Preparation and evaluation of a new releasable PEGylated tumor necrosis factor-α (TNF-α) conjugate for therapeutic application.

To design a releasable PEGylated TNF-α (rPEG-TNF-α), a cathepsin B-sensitive dipeptide (Val-Cit moiety) was inserted into conventional PEG-modified TNF-α (PEG-TNF-α), facilitating its clinical use for anti-tumor therapy. Comparative pharmacokinetic and pharmacodynamic studies showed that the half-lives of both PEGylated forms of TNF-α were ∼60-fold greater than that of unmodified TNF-α. In addition, the in vitro bioactivity of rPEG-TNF-α was greater than that of PEG-TNF-α with the same degree of PEG modification. Release of TNF-α from rPEG-TNF-α in vitro was dependent on the presence of cathepsin B and was inhibited by a cathepsin B inhibitor. Despite the potent cytotoxicity of unmodified TNF-α against normal cells, its PEGylated forms at higher TNF-α concentrations showed low cytotoxic activity against these cells. In contrast, both forms of PEGylated TNF-α showed potent cytotoxic activity against the B16 and L929 cell lines, with rPEG-TNF-α being 5- and 9-fold more potent, respectively, than PEG-TNF-α. Moreover, rPEG-TNF-α was a more potent in vivo antitumor agent than PEG-TNF-α.

Hyperthermic pelvic perfusion with tumor necrosis factor-α for locally advanced cancers: encouraging results of a phase II study.

PURPOSE: To assess the efficacy of isolated pelvic perfusion (IPP) with tumor necrosis factor (TNF)-α and melphalan in patients with locally advanced cancers in the pelvic and groin area requiring mutilating surgery. METHODS: A total of 27 patients were enrolled (carcinoma, n = 17; sarcoma/melanoma, n = 4; and endocrine tumor, n = 6). They were candidates for exarticulation (n = 3) or exenteration (n = 11) or were judged unresectable (n = 13). In installing IPP, tourniquets were positioned around both thighs, and an inflated pressure suit was placed at a subthoracic position. Tumor necrosis factor-α (300 µg) was injected in the perfusate, followed 5 minutes later by melphalan at 1.5 mg/kg. After 30 minutes, the remaining drugs were washed out. Leakage was assessed with technetium Tc 99m radiolabeled human serum albumin, and a pharmacokinetic study was performed. Efficacy was based on the complete response rate observed on magnetic resonance imaging. RESULTS: Pelvic/systemic ratios of melphalan/TNF/technetium Tc 99m were 14.2/7/3.6. Responses on magnetic resonance imaging were as follows: 30% complete, 30% partial, 19% no change, and 15% progression. Two patients were not evaluable because they did not receive the treatment. Pre-IPP/post-IPP median percentage of necrosis on magnetic resonance imaging was 10%/70%. Median follow-up was 43 months. Median overall survival was 17 months. Twelve-month survival rate, disease-free survival, and local and metastatic recurrence rates were 67%, 30%, 57%, and 26%, respectively. CONCLUSIONS: Isolated pelvic perfusion with TNF-α compares favorably with historical data, as it was observed in limb perfusion and could provide a chance to translate its successful combination with chemotherapy into treatment of locally advanced pelvic cancers.

Application of miniaturized immunoassays to discovery pharmacokinetic bioanalysis.

INTRODUCTION: Pharmacokinetic properties of biotherapeutics are an important aspect of preclinical drug development. The lead identification and optimization space is characterized by aggressive timelines, large sample numbers, a variety of species and matrices, and limited reagent and sample volumes all of which represent challenges for traditional microtiter plate assays. Since the Gyrolab immunoassay platform can accommodate small sample volumes and automated assay processing, we evaluated the workstation as an alternative to the plate-based assays. METHODS: Three representative example assays--a generic anti-human IgG, a target specific and an anti-drug capture assay--were investigated in detail for accuracy and precision performance and their application to bioanalytical support for preclinical pharmacokinetic studies. Different animal matrices were tested in the assays and during study support. RESULTS: Gyrolab procedures could be closely modeled after regular microtiter plate assays. The small reagent volumes necessary for Gyrolab allowed studying serial bleeds of transgenic mice with only 10µL of blood sample. During development and during study support, the Gyrolab performance was similar to what can be expected from plate-based systems with accuracy and precision within 100 ± 20% or less. DISCUSSION: Overall, the technology was well suited to support quantitation of biotherapeutics using small volume samples from different preclinical species. Limited operator involvement for assay processing allowed for reduced staffing and training. However, high instrument costs and a single source of reagent supplies represent risks when moving assays further into long-term applications such as clinical studies. Despite interest in the bioanalytical field, this is the first detailed investigation of bioanalytical applications of Gyrolab in pharmacokinetic studies.

