Developing Exposure/Response Models for Anticancer Drug Treatment: Special Considerations.

Anticancer agents often have a narrow therapeutic index (TI), requiring precise dosing to ensure sufficient exposure for clinical activity while minimizing toxicity. These agents frequently have complex pharmacology, and combination therapy may cause schedule-specific effects and interactions. We review anticancer drug development, showing how integration of modeling and simulation throughout development can inform anticancer dose selection, potentially improving the late-phase success rate. This article has a companion article in Clinical Pharmacology & Therapeutics with practical examples.

Changes in cortical, cerebellar and basal ganglia representation after comprehensive long term unilateral hand motor training.

We were interested in motor performance gain after unilateral hand motor training and associated changes of cerebral and cerebellar movement representation tested with functional magnetic resonance imaging (fMRI) before and after training. Therefore, we trained the left hand of strongly right-handed healthy participants with a comprehensive training (arm ability training, AAT) over two weeks. Motor performance was tested for the trained and non-trained hand before and after the training period. Functional imaging was performed for the trained and the non-trained hand separately and comprised force modulation with the fist, sequential finger movements and a fast writing task. After the training period the performance gain of tapping movements was comparable for both hand sides, whereas the motor performance for writing showed a higher training effect for the trained hand. fMRI showed a reduction of activation in supplementary motor, dorsolateral prefrontal cortex, parietal cortical areas and lateral cerebellar areas during sequential finger movements over time. During left hand writing lateral cerebellar hemisphere also showed reduced activation, while activation of the anterior cerebellar hemisphere was increased. An initially high anterior cerebellar activation magnitude was a predictive value for high training outcome of finger tapping and visual guided movements. During the force modulation task we found increased activation in the striate. Overall, a comprehensive long-term training of the less skillful hand in healthy participants resulted in relevant motor performance improvements, as well as an intermanual learning transfer differently pronounced for the type of movement tested. Whereas cortical motor area activation decreased over time, cerebellar anterior hemisphere and striatum activity seem to represent increasing resources after long-term motor training.

[True and virtual risks of travellers]. / Mort des voyageurs: risques réels, risques virtuels.

Evidence-based information on travel associated mortality is scarce. Perception, intuition and the availability of interventions such as vaccinations and chemoprophylaxis often guide pre-travel advice. Important risks including accidents and cardiovascular events are not routinely included in pre-travel consultations although they cause more fatalities and costs than infectious diseases. The increased risk of sustaining a road accident in poor economy countries should always be mentioned. The general practitioner is further best placed to discuss possible problems of travellers with chronic diseases before travel.

[Chronic pain therapy for patients with head and neck tumors]. / Therapie chronischer Schmerzen bei Patienten mit Kopf-Hals-Tumoren.

Patients with tumors of the head and neck region commonly suffer from chronic pain, which is often treated insufficiently. Pain management according to the WHO analgesic ladder can effectively reduce pain in most patients. For head and neck cancer, specific aspects of tumor localization and psychosocial factors must be taken into consideration.

Indole-3-acetic acid protein conjugates: novel players in auxin homeostasis.

Indole-3-acetic acid (IAA) is found in plants in both free and conjugated forms. Within the group of conjugated IAA there is a unique class of proteins and peptides where IAA is attached directly to the polypeptide structure as a prosthetic group. The first gene, IAP1, encoding for a protein with IAA as a prosthetic group, was cloned from bean (Phaseolus vulgaris). It was shown that the expression of IAP1 as a major IAA modified protein in bean seed (PvIAP1) was correlated to a developmental period of rapid growth during seed development. Moreover, this protein underwent rapid degradation during germination. Since further molecular analysis was difficult in bean, the IAP1 gene was transformed into Arabidopsis thaliana and Medicago truncatula. Expression of the bean IAP1 gene in both plant species under the control of its native promoter targeted protein expression to the seeds. In Arabidopsis no IAA was found to be attached to PvIAP1. These results show that there is specificity to protein modification by IAA and suggests that protein conjugation may be catalyzed by species specific enzymes. Furthermore, subcellular localization showed that in Arabidopsis PvIAP1 was predominantly associated with the microsomal fraction. In addition, a related protein and several smaller peptides that are conjugated to IAA were identified in Arabidopsis. Further research on this novel class of proteins from Arabidopsis will both advance our knowledge of IAA proteins and explore aspects of auxin homeostasis that were not fully revealed by studies of free IAA and lower molecular weight conjugates.

