Cytokines in the Treatment of Melanoma.

PURPOSE OF REVIEW: The use of cytokines in harnessing the immune system to eradicate cancer has been an important treatment modality. However, the dose-limiting toxicities of these cytokines limited their usage in clinic. Here, we review the basic biology of cytokines involved in the treatment of melanoma and discuss their therapeutic applications. Moreover, we describe several innovative technological approaches that have been developed to improve the pharmacokinetics, safety, and efficacy of these cytokines. RECENT FINDINGS: The safety and the anti-tumor activity of newly engineered cytokines including PEGylated IL-2 (NKTR-214), PEGylated IL-10 (AM0010), and IL-15 super agonist (ALT-803) have been evaluated in clinical trials with encouraging clinical activity and acceptable safety profile, both as single agents and in combination with immuno-oncology agents. A greater understanding of the mechanisms of action and effective dosing of these newly engineered cytokine together with determination of optimum combination therapy regimens may yield greater clinical benefits in the future.

Low-fluence laser-facilitated platelet-rich plasma permeation for treating MRSA-infected wound and photoaging of the skin.

Platelet-rich plasma (PRP) is rich in cytokines and growth factors and is a novel approach for tissue regeneration. It can be used for skin rejuvenation but the large molecular size of the actives limits its topical application. In this study, low-fluence laser-facilitated PRP was delivered to evaluate its effect on absorption through the skin, infection-induced wound, and photoaging. The PRP permeation enhancement was compared for two ablative lasers: fractional (CO2) laser and fully-ablative (Er:YAG) laser. In the Franz cell experiment, pig skin was treated with lasers with superficial ablation followed by the application of recombinant cytokines, growth factors, or PRP. The transport of interferon (IFN)-γ and tumor necrosis factor (TNF)-α was negligible in intact skin and stratum corneum (SC)-stripped skin. Both lasers significantly elevated skin deposition of IFN-γ and TNF-α from PRP, and fully-ablative laser showed a higher penetration enhancement. A similar tendency was found for vascular endothelial growth factor and epidermal growth factor. Er:YAG laser-exposed skin displayed 1.8- and 3.9-fold higher skin deposition of platelet-derived growth factor (PDGF)-BB and transforming growth factor (TGF)-ß1 from PRP, respectively. According to the confocal images, both laser interventions led to an extensive and deep distribution of IFN-γ and PDGF-BB in the skin. In the in vivo methicillin-resistant Staphylococcus aureus (MRSA) infection model, CO2 laser- and Er:YAG laser-assisted PRP delivery reduced bacterial load from 1.8 × 106 to 5.9 × 105 and 1.4 × 104 colony-forming units, respectively. The open wound induced by MRSA was closed by the laser-assisted PRP penetration. In the mouse photoaging model, elastin and collagen deposition were fully restored by combined PRP and full-ablative laser but not by PRP alone and PRP combined with fractional laser. Laser-facilitated PRP delivery even with a low fluence setting can be considered a promising strategy for treating some dermatological disorders.

A comparative review of intelectins.

Intelectin (ITLN) is a new type of glycan-binding lectin. It has been demonstrated to agglutinate bacteria probably due to its carbohydrate-binding capacity, suggesting its role in an innate immune response. It is involved not only in many physiological processes but also in some human diseases such as asthma, heart disease, inflammatory bowel disease, chronic obstructive pulmonary disease and cancer. Up to now, intelectin orthologs have been identified in placozoans, urochordatas, cephalochordates and several vertebrates, such as cyclostomata, fish, amphibians and mammals. Although the sequences of intelectins in different species are conserved, their expression patterns, quaternary structures and functions differ considerably among and within species. We summarize the evolution of the intelectin gene family, the tissue distribution, structure and functions of intelectins. We conclude that intelectin plays a role in innate immune response and there are still potential functions of intelectin awaiting discovery.

Update on Therapeutic Protein-Drug Interaction: Information in Labeling.