Introducción. Estructura química de etanercept, farmacocinética y mecanismo de acción / Introduction. Chemical structure of etanercept, pharmacokinetics and mechanism of action

Se presenta el contenido de esta monografía sobre el conocimiento actual de etanercept en relación con la psoriasis y la artritis psoriásica. Etanercept se empleó por primera vez en estudios clínicos humanos en 1992. Se hace referencia a las actuales indicaciones aprobadas por la Agencia Europea de Evaluación de Medicamentos; se describe su estructura química, se analizan los datos farmacocinéticos y su mecanismo de acción (AU)

The contents of this monography on the current knowledge of etanercept in relationship with psoriasis and psoriatic arthritis is presented. Etanercept was first used in clinical studies in humans in 1992. Reference is made to the current indications approved by the European Medicines Agency. Its chemical structure is described, and the pharmacokinetics data and its mechanism of action are analyzed (AU)

Transición de tratamientos sistémicos clásicos a etanercept / Transition of classical systemic treatments to etanercept

La transición de tratamientos sistémicos clásicos con etanercept es una estrategia atractiva y a priori útil en numerosas situaciones de la práctica clínica. Sin embargo, la experiencia publicada al respecto se encuentra limitada a casos aislados y pequeñas series. La transición entre metotrexato y etanercept parece segura y eficaz, recomendándose su puesta en práctica de forma paulatina hasta que etanercept alcance una respuesta suficiente (4-8 semanas), pudiendo incluso probarse el ajuste de dosis bajas de metotrexato a largo plazo en combinación. La estrategia será parecida para acitretino, siendo la limitación los efectos adversos propios de este último. La transición desde fototerapia de banda estrecha o ciclosporina resulta, a juzgar por la experiencia existente, eficaz, aunque debería limitarse a momentos puntuales de la enfermedad, debido a las incertidumbres acerca de la seguridad a largo plazo de estas asociaciones (AU)

The transition of classical systemic treatments to etanercept is an attractive strategy and is, a priori, useful in many clinical practice situations. However, the experience published in this regards has been limited to isolated cases and small series. Transition between methotrexate and etanercept seems to be safe and effective. It should be slowly introduced until etanercept has achieved a sufficient response (4-8 weeks). It even would be possible to try to adjust a long-term combination of low doses of methotrexate. The strategy would be similar for acitretine, the adverse effects characteristic of acitretine being its limitation. Transition from narrow band phototherapy or cyclosporine is, based on the existing experience, effective, although it should be limited to certain moments of the disease due to the uncertainty of the long-term safety of these associations (AU)

¿Qué tiene de singular adalimumab? / What is special about adalimumab?

Adalimumab es el primer anticuerpo monoclonal anti-factor de necrosis tumoral alfa (TNF-α) completamente humano que se une con una alta afinidad tanto a la forma soluble como a la forma unida a células del TNF-α, modulando su actividad biológica. Varios ensayos clínicos han demostrado la eficacia y rapidez de acción de adalimumab en pacientes con psoriasis, y la experiencia de uso en enfermedades como la artritis reumatoide, artritis psoriásica, espondilitis anquilosante y enfermedad de Crohn lo posicionan como un fármaco seguro, con un perfil de efectos secundarios similar a otros fármacos anti-TNF-α. Las peculiaridades específicas de esta molécula son revisadas en este artículo (AU)

Adalimumab is the first fully human anti-tumor necrosis factor (TNF-α) monoclonal antibody and it binds to both soluble and cell-bound TNF-α, modulating biological responses linked to this cytokine. Different trials have probed the efficacy of adalimumab even after one week, and accumulated experience in rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis and Crohn´s disease shows adalimumab as a safe drug sharing a similar adverse effects profile with the other anti-TNF-α molecules. Specific features of adalimumab are revised (AU)

Tratamiento de la hepatitis alcohólica / No disponible

No disponible

Enhancement of tumor thermal therapy using gold nanoparticle-assisted tumor necrosis factor-alpha delivery.

