A phase I/II study of siltuximab (CNTO 328), an anti-interleukin-6 monoclonal antibody, in metastatic renal cell cancer.

BACKGROUND: Serum interleukin (IL)-6 levels correlate with disease outcomes in renal cell carcinoma (RCC) patients. Siltuximab, a chimeric, murine-human mAb against IL-6, was evaluated in a three-part phase I/II study in patients with progressive metastatic RCC. METHODS: In part 1, 11 patients received 1, 3, 6, or 12mgkg-¹ at weeks 1, 4 and q2w × 2 thereafter; in part 2, 37 patients randomly received 3 or 6 mgkg-¹ q3w × 4; in part 3, 20 low-risk patients received 6mgkg-¹ q2w × 6. Modified WHO response criteria were assessed at weeks 7, 11, the 6-week follow-up, and when clinically indicated. RESULTS: Siltuximab was well tolerated overall, with no maximum tolerated dose or immune response observed. In all, 5 out of 11, 17 out of 37, and 9 out of 20 patients in parts 1, 2, and 3, respectively, received extended treatment beyond 4-6 initial infusions. In part 2, stable disease (SD) (≥11weeks) or better was achieved by 11 out of 17 (65%) 3 mgkg-¹ treated patients (one partial response (PR) ~8 months, 10 SD) and 10 out of 20 (50%) 6mgkg-¹ treated patients (10 SD). In part 3, documented complete or PR was not observed, but 13 out of 20 (65%) patients achieved SD. CONCLUSION: Siltuximab stabilised disease in >50% of progressive metastatic RCC patients. One PR was observed. Given the favourable safety profile of siltuximab and poor correlation of tumour shrinkage with clinical benefit demonstrated for other non-cytotoxic therapies, further evaluation of dose-escalation strategies and/or combination therapy may be considered for patients with RCC.

A clinical study assessing the tolerability and biological effects of infliximab, a TNF-alpha inhibitor, in patients with advanced cancer.

BACKGROUND: Tumour necrosis factor-alpha (TNF-alpha) is an important regulator of the chronic inflammation contributing to tumour progression. Infliximab, an anti-TNF-alpha monoclonal antibody was investigated in this trial of patients with advanced cancer. The primary objectives were to determine the safety profile and biological response of infliximab in a cancer population. Clinical response was a secondary objective. PATIENTS AND METHODS: Forty-one patients received infliximab at 5 mg/kg (n = 21) or 10 mg/kg (n = 20) i.v. at 0 and 2 weeks and then every 4 weeks. Post-treatment samples were measured for changes in plasma and serum TNF-alpha, CCL2, IL-6 and C-reactive protein (CRP). RESULTS: Infliximab was well tolerated with no dose-limiting toxic effects. At both doses of infliximab, neutralisation of serum TNF-alpha was observed after 1 h while plasma CCL2, IL-6 and serum CRP were decreased 24 and 48 h following infliximab administration. Seven patients experienced disease stablisation (range 10-50+ weeks). There was no evidence of disease acceleration in any patient. CONCLUSIONS: Infliximab treatment was safe and well tolerated in patients with advanced cancer. There was evidence of biological activity with baseline TNF-alpha and CCL2 being correlated with infliximab response.

Association of the anti-inflammatory activity of phosphodiesterase 4 (PDE4) inhibitors with either inhibition of PDE4 catalytic activity or competition for [3H]rolipram binding.