This review evaluated the significance of therapeutic protein (TP)-drug interactions and the current practices for assessing the interaction potential. We reviewed US FDA labels of approved TPs with drug-drug interaction (DDI) assessment. TP-drug interactions have been evaluated from in vitro studies, animal studies, and/or clinical settings. Of the 150 FDA-approved TPs as of May 2019, 49 TP labels contained pharmacokinetic (PK)-related DDI information derived from at least one study method. Our review found that more than half of the clinical PK DDI evaluations showed no interaction, and no dose adjustment has been recommended for any of the rest TPs. The results and trends observed in this review may further enhance and inform risk-based approaches to evaluating the potential for TP-drug interactions.

Natural Killer Cells may Exert Antidepressant-like Effects in Mice by Controlling the Release of Inflammatory Factors.

Depression or stress is reportedly related to the overflow of inflammatory factors in the body and T cells were reported to play important roles in balancing the release of inflammatory factors through vagus nerve circuit. However, few works have been conducted to find if natural killer (NK) cells can also exert the similar function in the reported vagus nerve circuit as T cells and if there was any relationship between depression and this function. In the present study, the behavioral tests on BALB/c mice indicated that the depressant-like symptoms could be improved and simultaneously the concentrations of inflammatory factors in peripheral blood could be reduced significantly by adoptively transferring NK cells into stressed BALB/c mice. The results revealed that NK cells could control the release of inflammatory factors secreted by macrophages and ß2-AR (ß2-adrenergic receptor) on the NK cells were of great importance. Behavioral tests on NCG mice indicated that the antidepressant-like effects of NK cells notably declined after adoptively transferring NK cells with ß2-AR deficiency or with ChAT (choline acetyltransferase) deficiency into stressed NCG mice. Simultaneously, the anti-inflammatory effects also declined significantly both in vivo and in vitro, which indicated that the antidepressant-like property of NK cells may be related to its ability of controlling the release of inflammatory factors. Taken together, we find that NK cells may balance the release of inflammatory factors in our body by transporting the information between the terminal vagal branches and macrophages, which is the mechanism that NK cells may exert antidepressant-like effects.

Challenges in the local delivery of peptides and proteins for oral mucositis management.

Oral mucositis, a common inflammatory side effect of oncological treatments, is a disorder of the oral mucosa that can cause painful ulcerations, local motor disabilities, and an increased risk of infections. Due to the discomfort it produces and the associated health risks, it can lead to cancer treatment restrains, such as the need for dose reduction, cycle delays or abandonment. Current mucositis management has low efficiency in prevention and treatment. A topical drug application for a local action can be a more effective approach than systemic routes when addressing oral cavity pathologies. Local delivery of growth factors, antibodies, and anti-inflammatory cytokines have shown promising results. However, due to the peptide and protein nature of these novel agents, and the several anatomic, physiological and environmental challenges of the oral cavity, their local action might be limited when using traditional delivering systems. This review is an awareness of the issues and strategies in the local delivery of macromolecules for the management of oral mucositis.

Prediction of the Optimal Dosing Regimen Using a Mathematical Model of Tumor Uptake for Immunocytokine-Based Cancer Immunotherapy.

Purpose: Optimal dosing is critical for immunocytokine-based cancer immunotherapy to maximize efficacy and minimize toxicity. Cergutuzumab amunaleukin (CEA-IL2v) is a novel CEA-targeted immunocytokine. We set out to develop a mathematical model to predict intratumoral CEA-IL2v concentrations following various systemic dosing intensities.Experimental Design: Sequential measurements of CEA-IL2v plasma concentrations in 74 patients with solid tumors were applied in a series of differential equations to devise a model that also incorporates the peripheral concentrations of IL2 receptor-positive cell populations (i.e., CD8+, CD4+, NK, and B cells), which affect tumor bioavailability of CEA-IL2v. Imaging data from a subset of 14 patients were subsequently utilized to additionally predict antibody uptake in tumor tissues.Results: We created a pharmacokinetic/pharmacodynamic mathematical model that incorporates the expansion of IL2R-positive target cells at multiple dose levels and different schedules of CEA-IL2v. Model-based prediction of drug levels correlated with the concentration of IL2R-positive cells in the peripheral blood of patients. The pharmacokinetic model was further refined and extended by adding a model of antibody uptake, which is based on drug dose and the biological properties of the tumor. In silico predictions of our model correlated with imaging data and demonstrated that a dose-dense schedule comprising escalating doses and shortened intervals of drug administration can improve intratumoral drug uptake and overcome consumption of CEA-IL2v by the expanding population of IL2R-positive cells.Conclusions: The model presented here allows simulation of individualized treatment plans for optimal dosing and scheduling of immunocytokines for anticancer immunotherapy. Clin Cancer Res; 24(14); 3325-33. ©2018 AACRSee related commentary by Ruiz-Cerdá et al., p. 3236.