Tumor necrosis factor-alpha (TNF-alpha) is a potent cytokine with anticancer efficacy that can significantly enhance hyperthermic injury. However, TNF-alpha is systemically toxic, thereby creating a need for its selective tumor delivery. We used a newly developed nanoparticle delivery system consisting of 33-nm polyethylene glycol-coated colloidal gold nanoparticles (PT-cAu-TNF-alpha) with incorporated TNF-alpha payload (several hundred TNF-alpha molecules per nanoparticle) to maximize tumor damage and minimize systemic exposure to TNF-alpha. SCK mammary carcinomas grown in A/J mice were treated with 125 or 250 microg/kg PT-cAu-TNF-alpha alone or followed by local heating at 42.5 degrees C using a water bath for 60 minutes, 4 hours after nanoparticle injection. Increases in tumor growth delay were observed for both PT-cAu-TNF-alpha alone and heat alone, although the most dramatic effect was found in the combination treatment. Tumor blood flow was significantly suppressed 4 hours after an i.v. injection of free TNF-alpha or PT-cAu-TNF-alpha. Tumor perfusion, imaged by contrast enhanced ultrasonography, on days 1 and 5 after treatment revealed perfusion defects after the injection of PT-cAu-TNF-alpha alone and, in many regions, complete flow inhibition in tumors treated with combination treatment. The combination treatment of SCK tumors in vivo reduced the in vivo/in vitro tumor cell survival to 0.05% immediately following heating and to 0.005% at 18 hours after heating, suggesting vascular damage-mediated tumor cell killing. Thermally induced tumor growth delay was enhanced by pretreatment with TNF-alpha-coated gold nanoparticles when given i.v. at the proper dosage and timing.

Isolated hepatic perfusion for the treatment of colorectal metastases confined to the liver: recent trends and perspectives.

Isolated hepatic perfusion (IHP) involves a method of complete vascular isolation of the liver to allow treatment of liver tumours with toxic systemic doses. The recent clinical studies mainly employed IHP with melphalan with or without tumour necrosis factor-alpha (TNF-alpha) and mild hyperthermia. The results of these studies show that high response rates and high survival rates can be achieved by IHP. In this article, the current status, recent developments and future perspectives of IHP are discussed.

FK778 attenuates lymphocyte-endothelium interaction after cardiac transplantation: in vivo and in vitro studies.

BACKGROUND: The malononitrilamide FK778 is a novel derivate of leflunomide and interacts with T- and B-cell function by inhibiting de novo pyrimidine synthesis. We investigated the effects of FK778 upon acute cardiac allograft rejection and upon adhesion molecule upregulation in experimental transplantation and by using in vitro cell culture. METHODS: Heterotopic, abdominal cardiac transplantations were performed in the Brown Norway (BN) to Lewis (Lew) rat model. The study groups received daily low- or high-dose FK778 immunosuppression. FK778 plasma levels were quantified by HPLC. Grafts were harvested on the fifth postoperative day for histologic and immunohistologic examinations using computerized morphometry. Purified BN aortic endothelial cell cultures were pretreated with low- or high-dose FK778 according to FK778 plasma levels and were stimulated with tumor necrosis factor (TNF)-alpha. Adhesion molecule expression was quantified by immunofluorescence, FACS analysis, and Western blotting. Lymphocyte-endothelium adhesion assays were performed using purified Lew lymphocytes and radiolabeled TNF-alpha was used for receptor binding assays. RESULTS: FK778 treatment dose-dependently reduced graft mononuclear infiltration of CD4(+), CD8(+), and ED1(+) cells, but only high-dose FK778 treatment significantly reduced early upregulation of ICAM-1 and VCAM-1 in vivo. FK778 also dose-dependently reduced TNF-alpha-stimulated endothelial adhesion molecule upregulation in vitro, whereas the effect on VCAM-1 was more dominant. We did not find evidence that FK778 interferes with surface receptor binding of TNF-alpha. Lymphocyte adhesion to endothelial cell monolayers was significantly attenuated by FK778. CONCLUSION: Besides its inhibitory effect on pyrimidine synthesis, FK778 directly reduces endothelial adhesion molecule upregulation and attenuates lymphocyte-endothelium interaction, which is a critical step in graft rejection.

