Assessment of nivolumab exposure and clinical safety of 480 mg every 4 weeks flat-dosing schedule in patients with cancer.

Background: A nivolumab monotherapy flat-dosing regimen of 480 mg every 4 weeks (Q4W) has been approved in several markets, including the United States, Canada, and European Union, as an alternative dosing regimen for several indications. Approvals of this Q4W regimen were based on population pharmacokinetic (PK) analyses, established flat exposure-response relationships, and clinical safety. The objective of this study was to compare the PK exposure of 480 mg Q4W with 3 mg/kg every 2 weeks (Q2W) and 240 mg Q2W using modeling and simulation, and to evaluate clinical safety of the Q4W regimen. Patients and methods: Nivolumab PK exposure for the 480 mg Q4W schedule was simulated for 3817 patients across multiple tumor types and compared with those for the 3 mg/kg Q2W and 240 mg Q2W schedules. The safety profile of the Q4W schedule was assessed by analysis of clinical data from 61 patients who transitioned to nivolumab 480 mg Q4W from 3 mg/kg Q2W during four phase III clinical trials. Results: Compared with 3 mg/kg Q2W, nivolumab 480 mg Q4W produced similar time-averaged concentration, approximately 16% lower trough concentration, and 45% higher peak concentration at steady state. The peak concentration for 480 mg Q4W was significantly lower than that of 10 mg/kg Q2W, a dose previously shown to have an acceptable tolerability and safety profile. Treatment-related adverse events (TRAEs) that started after transitioning from 3 mg/kg Q2W to 480 mg Q4W were reported in 14.8% of patients, with 1.6% of patients reporting grades 3-4 TRAEs. Pooled safety data for these patients are consistent with those for the 3 mg/kg Q2W schedules, and no new safety signals were identified. Conclusions: The time-averaged steady-state exposure and safety profile of nivolumab 480 mg Q4W are consistent with that of 3 mg/kg Q2W across multiple tumor types. Nivolumab 480 mg Q4W represents a new dosing schedule option, and in addition to 240 mg Q2W, provides convenience and flexibility for patient care. Clinical trial numbers: NCT01721772, NCT01668784, NCT01673867, NCT01642004.

Loss of DJ-1 impairs antioxidant response by altered glutamine and serine metabolism.

The oncogene DJ-1 has been originally identified as a suppressor of PTEN. Further on, loss-of-function mutations have been described as a causative factor in Parkinson's disease (PD). DJ-1 has an important function in cellular antioxidant responses, but its role in central metabolism of neurons is still elusive. We applied stable isotope assisted metabolic profiling to investigate the effect of a functional loss of DJ-1 and show that DJ-1 deficient neuronal cells exhibit decreased glutamine influx and reduced serine biosynthesis. By providing precursors for GSH synthesis, these two metabolic pathways are important contributors to cellular antioxidant response. Down-regulation of these pathways, as a result of loss of DJ-1 leads to an impaired antioxidant response. Furthermore, DJ-1 deficient mouse microglia showed a weak but constitutive pro-inflammatory activation. The combined effects of altered central metabolism and constitutive activation of glia cells raise the susceptibility of dopaminergic neurons towards degeneration in patients harboring mutated DJ-1. Our work reveals metabolic alterations leading to increased cellular instability and identifies potential new intervention points that can further be studied in the light of novel translational medicine approaches.

PPARγ population shift produces disease-related changes in molecular networks associated with metabolic syndrome.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of adipocyte differentiation and has an important role in metabolic syndrome. Phosphorylation of the receptor's ligand-binding domain at serine 273 has been shown to change the expression of a large number of genes implicated in obesity. The difference in gene expression seen when comparing wild-type phosphorylated with mutant non-phosphorylated PPARγ may have important consequences for the cellular molecular network, the state of which can be shifted from the healthy to a stable diseased state. We found that a group of differentially expressed genes are involved in bi-stable switches and form a core network, the state of which changes with disease progression. These findings support the idea that bi-stable switches may be a mechanism for locking the core gene network into a diseased state and for efficiently propagating perturbations to more distant regions of the network. A structural analysis of the PPARγ-RXRα dimer complex supports the hypothesis of a major structural change between the two states, and this may represent an important mechanism leading to the differential expression observed in the core network.