Effects of luteinizing hormone and human chorionic gonadotropin on corpus luteum cells in a spheroid cell culture system.

The human corpus luteum (CL) is a highly vascularized, temporarily active endocrine gland and consists mainly of granulosa cells (GCs), theca cells (TCs), and endothelial cells (ECs). Its cyclic growth and development takes place under the influence of gonadotropic hormones. If pregnancy does occur, human chorionic gonadotropin (hCG) takes over the function of luteinizing hormone (LH) and, in contrast to LH, extends the functional life span of the CL. In this study, we investigated the effects of hCG and LH in a spheroidal cell culture model of CL development. Our data indicate that GCs secrete factors under the control of hCG that increase sprout formation of EC-spheroids. We demonstrate that the most prominent of these factors is VEGF-A. Furthermore, we found that both LH and hCG decrease sprout formation of GC-spheroids. After forming EC-GC coculture spheroids and consequently bringing GCs and ECs in close contact, sprouting increased under the influence of hCG, however not under LH. These experiments provide evidence for an hCG dependent functional switch in the GCs after coming in contact with ECs. Moreover, it demonstrates the considerably different effects of hCG and LH on GCs although their signaling is transmitted via the same receptor.

Clinical relevance of circulating tumour cells in the bone marrow of patients with SCCHN.

BACKGROUND: Clinical outcome of patients with head and neck squamous cell carcinoma (SCCHN) depends on several risk factors like the presence of locoregional lymph node or distant metastases, stage, localisation and histologic differentiation of the tumour. Circulating tumour cells in the bone marrow indicate a poor prognosis for patients with various kinds of malignoma. The present study examines the clinical relevance of occult tumour cells in patients suffering from SCCHN. PATIENTS AND METHODS: Bone marrow aspirates of 176 patients suffering from SCCHN were obtained prior to surgery and stained for the presence of disseminated tumour cells. Antibodies for cytokeratin 19 were used for immunohistochemical detection with APAAP on cytospin slides. Within a clinical follow-up protocol over a period of 60 months, the prognostic relevance of several clinicopathological parameters and occult tumour cells was evaluated. RESULTS: Single CK19-expressing tumour cells could be detected in the bone marrow of 30.7% of the patients. There is a significant correlation between occult tumour cells in the bone marrow and relapse. Uni- and multivariate analysis of all clinical data showed the metastases in the locoregional lymph system and detection of disseminated tumour cells in the bone marrow to be statistically highly significant for clinical prognosis. CONCLUSION: The detection of minimal residual disease underlines the understanding of SCCHN as a systemic disease. Further examination of such cells will lead to a better understanding of the tumour biology, as well as to improvement of diagnostic and therapeutic strategies.

Solvent extraction employing a static micromixer: a simple, robust and versatile technology for the microencapsulation of proteins.