A insulina regula as vias de sinalização induzidas por LPS em macrófagos derivados de medula óssea / Insulin regulates LPS-induced signaling pathways in macrophages derived from bone marrow

Pacientes diabéticos apresentam alterações no sistema imunológico que promovem, em parte, maior suscetibilidade de infecções bacterianas. O tratamento com insulina melhora a sobrevida e reduz o número de infecções recidivas no paciente com diabetes mellitus do tipo 1 (DM1). Pouco se sabe sobre os efeitos do diabetes e a ação da insulina nos macrófagos. Neste trabalho, investigamos a proteína fosfatidilinositol-3-quinase (PI3K), proteína quinase B (Akt) e as quinases ativadas por mitógenos (MAPK) em macrófagos derivados de medula óssea (BMDM) e sua participação no estímulo por lipopolissacarídeo (LPS) na presença ou não do tratamento com insulina através da secreção dos mediadores inflamatórios fator de necrose tumoral (TNF)-α, interleucina (IL)-6 e IL-10. Observamos que os BMDM de animais com DM1 apresentam aumento da expressão da subunidade catalítica PI3K p110alpha com redução na subunidade reguladora PI3K p55 e maior expressão da fosforilação das proteínas Akt (Serina-473 e Treonina-308), quinase regulada por sinal extracelular (ERK) 1/2 e quinase ativada por estresse/quinase Jun-amino-terminal (SAPK/JNK) MAPK. Observou-se alteração na concentração das citocinas TNF-α, IL-6 e IL-10 no sobrenadante da cultura de BMDM dos animais diabéticos após estímulo com LPS, menor taxa de metabolismo mitocondrial, no entanto, sem resultar em morte celular, tampouco na expressão do receptor do tipo Toll 4 na membrana celular. Já o reestímulo destas células com LPS promoveu aumento na concentração de TNF-α sem alteração das demais citocinas. Além disto, o tratamento com insulina, simultaneamente ao estímulo com LPS, dos BMDM oriundos de animais diabéticos aumentou a concentração de TNF-α, IL-6, da fosforilação de p38, ERK 1/2 e SAPK/JNK MAPK, PI3K p55 e da Akt (Serina-473), o que não ocorreu nos BMDM dos animais não diabéticos sob a mesma condição. Este efeito foi abolido pela inibição farmacológica da PI3K e da ERK 1/2, resultando em novo aumento da concentração de TNF-α e IL-6. A análise conjunta destes resultados indica que a insulina, através da modulação das vias PI3K, Akt, ERK 1/2 e SAPK/JNK, amplifica o aumento da concentração de TNF-α e IL-6 sob estímulo com LPS

Diabetic patients present alterations in the immune system that promote in part a greater susceptibility of bacterial infections. Insulin treatment improves survival and reduces the number of recurrent infections in patients with type 1 diabetes mellitus (DM1). Little is known about the effects of diabetes and the action of insulin on macrophages. In this work we investigated the phosphatidylinositol-3-kinase (PI3K) / protein kinase B (Akt) and mitogen-activated kinase (MAPK) proteins in bone marrow-derived macrophages (BMDM) and their participation in lipopolysaccharide (LPS) or treatment with insulin through the secretion of inflammatory mediators tumor necrosis factor (TNF) -α, interleukin (IL) -6 and IL-10. We observed that BMDM of animals with DM1 increased PI3K p110alpha catalytic subunit expression with a reduction in the PI3K p55 regulatory subunit and increased expression of the phosphorylation of the Akt (Serine-473 and Threonine-308), extracellular signal regulated kinase (ERK) 1/2 and Jun-amino-terminal stress-kinase / kinase (SAPK / JNK) MAPK. Changes in the concentration of TNF-α, IL-6 and IL-10 cytokines in the supernatant of the BMDM culture of diabetic animals after stimulation with LPS were observed, possibly due to a lower rate of mitochondrial metabolism, however, without resulting in cell death , so little in the expression of the Toll 4 receptor on the cell membrane. The re-stimulation of these cells with LPS promoted an increase in TNF-α concentration without alteration of the other cytokines. In addition, insulin and simultaneously LPS stimulation of BMDM from diabetic animals increased the concentration of TNF-α, IL-6, phosphorylation of p38, ERK 1/2 and SAPK / JNK MAPK, PI3K p55 and Akt (Serine-473), which did not occur in the BMDM of non-diabetic animals under the same condition. This effect was abolished by pharmacological inhibition of PI3K and ERK 1/2, resulting in a further increase in the concentration of TNF-α and IL-6. The analysis of these results indicate that insulin by modulating the PI3K, Akt, ERK 1/2 and SAPK / JNK pathways amplifies the concentration levels of TNF-α and IL-6 under stimulation with LPS