The proline 12 alanine substitution in the PPARgamma2 gene is associated with increased extent of coronary artery disease in men.

OBJECTIVE: To determine whether there is an independent association between the Pro12Ala polymorphism in the peroxisome proliferator-activated-receptor gamma2 (PPARgamma2)-gene and the extent of coronary artery disease in men. RESEARCH DESIGN AND METHODS: We determined the Pro12Ala polymorphism in the PPARgamma2 gene in 240 male patients undergoing elective coronary angiograpy, and quantitated the degree of CAD by evaluating the extent-score which better correlates with known risk factors than other measures of CAD. RESULTS: The presence of the 12Ala allele was significantly associated with higher CAD extent (r=0.27, p<0.01). CAD extent was also correlated with the extent of insulin resistance (HOMA, r=0.22, p<0.01), and age (r=0.16, p<0.05). Multivariate analysis revealed an independent association between the 12Ala allele PPARgamma2 with extent-score (beta=0.32, p<0.01). CONCLUSIONS: The 12Ala allele in PPARgamma2 correlates with a significantly increased CAD extent in men, which suggest that lower activity of the transcription factor PPARgamma2 is associated with more severe CAD.

Postprandial mononuclear NF-kappaB activation is independent of the AGE-content of a single meal.

OBJECTIVE: Dietary uptake of Advanced Glycation Endproducts (AGE) is supposed to potentially contribute to inflammatory reactions linked to vascular dysfunction and late diabetic complications. One mechanism by which dietary AGE might exert these effects is by activation of the proinflammatory transcription factor NF-kappa-B. The aim of this study was to analyze the postprandial effects of a casein meal with low or high AGE content on postprandial NF-kappaB activation in peripheral blood mononuclear cells (pBMC) of healthy volunteers. RESEARCH DESIGN AND METHODS: Casein was heated for 40 h at 50 degrees C in the presence of sorbitol or glucose, resulting in either minimal (Sorbitol [S]-casein) or large (glucose [G]-casein) amounts of AGE-modified casein. Nine healthy volunteers ate 250 g of both types of casein, whereas both meals were separated at least by 2 weeks. Plasma and pBMC were taken before and 2 h after each meal. Thereafter, the defined AGE carboxymethyllysine (CML) was determined by ELISA and Western blot. NF-kappaB activation in pBMC was assayed using Electrophoretic Mobility Shift Assays (EMSA) and Western blot analysis. RESULTS: S-casein contained only minor amounts of CML and no pentosidine, while G-casein contained large amounts of both. 2 h after ingestion, the S-casein or the G-casein-meal, both, resulted in a non-significant increase in plasma CML and in the intracellular CML-content of pBMC. This was paralleled by a highly significant increase in postprandial mononuclear NF-kappaB-binding activity. Remarkably, neither the extent of NF-kappaB induction (178% for S-casein, 188% for G-casein), nor composition of the NF-kappaB heterodimer (mainly consisting of NF-kappaB p50/p65) were significantly different after intake of S-casein or G-casein. Consistently, Western blots confirmed an increased NF-kappaBp65 nuclear translocation and a decrease of NF-kappaBp65 in the cytoplasm, while no difference in postprandial NF-kappaB nuclear translocation was observed following intake of S-casein or G-casein. CONCLUSION: Postprandial mononuclear NF-kappaB activation after a single meal is independent of the AGE-content of the ingested protein.

A 63bp deletion in the promoter of rage correlates with a decreased risk for nephropathy in patients with type 2 diabetes.

Several polymorphisms have been identified in the RAGE-promoter region that might modulate the outcome of disease. Here we analyse the association of a 63bp deletion (delta63) spanning from bp - 407 to bp - 345 with diabetic nephropathy. The deletion was determined using the polymerase chain reaction (PCR) in a cross-sectional study with 1087 patients with type 1 diabetes (n = 559) and type 2 diabetes (n = 528). 475 patients with osteoporosis served as disease independent control. The prevalence of the heterozygous genotype did not significantly differ between the three groups (type 1: 2.15 %, type 2: 2.27 %, controls: 1.47 %), indicating that heterozygous delta63 is not related to the manifestation of diabetes. Homozygous carriers were not identified in this study. The heterozygous delta63 genotype, was associated with a reduced prevalence of diabetic nephropathy in patients with type 2 diabetes (OR = 0.06; 95 % CI: [0.05, 0.07]), but not in patients with type 1 (OR = 1.49; 95 % CI: [1.14, 1.94]). We conclude, that patients with type 2 diabetes and the 63bp deletion in the promoter of RAGE seem to be protected from diabetic nephropathy. The observed difference between type 1 and type 2 diabetes might point to diverse pathomechanisms of nephropathy in both types of diabetes.

