceptors, (ii) binding of S-1'-[18F]fluorocarazolol to atypical or non-beta-adrenergic sites is negligible, (iii) uptake of radioactive metabolites in the brain is less than 25% of total radioactivity, 60 min after injection, (iv) in vivo measurements of receptor densities (Bmax) in cortex, cerebellum, heart, lung and erythrocytes are within range of densities determined from in vitro assays, (v) binding of S-1'-[18F]fluorocarazolol can be displaced. In conclusion, S-1'-[18F]fluorocarazolol seems to possess the appropriate characteristics to visualize and quantify beta-adrenoceptors in vivo in the central nervous system using PET.

Carbon-11-labeled daunorubicin and verapamil for probing P-glycoprotein in tumors with PET.

UNLABELLED: One of the mechanisms for multidrug resistance (MDR) of tumors is an overexpression of the P-glycoprotein (P-gp). The cytostatic agent daunorubicin and the modulator verapamil were labeled with 11C to probe P-gp with PET. METHODS: Carbon-11-daunorubicin was prepared from 11CCH2N2 with an aldehyde precursor, followed by hydrolysis. Carbon-11-verapamil was synthesized by 11C-methylation. Both tracers were evaluated by investigating pharmacokinetics in rats and in vitro cell kinetics using human ovarian carcinoma cells. RESULTS: Amounts of 111 MBq 11C-daunorubicin were prepared. Biodistribution studies of 11C-daunorubicin in male Wistar rats showed dose-dependent pharmacokinetics, whereas with 11C-verapamil the pharmacokinetics were dose independent. In in vitro experiments with cells, the ratio of accumulation of 11C-daunorubicin in drug sensitive/resistant cell lines was 16. Addition of verapamil resulted in increased accumulation of 11C-daunorubicin in the resistant cell line. The ratios of 11C-verapamil accumulation in drug-sensitive versus the MDR counterpart were 4-5. CONCLUSION: Carbon-11-daunorubicin and 11C-verapamil both have potential for in vivo probing of P-glycoprotein with PET.

Quantification of the beta-adrenoceptor ligand S-1'-[18F]fluorocarazolol in plasma of humans, rats and sheep.

Myocardial and pulmonary beta-adrenoceptors can be imaged with 2-(S)-(-)-(9H-carbazol-4-yl-oxy)-3-[1-(fluoromethyl)ethyl]amino-2- propanol (S-1'-[18F]fluorocarazolol, I). Quantification of unmodified fluorocarazolol in plasma is necessary for analysis of PET images in terms of receptor densities. We have determined I and its radioactive metabolites in rat, sheep and human plasma, using (1) solid-phase extraction (C18) followed by reversed-phase HPLC and (2) direct injection of untreated plasma samples on an internal-surface reversed-phase (ISRP) column. The two methods were in good agreement. Unmodified I decreased from over 99% initially to less than 5%, 5-10% and 20% at 60 min post-injection in rats, sheep and human volunteers, respectively. Protein binding in sheep and human plasma was determined by ultrafiltration. The fraction of total plasma radioactivity bound to protein and the fraction representing unmodified radioligand were linearly correlated, suggesting that fluorocarazolol was more than 70% protein-bound, whereas its metabolites showed negligible protein binding. Direct injection of plasma on an ISRP column seems a convenient method for quantification of lipophilic radioligands such as fluorocarazolol.

Quantification of an 11C-labelled beta-adrenoceptor ligand, S-(-)CGP 12177, in plasma of humans and rats.

beta-Adrenoceptors in human lungs and heart can be imaged with the radioligand 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3- dihydro-2H-benzimidazol-2-11C-one (CGP 12177, [11C]I). For quantification of receptor density with compartment models by adjustment of rate constants, an 'input function' is required which consists of the integral of the concentration of unmodified ligand in arterial plasma over time. A discrepancy in the literature regarding metabolic stability of [11C]I prompted us to study metabolism in rats by reversed-phase HPLC (RP-HPLC) of trichloroacetic acid extracts of arterial plasma after i.v. injection of [11C]I (> 11.1 TBq/mmol, 11 MBq/kg). Some plasma samples were also directly applied to an internal-surface reversed-phase (ISRP) column. In parallel experiments, tritiated [11C]I was employed and methanol extracts of arterial plasma were analyzed by straight-phase TLC. The three methods were in excellent agreement. Unmodified [11C]I decreased from > 98.5% (3H) or > 99.9% (11C) initially to 57 +/- 7% at 80 min post injection due to formation of two polar metabolites. Using the RP-HPLC method, no metabolism was detectable in humans up to 30 min after injection of [11C]I (1851 MBq). Deproteinization of plasma with acetonitrile resulted in the formation of a radioactive species (artifact) which eluted immediately after the void volume in RP-HPLC and which could be mistakenly interpreted as a metabolite. Plasma protein binding was low (ca. 30%) in both humans and rats.(ABSTRACT TRUNCATED AT 250 WORDS)