Nanofibers as novel drug carrier--An overview.

Presently polymer nanofibers have received much attention due to their unique properties such as large surface area, high porosity, small pore size, superior mechanical properties and ease of addition of surface functionalities compared with any other material. Nanofibers particularly polymeric nanofiber prepared by electrospinning process can be used as carriers for the controlled drug delivery of bioactive molecules such as cytokines, growth factors, anticancer drugs, enzymes and certain vitamins. This article presents an overview of nanofibers, various techniques involved in fabrication of nanofibers, their characterization, parameters affecting electrospinning process and their applications.

Potential pharmacokinetic interactions of therapeutic cytokines or cytokine modulators on small-molecule drugs: mechanistic understanding via studies using in vitro systems.

The potential pharmacokinetic interactions between macromolecules and small-molecule drugs have received more and more attention with the increasing development of macromolecule therapeutics. Studies have shown that cytokines can differentially modulate drug-metabolizing enzymes and transporters, which raises concerns on the potential interactions of therapeutic cytokines and cytokine modulators on the disposition of small-molecule drugs. Although many in vitro studies have been conducted to characterize the effects of cytokines on drug-metabolizing enzymes and transporters, these studies were limited to only a handful of cytokines, such as interleukin-1 (IL-1), IL-6, tumor necrosis factor-α, and interferon. It is also challenging to translate these in vitro results to in vivo. In addition, information on the impact of cytokine modulators on drug-metabolizing enzymes and transporters is rather limited. More research is needed in this area. The present review is to provide a summary of the in vitro findings on the pharmacokinetic interactions of therapeutic cytokines and cytokine modulators on small-molecule drugs. Discussion on current challenges in assessing these interactions is also included.

Subfornical organ mediates sympathetic and hemodynamic responses to blood-borne proinflammatory cytokines.

Proinflammatory cytokines play an important role in regulating autonomic and cardiovascular function in hypertension and heart failure. Peripherally administered proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), act on the brain to increase blood pressure, heart rate, and sympathetic nerve activity. These molecules are too large to penetrate the blood-brain barrier, and so the mechanisms by which they elicit these responses remain unknown. We tested the hypothesis that the subfornical organ (SFO), a forebrain circumventricular organ that lacks a blood-brain barrier, plays a major role in mediating the sympathetic and hemodynamic responses to circulating proinflammatory cytokines. Intracarotid artery injection of TNF-α (200 ng) or IL-1ß (200 ng) dramatically increased mean blood pressure, heart rate, and renal sympathetic nerve activity in rats with sham lesions of the SFO (SFO-s). These excitatory responses to intracarotid artery TNF-α and IL-1ß were significantly attenuated in SFO-lesioned (SFO-x) rats. Similarly, the increases in mean blood pressure, heart rate, and renal sympathetic nerve activity in response to intravenous injections of TNF-α (500 ng) or IL-1ß (500 ng) in SFO-s rats were significantly reduced in the SFO-x rats. Immunofluorescent staining revealed a dense distribution of the p55 TNF-α receptor and the IL-1 receptor accessory protein, a subunit of the IL-1 receptor, in the SFO. These data suggest that SFO is a predominant site in the brain at which circulating proinflammatory cytokines act to elicit cardiovascular and sympathetic responses.