Endothelin-1 levels in patients with disorders of the thyroid gland.

The endothelium derived peptide endothelin-1 (ET-1) is the major isoform of the endothelin peptide family, which is produced and secreted in the endothelial cell system. We measured plasma levels in patients with thyroid diseases and investigated associations between laboratory and clinical markers of thyroid metabolism and ET-1 plasma levels. ET-1 plasma levels were determined in patients with Graves' disease (n = 54), endemic goiter (n = 26), patients with Hashimoto's thyroiditis (n = 21) and compared to healthy controls (n = 60). ET-1 plasma levels were significantly elevated in patients with Hashimoto's thyroiditis (p < 0.0001) and in patients with Graves' disease (p = 0.003), when compared to healthy controls. In patients with endemic goiter, no significant differences were found compared to healthy controls (p = 0.298) and when compared to patients with Graves' disease (p = 0.16). We did not observe an association between ET-1 plasma levels and parameters of thyroid disease (e.g. thyroidea-stimulating hormone, thyroxine, volume of the thyroid). Furthermore, patients with and without endocrine thyroid disease showed no significantly different ET-1 plasma levels (p = 0.78). These data suggest that the autoimmunologically induced inflammatory response of the thyroid gland in Hashimoto's thyroiditis and Graves' disease is responsible for increased ET-1 plasma levels. Furthermore, our data do not support a role for ET-1 as a valid quantitative indicator for stage or progression in endemic goiter, Graves' disease or Hashimoto's thyroiditis.

Recurrent deep venous thrombosis caused by congenital interruption of the inferior vena cava and heterozygous factor V Leiden mutation.

A case of a 44-year-old patient with recurrent deep venous thrombosis (DVT) caused by congenital dysgenesis of the inferior vena cava (IVC) in coincidence with heterozygous factor V Leiden mutation is presented. The IVC malformation was a fortuitous finding because the vascular malformation of the collateral draining thoracic veins were suspected to be a malignant mass in chest X-ray. This vascular abnormality is a rare finding but recent epidemiological research suggests that there may be an association between the congenital absence of the IVC and DVT. In our case, the patient is even at higher risk combining the malformation probably affecting venous blood flow and the hypercoagulabilic state by heterozygous presence of the factor V Leidenmutation.

Low hepatic lipase activity is a novel risk factor for coronary artery disease.

BACKGROUND: The crucial function of hepatic lipase (HL) in lipid metabolism has been well established, but the relationship between HL activity and coronary artery disease (CAD) is disputed. METHODS AND RESULTS: We measured HL activity in the postheparin plasma of 200 consecutive men undergoing elective coronary angiography and determined the degree of CAD with the extent score, which has been shown to be better correlated with known risk factors than other measures of CAD extent. We found a significant inverse correlation between HL activity and the extent of CAD (r=-0.19, P<0.01). This association was mainly due to patients with HDL levels >0.96 mmol/L (n=94, r=-0.30, P<0.005). HL activity was lower in 173 patients with CAD than in 40 controls with normal angiograms (286+/-106 versus 338+/-108 nmol. mL(-1). min(-1), P<0.01). To correct for potential confounding factors, we performed multivariate analyses that confirmed the independent association of HL activity with CAD extent. In addition, the presence of the T allele at position -514 in the HL promoter, which leads to a reduced HL promoter activity, was associated with lower HL activity (r=0.30, P<0.001) and higher CAD extent (42.2+/-20.8 versus 35.3+/-23.6 [extent score], P<0.05). In patients with heterozygous familial hypercholesterolemia, calcified lesions in ECG-gated spiral computed tomography were higher in patients with low HL activity (6.3+/-6.8 versus 1.5+/-3.1, P=0.01). CONCLUSIONS: Our data show that low HL activity is associated with CAD. Therefore, HL might be useful for CAD risk estimation and might be a target for pharmacological intervention.