Hyperthermic isolated perfusion with low-dose TNFalpha and doxorubicin in patients with locally advanced soft tissue limb sarcomas.

We report here the results of 27 patients who underwent hyperthermic isolated limb perfusion with low-dose TNFalpha (1 mg) and doxorubicin (8.5 mg/l of limb volume) for locally advanced soft tissue sarcomas. A tumor response was observed in 85% of cases. After a median follow-up of 30 months, limb salvage and local disease control were achieved in 82 and 85% of patients, respectively. Locoregional toxicity was low or mild in 14 patients, while 2 patients had severe limb toxicity. Systemic side effects were negligible. The perfusate/plasma area under the curve (AUC) ratio for TNFalpha was 56. HILP with low-dose TNFalpha and DXR proved to be an active neoadjuvant drug regimen against limb-threatening STS.

Continuous leakage measurement during hyperthermic isolated limb perfusion.

BACKGROUND: Continuous measurement of perfusate leakage into the systemic circulation is of the utmost importance and can be performed with the help of radioactive tracers. The purpose of this study was to assess changes in the perfusion leakage rate between two periods: 1977-1990 and 1991-2000, and to determine the factors responsible for these changes. METHODS: During the 1991-2000 period, 119 patients underwent HILP mainly for locally recurrent melanoma or locally advanced soft tissue sarcoma. HILP was performed with melphalan (33%) or in combination with TNFalpha (65%). There were 67 iliacal, 12 femoral, 25 popliteal, and 15 axillary perfusions performed. Leakage into the systemic circulation was monitored continuously with the help of 131I-albumin and a stationary scintillation detector placed above the heart. RESULTS: The median maximum leakage was 2.7% (range 0%-21%) which is significantly less than the previous period (1977-1990) where leakage of 8% (range 0%-30%) was reported (P < .05). A statistical difference in leakage was detected among perfusion locations where the iliac and femoral vessels showed more leakage than the axillary and popliteal vessels (P < .05). Furthermore, there appeared to be significantly less leakage when TNFalpha was used than when melphalan was the sole drug (P < .05). CONCLUSIONS: Nowadays leakage from isolated perfusions into the systemic circulation is further minimized compared with the days when melphalan was the sole drug used. Increased awareness about TNFalpha leakage, continuous external monitoring with 131I-albumin as the main isotope, flow rate regulation in the perfusion circuit, and regulation of the patient's systemic blood pressure have all been major contributors to this improvement.

Regional transport of TNF-alpha across the blood-brain barrier in young ICR and young and aged SAMP8 mice.

The blood-brain barrier (BBB) controls the exchange of regulatory substances, including tumor necrosis factor-alpha (TNF), between the brain and the blood. Transport across the BBB of some regulatory substances is altered with aging. Here, we measured the blood to brain unidirectional influx rate (Ki) for whole brain and 10 brain regions for radioactively labeled TNF in three groups of mice: young (2 mo old) ICR (the standard outbred albino laboratory mouse also termed CD-1), young SAMP8 (a strain which develops impaired learning and memory with aging that correlates with an age-related increase in brain levels of amyloid beta protein), and aged (17 mo) SAMP8 mice. In ICR mice, the hypothalamus had the fastest (1.73 microl/g-min) and the parietal cortex the slowest (0.189 microl/g-min) rates of uptake, a regional difference of about 9 fold. No differences in transport into whole brain or brain regions occurred between the ICR and young SAMP8, showing a lack of differences between strains. Transport was higher for the occipital cortex, midbrain, and striatum in aged SAMP8 mice. These results show blood-borne TNF enters some regions of the brain much more readily than others and TNF transport is increased into some brain regions of the SAMP8 mice at an age when learning and memory are impaired.

Isolated hyperthermic liver perfusion with high dose tumor necrosis factor alpha in pigs: an experimental study in preparation of clinical Use.