The potential of a static micromixer for the production of protein-loaded biodegradable polymeric microspheres by a modified solvent extraction process was examined. The mixer consists of an array of microchannels and features a simple set-up, consumes only very small space, lacks moving parts and offers simple control of the microsphere size. Scale-up from lab bench to industrial production is easily feasible through parallel installation of a sufficient number of micromixers ('number-up'). Poly(lactic-co-glycolic acid) microspheres loaded with a model protein, bovine serum albumin (BSA), were prepared. The influence of various process and formulation parameters on the characteristics of the microspheres was examined with special focus on particle size distribution. Microspheres with monomodal size distributions having mean diameters of 5-30 micro m were produced with excellent reproducibility. Particle size distributions were largely unaffected by polymer solution concentration, polymer type and nominal BSA load, but depended on the polymer solvent. Moreover, particle mean diameters could be varied in a considerable range by modulating the flow rates of the mixed fluids. BSA encapsulation efficiencies were mostly in the region of 75-85% and product yields ranged from 90-100%. Because of its simple set-up and its suitability for continuous production, static micromixing is suggested for the automated and aseptic production of protein-loaded microspheres.

Comparison of CXC chemokines ENA-78 and interleukin-8 expression in Helicobacter pylori-associated gastritis.

Colonization of the gastric mucosa with Helicobacter pylori is associated with a dense infiltration of granulocytes into the lamina propria in the active phase of gastritis. In this study, we investigated the involvement of epithelial cell-derived neutrophil-activating protein 78 (ENA-78) in development of H. pylori-associated gastritis. Antral biopsies from 27 patients with H. pylori-associated gastritis and 25 from H. pylori-negative individuals were first analyzed for ENA-78 and interleukin-8 (IL-8) mRNA by semiquantitative reverse transcription (RT)-PCR. In H. pylori-positive patients, significantly elevated levels were found for both chemokines (P<0.05). Only IL-8 mRNA levels differed significantly (P<0.05) in H. pylori-infected individuals who had serum antibodies for cytotoxin-associated protein CagA versus H. pylori-infected CagA-negative persons. Quantification of ENA-78 transcript levels by competitive RT-PCR yielded a significant 45-fold upregulation for ENA-78 transcripts in biopsies of H. pylori-positive versus H. pylori-negative patients (P<0.05). In contrast to earlier findings with IL-8, the degree of ENA-78 mRNA upregulation was independent of the grade of activity of gastritis. Immunofluorescence studies on tissues of antral biopsies localized ENA-78 protein expression mainly to the gastric epithelium of H. pylori-positive patients, while control tissues were negative. Upregulation of ENA-78 and IL-8 mRNA and protein expression was also observed in an in vitro system using a gastric adenocarcinoma cell line. Only viable H. pylori yielded a strong ENA-78 and IL-8 induction, while H. pylori outer membrane proteins or water-soluble proteins had no significant effect. These data provide evidence for the importance of both IL-8 and ENA-78 in the development and perpetuation of H. pylori-associated gastritis.

TNFalpha contributes to the antitumor activity of a bispecific, trifunctional antibody.

Immunological cancer therapies focus on the activation of immune effector cells yielding a specific antitumor activity. Disseminated tumor cells are regarded as the origin of metastases and consequently their elimination is the central objective of adjuvant immune therapies. The use of bispecific antibodies is an approach that is regarded as promising in order to fight those disseminated tumor cells. Unfortunately, the efficiency of these antibodies is limited by the fact that they usually activate a single class of effector cell, thus not yielding optimal immune response. In addition, tumor cells may down-regulate the antibody's target molecule and escape recognition. We have recently described results with an intact bispecific molecule, BiUII, that represents a new class of intact antibodies. These antibodies, termed "triomab", provide an excellent antitumor activity in vitro, a fact that most probably is attributable to the simultaneous activation of different classes of immune effector cells. We have now investigated this antitumor activity in more detail and demonstrate here that at least a dual mechanism accounts for triomab-mediated killing of tumor cells: besides direct cell-mediated killing, triomab induces the production of TNFalpha in PBMCs at concentrations that induce apoptosis in target cells. This bystander effect may be of special interest for the clinical application of triomab in terms of killing of target antigen-negative tumor cells.

Synthesis of 8-methoxy-1-methyl-1H-benzo[de][1,6]naphthyridin-9-ol (Isoaaptamine) and analogues.