Novel site in lipoprotein lipase (LPL415;-438) essential for substrate interaction and dimer stability.

LPL, like other lipases, has the ability to hydrolyze water-insoluble lipid substrates, but the mechanism is incompletely understood. We previously demonstrated a 22-amino acid loop in the amino-terminal domain of LPL to be essential for interaction with lipid substrates (Dugi, K. A., H. L. Dichek, G. D. Talley, H. B. Brewer, Jr., and S. Santamarina-Fojo. 1992. J. Biol. Chem. 267: 25086-25091) and mediation of substrate specificity (Dugi, K. A., H. L. Dichek, and S. Santamarina-Fojo. 1995. J. Biol. Chem. 270: 25396-25401). The carboxy-terminal domain, LPL415-438, contains two highly conserved hydrophobic stretches, and represents a candidate region for substrate interactions. Specific point mutations or deletion of the region between the hydrophobic stretches (LPL419-430) caused up to 90% selective loss of hydrolyzing activity against water-insoluble triolein, but not against water-soluble tributyrin, implicating a crucial function for LPL419-430 in the interaction with lipid substrates. In contrast, mutations introduced into the hydrophobic regions led to concomitant changes in tributyrin and triolein activities. The presence of an additional positive charge at position 416 yielded a gain of function mutant with 3-fold increased activity. This mutant was about three times more stable at 37 degrees C than wild-type LPL, suggesting an important role for the hydrophobic regions in LPL dimer stability. In summary, our data demonstrate that the carboxy-terminal region LPL415-438 plays an important role in both the interaction of LPL with lipid substrates and the stability of the LPL homodimer.

Factors affecting the mobilization of primitive and committed hematopoietic progenitors into the peripheral blood of cancer patients.

Rapid hematopoietic reconstitution following peripheral blood progenitor cell (PBPC) autotransplantation is thought to result from reinfusion of committed progenitor cells. This has raised concern that PBPC autografts might be rich in committed hematopoietic progentors responsible for early engraftment, but deficient in more primitive progenitors required for long-term hematopoietic reconstitution. The granulomonocytic colony-forming unit (CFU-GM) assay measures committed progenitors responsive to a single species of colony-stimulating activity such as granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas the pre-CFU assay identifies more primitive progenitors by measuring interleukin-3 (IL-3) and kit ligand (KL) induced generation of secondary CFU-GM from CD34+, 4-hydroperoxycyclophosphamide resistant progenitors that require multiple cytokine stimuli. Paired bone marrow (BM) and PBPC samples from 17 breast and ovarian cancer patients participating in four separate clinical trials were compared in these assay systems. In seven of nine patients, PBPC autografts mobilized with cyclophosphamide rebound and G-CSF compared favorably with paired BM autografts in both committed and primitive progenitor capacity. Failure to mobilize substantial primitive progenitor cell numbers occurred in two of nine patients undergoing this mobilization regimen and could not have been predicted by either circulating CFU-GM or CD34+ cell number. Prior myelosuppressive treatment experiences reduced peripheral progenitor yields somewhat, but still allowed for the collection of PBPC autografts which compared favorably with BM autografts in total CFU-GM and Pre-CFU. Mobilization of PBPC with G-CSF or GM-CSF alone in patients who had received prior myelosuppressive therapies produced autografts which were relatively deficient in committed progenitors, but absolutely deficient in primitive progenitors. We conclude that optimization of patient characteristics and mobilization parameters can achieve PBPC autografts rich in both the primitive and committed hematopoietic progenitor cells.

Intraperitoneal chemotherapy.

Despite high initial tumor response rates to systemically administered platinum-based chemotherapy, fewer than one quarter of advanced-stage ovarian cancer patients currently experience prolonged disease-free survival. Because persistent and relapsed disease tends to remain confined to the peritoneal cavity, the strategy of enhancing drug delivery to tumor via the direct intraperitoneal route has been the subject of much preclinical and clinical research. This review summarizes the pharmacokinetic rationale, logistical feasibility, and clinical experience associated with intraperitoneal chemotherapy for ovarian cancer.