Phosphodiesterase 4 (PDE4) inhibitors are novel anti-inflammatory compounds. Unfortunately, the archetypal PDE4 inhibitor rolipram produces central nervous system and gastrointestinal side-effects. To exploit these agents, we need to identify PDE4 inhibitors that retain the anti-inflammatory activity with a reduced potential to elicit unwanted side-effects. PDE4 possesses both cyclic AMP catalytic activity that is inhibitable by rolipram and a high affinity binding site for rolipram. The function of this high affinity rolipram binding site is unclear; however, certain pharmacological effects of PDE4 inhibitors are associated with competition for this site. Since PDE4 inhibitors suppress both monocyte and neutrophil activation, the present experiments were carried out to establish a correlation between suppression of monocyte activation [tumor necrosis factor alpha (TNF alpha) formation] or suppression of neutrophil activation (degranulation) with inhibition of either PDE4 catalytic activity or [3H] rolipram binding. Suppression of TNF alpha formation demonstrated a strong correlation with inhibition of PDE4 catalytic activity (r=0.87; P<0.01; Spearman's Rho = 0.79, P<0.05), whereas there was no correlation with inhibition of [3H]rolipram binding(r=0.21, P>0.5; Spearman's Rho=0.16, P>0.5). Suppression of neutrophil degranulation was not associated with inhibition of PDE4 catalytic activity (r=0.25, P>0.4; Spearman's Rho=0.33, P>0.2), but was associated with inhibition of [3H]rolipram binding (r=0.68, P<0.05; Spearman's Rho=0.6, P=0.06). These results indicate that anti-inflammatory effects of PDE4 inhibitors can be associated with either inhibition of PDE4 catalytic activity or high affinity rolipram binding.

Protein kinase C regulates TNF-alpha production by human monocytes.

Tumor Necrosis Factor alpha (TNF alpha) is a cytokine mediator that is produced primarily by activated monocytes/macrophages in response to endotoxin/lipopolysaccharide (LPS) as well as other stimuli. The second messenger systems that regulate the synthesis and release of TNF alpha are not clearly defined. In the present study, the role of protein kinase C (PKC) in the production of TNF alpha was investigated in human peripheral blood monocytes stimulated with either LPS or zymosan. Two broad spectrum protein kinase inhibitors (staurosporine and K252a) and two PKC specific inhibitors (calphostin C and chelerythrine), were used as probes to delineate the involvement of PKC in the production of TNF alpha. The results indicate that inhibition of PKC diminished LPS- or zymosan- induced TNF alpha production in a concentration-dependent manner. The IC50 values for the inhibition of TNF alpha production were 0.2 nM for staurosporine, and 20 nM for K252a, Calphostin C and chelerythrine. Furthermore, long term PMA treatment of these cells (to abrogate PKC-mediated responses) resulted in a significant reduction of stimuli-induced TNF alpha production. LPS and zymosan also induced an increase in membrane associated PKC activity in human monocytes, which could be inhibited by pretreatment of the cells with calphostin C. Finally, western blot analysis with PKC isoform-specific antibodies demonstrates that the alpha and xi are the predominent isoforms expressed in human monocytes. These data strongly suggest that an initial step in TNF alpha production by human monocytes challenged with physiological stimulants, such as LPS and zymosan, involves a PKC-dependent mechanism.

Inhibition of CD44, CD45 and LFA-3 mediated cytokine release from human monocytes by SK&F 86002 and pentoxifylline.

Compounds from two distinct pharmacological classes namely, SK&F 86002 and pentoxifylline, were examined for their effects on TNF alpha and IL-1 beta release by human monocytes stimulated with LPS or monoclonal antibodies to three cell surface glycoproteins, CD44, CD45 and LFA-3 (LFA-3 is also known as CD58). SK&F 86002, an inhibitor of 5-LO and CO in arachidonic acid metabolism, inhibited LPS-induced release of TNF alpha and IL-1 beta with an IC50 of 1 microM. At this dose, it also inhibited by > 50%, release of both cytokines induced by the three monoclonal antibodies. Pentoxifylline, a methylxanthine derivative with phosphodiesterase inhibitory activity, selectively inhibited LPS-induced TNF alpha release with an IC50 of 100 microM. TNF alpha and IL-1 beta release mediated by the monoclonal antibodies were inhibited by less than 30% in the presence of 100 microM pentoxifylline. These results suggest that (a) LPS induced cytokine release shares a common step with the physiologically relevant stimuli (involving cross-linking of cell surface receptors), and that this pathway is sensitive to inhibition by SK&F 86002 and, (b) SK&F 86002 is more potent than pentoxifylline in inhibiting TNF alpha and IL-1 beta release induced by both stimuli.

Upregulation of osteopontin in ischemia-induced renal failure in rats: a role for ET-1?