8-Methoxy-1-methyl-1H-benzo[de][1,6]naphthyridin-9-ol, isoaaptamine, a PKC inhibitor isolated from sponge was synthesized. The synthesis parallels a synthesis of 8,9-dimethoxybenzo[de][1,6]naphthyridine, aaptamine, but uses a nitromethyl substituent as a precursor of the key 5-(2-aminoethyl)-1H-quinolin-4-one intermediate. The quinolone intermediates were prepared by thermolysis (220-240 degrees C) of anilinomethylene derivatives of Meldrum's acid. The quinolone intermediates were N-methylated prior to cyclization to the benzo[de][1,6]naphthyridine derivatives. Aaptamine and several analogues of aaptamine and isoaaptamine were prepared including 9-demethylaaptamine, 1-methyl-8-demethylaaptamine, 1-methylaaptamine, and the 8,9-methylenedioxy analogues of aaptamine and 1-methylaaptamine.

The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR+ CXC chemokine-induced angiogenic activity.

We have previously shown that members of the ELR(+) CXC chemokine family, including IL-8; growth-related oncogenes alpha, beta, and gamma; granulocyte chemotactic protein 2; and epithelial neutrophil-activating protein-78, can mediate angiogenesis in the absence of preceding inflammation. To date, the receptor on endothelial cells responsible for chemotaxis and neovascularization mediated by these ELR(+) CXC chemokines has not been determined. Because all ELR(+) CXC chemokines bind to CXC chemokine receptor 2 (CXCR2), we hypothesized that CXCR2 is the putative receptor for ELR(+) CXC chemokine-mediated angiogenesis. To test this postulate, we first determined whether cultured human microvascular endothelial cells expressed CXCR2. CXCR2 was detected in human microvascular endothelial cells at the protein level by both Western blot analysis and immunohistochemistry using polyclonal Abs specific for human CXCR2. To determine whether CXCR2 played a functional role in angiogenesis, we determined whether this receptor was involved in endothelial cell chemotaxis. We found that microvascular endothelial cell chemotaxis in response to ELR(+) CXC chemokines was inhibited by anti-CXCR2 Abs. In addition, endothelial cell chemotaxis in response to ELR(+) CXC chemokines was sensitive to pertussis toxin, suggesting a role for G protein-linked receptor mechanisms in this biological response. The importance of CXCR2 in mediating ELR(+) CXC chemokine-induced angiogenesis in vivo was also demonstrated by the lack of angiogenic activity induced by ELR(+) CXC chemokines in the presence of neutralizing Abs to CXCR2 in the rat corneal micropocket assay, or in the corneas of CXCR2(-/-) mice. We thus conclude that CXCR2 is the receptor responsible for ELR(+) CXC chemokine-mediated angiogenesis.

Quantification of active caspase 3 in apoptotic cells.

We describe an enzyme-linked immunosorbent assay (ELISA) for quantifying relative amounts of active caspase 3 in apoptotic cells. Covalent modification of caspase 3 active sites with a biotinylated inhibitor differentiates active from latent caspases. Capture on an ELISA plate with an antibody specific for caspase 3 makes the assay specific for caspase 3. Detection is with horseradish peroxidase (HRP)-conjugated streptavidin that binds to the biotinylated inhibitor covalently bound to caspase 3. Using the assay we detected 6.6 ng active caspase 3 per 10(6) apoptotic staurosporine-treated Jurkat cells. Specificity of the assay for caspase 3 was demonstrated by lack of signal with purified caspases 2, 7, 8, and 10 that were modified by a biotinylated inhibitor. Specificity was also demonstrated by lack of signal with apoptotic MCF-7 cells which do not express caspase 3. The ability to discriminate between active and latent caspase 3 was shown by Western blotting with HRP-streptavidin and anti-caspase 3. Although latent caspase 3 was captured it was not covalently modified with the biotinylated inhibitor. The basic principle of using a covalent inhibitor to identify active enzymes and an antibody to differentiate between enzymes with similar activities has potential for quantifying active members of many classes of enzymes.