Beneficial impact of peripheral blood progenitor cells in patients with metastatic breast cancer treated with high-dose chemotherapy plus granulocyte-macrophage colony-stimulating factor. A randomized trial.

BACKGROUND: This study compared the efficacy of granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or in combination with peripheral blood-derived hematopoietic progenitor cells (PBP) as support for patients receiving high-dose chemotherapy and assessed the adequacy of these strategies as alternatives to autologous bone marrow rescue. METHODS: The authors studied patients with metastatic breast carcinoma who had a major response to conventional chemotherapy or had achieved a complete remission by surgical resection of all known metastases. They were treated with carboplatin 1500 mg/m2, etoposide 1200 mg/m2, and cyclophosphamide 5.0 g/m2. Before this high-dose chemotherapy, the patients had been randomly assigned to one of two hematopoietic support strategies: GM-CSF alone (Group 1) or GM-CSF-primed PBP and GM-CSF (Group 2). Autologous bone marrow was harvested from all patients for use only in the event of persistent pancytopenia with marrow aplasia on day 15. RESULTS: A total of 18 patients were treated. Randomization was halted after the initial 10 patients because of the significant advantages for patients in Group 2 in comparison with those in Group 1 in regard to (1) the median number of days to absolute neutrophil count 0.5 x 10(9)/l (12 versus 21) and platelet count to 50 x 10(9)/l (13 versus 23), (2) platelet transfusions (3 versus 15.5), and (3) episodes of neutropenic sepsis (0 versus 4, respectively). One patient in Group 1 died from treatment-related complications. All patients in Group 1 required bone marrow reinfusion. No patient in Group 2 required bone marrow reinfusion, and no early mortality was observed in this group. Eight subsequent patients were treated with PBP and GM-CSF (Group 3). This group was more heavily pretreated than Groups 1 or 2 and had a slower hematologic recovery than Group 2. However, none of these patients required bone marrow reinfusion. The four patients in Group 1 that did not have early bone marrow rescue all had neutrophil counts of 0.0 on day 15. For Groups 2 and 3, the neutrophil counts on day 15 ranged from 0.3-2.1 x 10(9)/l (median, 1.9) and from 0.2-2.1 x 10(9)/l (median 0.6), respectively. CONCLUSIONS: The use of PBP plus GM-CSF accelerated hematologic recovery after this chemotherapeutic regimen compared with GM-CSF alone; there were reduced morbidity and platelet transfusion requirements. Recovery was sufficiently rapid that PBP were an acceptable alternative to autologous bone marrow transplantation in patients receiving high-dose carboplatin, etoposide, and cyclophosphamide.

Interactions among colony-stimulating factors, IL-1 beta, IL-6, and kit-ligand in the regulation of primitive murine hematopoietic cells.

We have investigated the regulation of primitive murine hematopoietic progenitors by the cytokines interleukin 1 (IL-1), interleukin 6 (IL-6), and kit-ligand (KL). Individually these cytokines have a limited ability to stimulate the growth of high proliferative potential colony-forming cells (HPP-CFC) from 5-fluorouracil (5-FU)-purged bone marrow, but in combination these cytokines demonstrate synergism in promoting the growth of HPP-CFC. Furthermore, IL-1, IL-6, and KL, alone or in combination, synergized with the colony-stimulating factors (CSFs) granulocyte CSF (G-CSF), macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF), or interleukin 3 (IL-3) in clonal and liquid cultures of 5-FU-purged bone marrow. The pattern of HPP-CFC growth that was observed with 40 different cytokine combinations demonstrated the unique roles of IL-1, IL-6, and KL in the regulation of HPP-CFC proliferation. Short-term liquid cultures (delta-cultures), with secondary recloning, of 5-FU-purged bone marrow were stimulated to greatly expand the numbers of progenitor cells generated in response to cytokine stimulation. The greatest expansion, over 1800-fold, of the more mature progenitor compartments took place in delta-cultures stimulated with IL-1, IL-6, and KL plus IL-3. However, the combination of IL-1 and IL-6 plus KL was optimal in expanding HPP-CFC, increasing their numbers by 700-fold. The ability to expand early progenitor cells in delta-cultures was further demonstrated by the greater than 100-fold expansions of day-12 spleen colony-forming units (CFU-S) by the synergistic interactions of IL-1 with IL-3 or KL.