Isolated hyperthermic perfusion of the liver was performed for 45 min in 27 pigs via hepatic artery and portal vein at mean inflow temperatures between 40.7 and 41.2 degrees C. In two study groups B and C (n = 9 pigs each) 50 microg recombinant human tumor necrosis factor-alpha (rhTNFalpha) per kg body weight were added to the perfusate, whereas in a control group A liver perfusion was done without rhTNFalpha. Before reperfusion the livers were washed out with Ringer's solution in all groups followed by a protein solution in group C. At 30 and 60 min after reperfusion the maximum systemic rhTNFalpha concentrations were significantly higher in group B with 68 and 61 ng/ml compared to 14.5 and 14.9 ng/ml in group C (p < 0. 05). Mean systemic porcine TNFalpha concentration was significantly higher in group B (217 pg/ml) compared to group C (50 pg/ml) 30 min after reperfusion (p = 0.012). Survival was 7/9 in group A and C and only 2/9 in group B with 6/7 pigs dying due to severe cardiopulmonary failure within 12 h after operation. In surviving pigs of group A and C only mild and transient hepatotoxicity was registered. The presented study underlines the feasibility of high dose rhTNFalpha application in an isolated hyperthermic liver perfusion system. Washout of the liver with a protein solution before reperfusion reduces systemic TNFalpha levels as well as associated lethal cardiocirculatory and hepatotoxic side effects.

Observations on the role of tumor necrosis factor-alpha in a murine model of shock due to Streptococcus pyogenes.

OBJECTIVE: To examine the effect of a monoclonal antibody to tumor necrosis factor-alpha (TNF-alpha) in a murine model of shock due to Streptococcus pyogenes. DESIGN: Prospective, multiexperimental, randomized, controlled trial. SETTING: University hospital research laboratory. INTERVENTIONS: An LD90 murine model of Gram-positive shock using S. pyogenes, associated with the presence of significant concentrations of TNF-alpha in the circulation. Prophylactic administration of antibody with concomitant saline controls. A 500-micrograms TN3-19.12 (hamster monoclonal antibody to recombinant murine TNF), or saline, by intravenous injection was administered. MEASUREMENTS AND MAIN RESULTS: Administration of 0.3 mL of 6 x 10(8) colony-forming units/mL of S. pyogenes H250 to mice resulted in 90% to 100% mortality rates in 72 hrs. Serum TNF-alpha concentrations peaked at 2 hrs after bacterial challenge and were 67.7 +/- 18.6 ng/mL. Treatment with anti-TNF-alpha monoclonal antibody abolished the serum TNF-alpha concentrations but did not affect the mortality rate. Serum endotoxin concentrations were < 50 pg/mL before challenge and at 0.5, 1, 2, 5, and 24 hrs after challenge. CONCLUSIONS: Pretreatment with an anti-TNF monoclonal antibody was not protective in this model of S. pyogenes sepsis, despite the presence of significant amounts of TNF in the circulation. These data suggest that TNF-alpha may not play such a crucial role in the pathogenesis of shock due to S. pyogenes.

Tumor necrosis factor and cardiac function.

The direct effect of tumor necrosis factor (TNF), a product of activated macrophages, on myocardial performance was determined using an isolated papillary muscle technique and a modified Langendorff preparation. Papillary muscle was obtained from male adult rats 4-5 hours after they received either 100 ng/kg TNF (group A), or 100 micrograms/kg TNF (group B) or saline (control). Group B animals exhibited significantly greater peak tension development and velocity of contraction compared with controls (p less than 0.05). In group A animals these variables were not significantly different from those of the controls (p greater than 0.05). Electrophysiologic measurements revealed a significant decrease in resting membrane potential in both group A and group B animals compared with the controls (p less than 0.05). Whole hearts perfused with serum from animals treated with TNF 18-22 hours earlier exhibited significant impairment of contractility, decreased rate of systolic pressure development, and decreased rate of relaxation compared with the controls (p less than 0.05). Coronary flow and myocardial water content were similar for both groups of perfused hearts. These data suggest that tumor necrosis factor stimulates an early beneficial effect on myocardial function, which 18-22 hours later is associated with impairment of myocardial performance. This effect appears to be serum transferable.