The Fc-region of a new class of intact bispecific antibody mediates activation of accessory cells and NK cells and induces direct phagocytosis of tumour cells.

Bispecific antibodies (bsAb) are considered as promising tools for the elimination of disseminated tumour cells in a minimal residual disease situation. The bsAb-mediated recruitment of an immune effector cell in close vicinity of a tumour cell is thought to induce an antitumoural immune response. However, classical bispecific molecules activate only a single class of immune effector cell that may not yield optimal immune responses. We therefore constructed an intact bispecific antibody, BiUII (anti-CD3 x anti-EpCAM), that not only recognizes tumour cells and T lymphocytes with its two binding arms, but also binds and activates Fcgamma-receptor positive accessory cells through its Fc-region. We have demonstrated recently that activated accessory cells contribute to the bsAb-induced antitumoural activity. We now analyse this stimulation in more detail and demonstrate here the BiUII-induced upregulation of activation markers like CD83 and CD95 on accessory cells and the induction of neopterin and biopterin synthesis. Experiments with pure cell subpopulations revealed binding of BiUII to CD64+ accessory cells and CD16+ NK cells, but not to CD32+ B lymphocytes. We provide further evidence for the importance of the Fc-region in that this bispecific molecule stimulates Fcgamma-R-positive accessory cells to eliminate tumour cells in vitro by direct phagocytosis.

Differential expression of chemokines in normal pancreas and in chronic pancreatitis.

BACKGROUND & AIMS: Cellular infiltrates are present already in early stages of chronic pancreatitis. The mechanisms responsible for their recruitment are unknown. Hence, we determined the differential expression of chemokine genes and their cellular sources in normal and affected pancreatic tissues. METHODS: Pancreatic tissues from 23 patients with chronic pancreatitis and from 4 normal controls were subjected to in situ hybridization for detecting messenger RNA (mRNA) of the chemokine genes interleukin 8, ENA-78, MIG, MCP-1, and I-309. RESULTS: Normal pancreatic tissues lack cells expressing mRNA for IL-8, ENA-78, MIG, and MCP-1. In contrast, pancreatic lobuli with mild to moderate signs of tissue alterations strongly expressed MCP-1 mRNA in centroacinar ducts, endothelia, fibroblasts, macrophages, T cells, and occasionally in nerves. Interleukin 8 and ENA-78 mRNA is preferentially detected in centroacinar ducts of pancreatic lobuli with more advanced alterations. Variable numbers of pancreas-infiltrating T cells express MIG mRNA. I-309 mRNA, however, is consistently observed in normal acini and in tissue with mild to moderate signs of tissue alterations. CONCLUSIONS: The observed differential expression of distinct chemokine genes in pancreatic parenchyma and infiltrates from patients with chronic pancreatitis strongly suggests an involvement of distinct chemokines in the initiation and perpetuation of disease.

The role of an epithelial neutrophil-activating peptide-78-like protein in rat adjuvant-induced arthritis.

The chemokine, epithelial neutrophil-activating peptide-78 (ENA-78), is a potent neutrophil chemotaxin whose expression is increased in inflamed synovial tissue and fluid in human rheumatoid arthritis compared with osteoarthritis. Since ENA-78 has been implicated in the pathogenesis of RA, we examined the expression of an ENA-78-like protein during the development of rat adjuvant-induced arthritis (AIA). Using an ELISA assay, we found increased levels of antigenic ENA-78-like protein in the sera of AIA animals compared with control normal animals by day 7 postadjuvant injection. ENA-78-like protein levels continued to increase as AIA developed. ENA-78-like protein levels in joint homogenates were increased in AIA animals later in the development of the disease, by day 18 during maximal arthritis, compared with control animals. Expression of ENA-78-like protein in both the AIA serum and joint correlated with the progression of inflammation of the joints. Anti-human ENA-78 administered before disease onset modified the severity of AIA, while administration of anti-ENA-78 after clinical onset of AIA did not modify the disease. These data support a role for an ENA-78-like protein as an important chemokine in the progression and maintenance of AIA.