In this study, the involvement of osteopontin in a rat model of ischemia-induced acute renal failure (ARF) was evaluated. In unilaterally nephrectomized Sprague Dawley rats where the left artery was occluded for 30 min., plasma creatinine levels increased significantly within two hours following reperfusion indicating the onset of renal failure. Northern analysis of kidney cortical RNA from these rats showed a time-dependent increase in osteopontin mRNA expression that was significantly higher than sham-operated rats. Since endothelin-1 (ET-1) is implicated as a mediator of acute renal failure, we evaluated its effects on osteopontin expression in a rat mesangial cell-line. Data from in vitro studies indicated that endothelin-1 (ET-1) caused a modest but reproducible increase in osteopontin mRNA in these cells. While the signal for osteopontin upregulation in the rat model is not known, ET-1, which is known to be increased during ischemia, may contribute at least in part to this process.

Inhibition of interleukin-1 (IL-1) and tumor necrosis factor (TNF) production by pyridinyl imidazole compounds is independent of cAMP elevating mechanisms.

Exposure of human monocytes (HM) to E. coli lipopolysaccharide (LPS) results in measurable production of both IL-1 beta and TNF alpha in culture supernatants. It has previously been reported that the elevation of cAMP levels in HM selectively suppresses the LPS-induced TNF alpha but not IL-1 beta production. In this study we investigated whether the novel anti-inflammatory drug, SK&F 86002 [5-4(-pyridyl)-6(4-fluorophenyl)-2,3-dihydroimidazole(2,1-b)thi azol] and related analogs of the pyridinyl imidazole class, inhibit IL-1 and TNF production via a cAMP-dependent mechanism. These compounds, when added together with LPS result in inhibition of IL-1 and TNF production with equal-rank-order potency. Although the pyridinyl imidazole compounds were found to be generally weak phosphodiesterase inhibitors, they did not affect cAMP levels in HM, alone or in the presence of LPS. In contrast, PGE2, which significantly elevated intracellular cAMP levels, inhibited TNF but not IL-1 production at the transcriptional level. Taken together, these results suggest that the pyridinyl imidazoles inhibit the production of IL-1 beta and TNF alpha through pathways independent of cAMP elevating mechanisms.

Identification and characterization of endothelin receptors on rat osteoblastic osteosarcoma cells: down-regulation by 1,25-dihydroxy-vitamin D3.

Endothelins (ETs) (ET-1, ET-2, and ET-3), a family of 21-amino acid peptides, mediate a host of biological responses by binding to specific cell surface receptors termed ETA and ETB. Because a role for ET in bone remodeling has been suggested, the present study was undertaken (a) to characterize ET receptors and their responses in the rat osteosarcoma cell line ROS 17/2.8 and (b) to study their regulation by 1,25-dihydroxy-vitamin D3. Binding studies using 125I-ET-1 (a nonselective agonist) and 125I-IRL-1620 (an ETB receptor-selective agonist) indicated that these cells display high affinity ETA and ETB receptors in the ratio of 3:1. Addition of ET-1 or sarafotoxin 6c to myo-[3H]inositol-labeled cells resulted in an increase in inositol phosphate accumulation as well as in intracellular Ca2+ release, suggesting that these receptors are coupled to phospholipase C. In addition, ET-1 but not sarafotoxin 6c induced a modest increase in the expression of osteocalcin protein that was completely blocked by BQ123 (an ETA receptor-selective antagonist), indicating that activation of ETA receptors plays a role in the induction of osteocalcin. Treatment of ROS osteoblasts with 10 nM 1,25-dihydroxy-vitamin D3 for 14 hr resulted in a significant (> 50%) decrease in 125I-ET-1 and 125I-IRL-1620 binding. This decrease in binding was shown to be due to a decrease in the number of ET receptors, with no change in affinity. Although both ETA and ETB receptors were down-regulated in response to 1,25-dihydroxy-vitamin D3, only ETA receptor mRNA levels were significantly decreased, with very little change in ETB mRNA levels. These data indicate that ROS osteoblasts display both ETA and ETB receptors that are functional. Induction of osteocalcin was primarily mediated by ETA receptors, and these receptors were also down-regulated at the mRNA level by 1,25-dihydroxy-vitamin D3.

Inhibition of LPS-induced TNF alpha production in human monocytes by adenosine (A2) receptor selective agonists.

We examined the effects of adenosine A1 and A2 receptor agonists on LPS-stimulated TNF alpha production by human monocytes isolated from peripheral blood. We have demonstrated that CGS-21680, a highly selective A2 agonist inhibited production of TNF alpha at the protein level by 75% whereas the A1 selective agonist N6 reduced TNF alpha production by only 25%. The action of CGS-21680 was mediated via the A2 receptors since its effect on TNF production was blocked by 3,7-dimethyl-1-propargylxanthine (DMPX) but not by xanthine amine cogener (XAC), antagonists selective for the A2 and A1 receptors, respectively. Thus intervention with A2-selective agonists or compounds that can elevate endogenously released adenosine may be beneficial in TNF alpha-mediated diseases.

Down regulation of kidney neutral endopeptidase mRNA, protein and activity during acute renal failure: possible mechanism for ischemia-induced acute renal failure in rats?

Neutral endopeptidase (NEP, 24.11) is an ectoenzyme involved in the degradation of peptide hormones such as endothelin (ET), atrial natriuretic factor and enkephalins. The current study was designed to assess the involvement of NEP in ischemia-induced acute renal failure (ARF). In unilaterally nephrectomized Sprague-Dawley rats, the left renal artery was occluded for 30 min under pentobarbital anesthesia (40 mg/kg, i.p.) at 37 degree C. In addition to plasma creatinine levels, NEP activity was determined in renal cortical membranes at 0, 2, 5, and 24 h following reperfusion. Plasma creatinine levels significantly increased at 2, 5 and 24 h. There was a significant decrease in NEP activity as early as 2 h following reperfusion that was maintained up to 24 h (57.9 +/- 4%) with a concomitant loss of enzyme protein shown by Western analysis. Northern analysis of kidney cortical RNA, probed with an NEP cDNA, showed a 45% decrease in NEP mRNA level by the end of the ischemic period and decreased further during reperfusion. Thus, decrease in NEP mRNA levels preceded the changes in protein level, enzyme activity and plasma creatinine levels. These data, along with the reported increase in the tissue level of ET in kidney cortex, and the beneficial effect of ET antibody as well as ET receptor antagonist in ARF, suggest that down regulation of NEP, one of the mechanisms leading to increased tissue level of ET, may be a contributing factor to ARF.

Characterization of cAMP-dependent inhibition of LPS-induced TNF alpha production by rolipram, a specific phosphodiesterase IV (PDE IV) inhibitor.

Bacterial endotoxins (lipopolysaccharide or LPS) provoke shock and tissue injury by eliciting the release of toxic factors from reticuloendothelial cells. One of the principal endogenous factors involved in this process is tumor necrosis factor alpha (TNF alpha). In this study, inhibitors selective for different classes of phosphodiesterases (PDE), were examined for their effects on LPS-induced TNF alpha production by human monocytes. The selective cAMP-PDE IV inhibitors, rolipram and RO-20-1724 were capable of inhibiting LPS-induced TNF alpha production by human monocytes in a concentration-dependent manner. Rolipram was used to examine further the cellular pharmacology of PDE IV inhibitors on cytokine production. The IC50 for inhibition of LPS-induced TNF alpha production by rolipram was 0.1 microM, whereas production of IL-1 beta or IL-6 was unaffected. Furthermore, rolipram was equally effective in inhibiting TNF alpha production by a number of other stimuli. Inhibition of TNF alpha production by rolipram was associated with an elevation of intracellular cAMP, consistent with a mechanism involving phosphodiesterase inhibition. Rolipram was efficacious in suppressing LPS-induced TNF alpha mRNA expression, and at the protein level was also active when added to cultures post-stimulated with LPS. This indicates that rolipram may act at both the transcriptional and translational levels. Rolipram inhibited TNF alpha production in vivo in a rat endotoxemia model. Collectively, these data suggest that the prototypic inhibitor of PDE IV isozyme, rolipram, can effectively and selectively inhibit LPS-induced TNF alpha production through elevation of intracellular cAMP.

SB 207499 (Ariflo), a potent and selective second-generation phosphodiesterase 4 inhibitor: in vitro anti-inflammatory actions.