Evaluation of Fc-receptor positive (FcR+) and negative (FcR-) monocyte subsets in sepsis.

The monocyte/macrophage (Mphi is central in the regulation of the immune response in states of trauma and sepsis. Because monocyte subsets, characterized by expression of the Fc-receptor (FcR), were shown to play distinct immunologic roles in trauma, it was the objective of this study to assess insights into the functional role of FcR positive (FcR+) and negative (FcR-) subclasses in surgical sepsis. In a prospective study, peripheral blood Mphi from 20 septic patients and 10 healthy volunteers were evaluated on consecutive days after the onset of sepsis. FcR+/- subsets were separated by rosetting with antibody-coated human erythrocytes. Receptor expression and synthesis of proinflammatory cytokines were used to evaluate the functional role of these cells. We demonstrated a significant monocytosis (350%; p<.01) and suppression of human lymphocyte antigen (HLA-DR) expression (35%; p<.05). Synthesis of Interleukin-1beta (IL-1beta; e.g., Day 1: 230+/-30 pg/mL) and Interleukin-6 (IL-6; e.g., Day 1: 1920+/-350 U/mL) were significantly higher (p<.05) in FcR+ subsets than in controls (IL-1beta: 100+/-5 pg/mL; IL-6: 353+/-75 U/mL). Tumor necrosis factor-alpha (TNF-alpha) was elevated in FcR+ monocytes but did not reach a significant value. Interleukin-8 (IL-8) synthesis showed only on Day 1 and in controls significant differences in FcR+ and FcR- cells (Day1: FcR-: 19.6+/-4.1 nM; FcR+: 9+/-4.3 nM). Sepsis results in a significant shift toward FcR+ monocytes. This cell population is characterized by high proinflammatory cytokine synthesis. The extent of this shift seems to identify a group of high risk septic patients that might benefit from immunomodulatory therapy.

Amino-terminal processing of chemokine ENA-78 regulates biological activity.

Epithelial cell-derived neutrophil-activating protein-78 (ENA-78) is a potent stimulator of neutrophils, inducing a variety of biological responses such as chemotaxis, enzyme release, up-regulation of surface receptors, and intracellular calcium mobilization. Proteolysis of ENA-78 with cathepsin G and chymotrypsin yielded a time-dependent increase in elastase-releasing activity, predicting the formation of truncation products with higher potency than native ENA-78. To investigate the biological implications of progressive truncation of ENA-78, the N-terminal variants ENA(5-78), ENA(9-78), and ENA(10-78) were cloned and expressed in E. coli. When tested in the neutrophil elastase release assay, the variants ENA(5-78) and ENA(9-78) had a 2-3-fold higher potency than full-length ENA-78, while ENA(10-78) was 3-fold less potent. In the chemotaxis assay, the variant ENA(5-78) exhibited an 8-fold and ENA(9-78) a 2-fold higher potency than native ENA-78. ENA(10-78), conversely, was 10-fold less potent, but reached a comparable efficacy to ENA-78 at 10(-)7 M concentration. In summary, the rank order in potency with respect to elastase release was ENA(9-78) > ENA(5-78) > ENA-78 > ENA(10-78), while for chemotaxis it was ENA(5-78) > ENA(9-78) > ENA-78 > ENA(10-78). Variant ENA(5-78) had a higher overall potency and efficiency for chemotaxis than interleukin-8 (IL-8), while ENA(9-78) exhibited a higher efficiency at concentrations of 1-100 nM. The fact that neutrophil cathepsin G produces the stable ENA(9-78) variant in vitro strongly suggests a role for this N-terminal proteolysis during inflammatory processes in vivo.