First-generation phosphodiesterase 4 (PDE4) inhibitors, such as rolipram, inhibit the activation of immune and inflammatory cells. The clinical use of these compounds is limited by gastrointestinal side effects, such as increased acid secretion and nausea. Consequently, the challenge has been to design novel PDE4 inhibitors that maintain the anti-inflammatory actions of rolipram while achieving an improved side effect profile. Among the first of this new class of PDE4 inhibitors specifically designed to have an improved therapeutic index relative to earlier compounds is SB 207499 (Ariflo) [c-4-cyano-4-(3-cyclopentyloxy-4-methoxy-phenyl)-r-1-cyclohexanecarboxyl ic acid]. In this study, we compared the anti-inflammatory and gastric secretogogue activities of SB 207499 with those of rolipram. The cellular models used were (1) histamine release from human basophils, (2) tumor necrosis factor-alpha generation in human monocytes, (3) degranulation of human neutrophils, (4) antigen-driven proliferation and cytokine synthesis from human T cells and (5) acid secretion from isolated rabbit gastric glands. SB 207499 inhibited the activation of a variety of immune and inflammatory cells in a concentration-dependent manner: (1) histamine release in basophils [-log IC25 = 6.6 +/- 0.3 vs. 8.0 for (R)-rolipram], (2) lipopolysacchride-induced TNF-alpha formation in monocytes [-log IC50 = 7.0 +/- 0.1 vs. 7.2 +/- 0.1 for (R)-rolipram], (3) fMLP-induced degranulation in neutrophils [-log IC15 = 7.1 +/- 0.2 vs. 6.4 +/- 0.5 for (R)-rolipram], (4) house dust mite induced-proliferation of peripheral blood mononuclear cells [-log IC40 = 6.5 +/- 0.3 vs. 6.4 +/- 0.3 for (R)-rolipram] and (5) ragweed-induced production of interferon-gamma [-log IC50 = 5.4] and interleukin-5 [-log IC50 = 5.0]. Although SB 207499 inhibits the activation of a variety of immune and inflammatory cells with a potency equal to that of rolipram, it is > 100-fold less potent than the latter compound as an acid secretagogue [-log EC50 = 6.1 +/- 0.1 vs. 8.3 +/- 0.2 for (R)-rolipram]. Collectively, these data indicate that SB 207499 retains the anti-inflammatory activity of the prototypical PDE4 inhibitor rolipram but is substantially less likely to stimulate gastric acid secretion.

A novel human bone marrow stroma-derived cell line TF274 is highly osteogenic in vitro and in vivo.

A novel, immortalized, human bone marrow stroma-derived cell line TF274 is described which has the ability to form bone both in vitro and in vivo. Under basal conditions these cells expressed alkaline phosphatase (ALP) and type I collagen genes which are characteristic of the osteoblast phenotype. ALP levels were upregulated in the presence of osteotropic agents such as parathyroid hormone (PTH), transforming growth factor beta (TGF-beta), and BMP-2. In addition, PTH also increased cAMP levels in these cells. The capacity of these cells to form bone in vitro was evaluated by culturing them in the presence of L-ascorbic acid and beta-glycerophosphate. Matrix mineralization in these cultures was assessed by Alizarin Red staining and increased 45Ca uptake. Under these conditions mineralized nodule formation was observed in less than 2 weeks. Northern analysis of TF274 cells at various times during the mineralization process indicated a temporal expression of the osteocalcin gene that is typically associated with differentiating osteoblasts. The osteogenic nature of TF274 cells was confirmed in vivo using the severe combined immunodeficient (SCID) mouse model. Antibodies to human leukocyte antigens (HLA), class I antigens, and human OKa blood group antigen were used to demonstrate that the lesions formed were of human origin. By 21 days, the lesion consisted of a homogeneous focus of ALP-positive cells containing areas of mineralized bone lined with tartarate-resistant acid phosphatase (TRAP) positive osteoclasts. Thus, the TF274 cells exhibit osteogenic potential both in vitro and in vivo. This immortalized cell line represents a consistent source of cells that can be used to study human osteoblast differentiation both in vitro and in vivo.