Immunocytokines: a novel class of potent armed antibodies.

Several cytokines have been investigated in clinical trials, based on their potent therapeutic activity observed in animal models of cancer and other diseases. However, substantial toxicities are often reported at low doses, thus preventing escalation to therapeutically active regimens. The use of recombinant antibodies or antibody fragments as delivery vehicles promises to enhance greatly the therapeutic index of pro-inflammatory and anti-inflammatory cytokines. This review surveys preclinical and clinical data published in the field of antibody-cytokine fusions (immunocytokines). Molecular determinants (such as molecular format, valence, target antigen), which crucially contribute to immunocytokine performance in vivo, are discussed in the article, as well as recent trends for the combined use of this novel class of biopharmaceuticals with other therapeutic agents.

Kinetics of plasmatic cytokines and cystatin C during and after hemodialysis in septic shock-related acute renal failure.

INTRODUCTION: Cystatin C could be a relevant residual glomerular filtration rate marker during hemodialysis (HD), and a high cytokine plasma (p) rate is associated with an increase in mortality during sepsis. To the best of our knowledge, cytokines and cystatin C kinetics during and after HD during sepsis have never been studied. In this study, we described p cytokines and cystatin C variations during and after hemodialysis in septic-shock patients with acute kidney injury (AKI). METHODS: Ten patients, from two tertiary ICUs, with septic shock-related AKI, according to RIFLE class F, were studied. In this prospective observational study, blood samples were collected at the start, after 1 hour, 2 hours, and at the end of HD with a polymethymethacrylate (PMMA) hemodialyzer (D0, D1, D2, and endD), and 30, 60, 90, 120, and 180 min after HD (postD0.5, postD1, postD1.5, postD2, and postD3). We measured p interleukins (IL)-6, IL-8, IL-10, cystatin C, and albumin. Results are expressed as variations from D0 (mean +/- SD). RESULTS: During HD, p[IL-6] did not vary significantly, whereas p[IL-8] and p[IL-10] reductions by D1 were 31.8 +/- 21.2% and 36.3 +/- 26%, respectively (P < 0.05 as compared with D0). At postD3, p[IL-8] and p[IL-10] returned to their initial values. p[Cystatin C] was significantly reduced from D1 to postD1, with a maximal reduction of 30 +/- 6.7% on D2 (P < 0.05). Norepinephrine infusion rate decreased from D0 to postD3 (0.65 +/- 0.39 to 0.49 +/- 0.37 microg/kg/min; P < 0.05). CONCLUSIONS: HD allows a transient and selective decrease in p cytokines, which are known as being correlated with mortality during septic shock. Because of a significant decrease in p cystatin C during HD, this should not be considered as an accurate marker for residual glomerular filtration rate during septic acute renal failure when receiving HD with a PMMA hemodialyzer.

Antitumor activity and pharmacokinetic properties of ARS-interacting multi-functional protein 1 (AIMP1/p43).

Although AIMP1 was identified as a component of the macromolecular aminoacyl tRNA synthetase complex involved in the cellular translation process, it was also found to be secreted as a cytokine having complex physiological functions. Among these, AIMP1's angiostatic and immune stimulating activities suggest its potential use as a novel antitumor therapeutic protein. Here we evaluated its antitumor efficacy in a mouse xenograft model bearing human stomach cancer cells. Intravenous injection of recombinant AIMP1 for 6 days resulted in significant decreases in both tumor volume and weight. Tumor volume decreased 31.1% and 54.0% when treated with AIMP1 at a concentration of 2mg/kg and 10mg/kg, respectively. Tumor weight decreased 29.1% and 52.2% when treated with AIMP1 at a concentration of 2mg/kg and 10mg/kg, respectively. Proliferating cell nuclear antigen (PCNA) staining of tumor tissues from AIMP1-treated mice (at both 2mg/kg and 10mg/kg) showed a 53% reduction of cells exhibiting an active cell cycle progression. Blood levels of tumor-suppressing cytokines such as TNF-alpha and IL-1beta increased in AIMP1-treated mice, whereas IL-12p40 and IFN-gamma levels remained unaltered. Thus, this work suggests that AIMP1 may exert its antitumor activity by inducing tumor-suppressing cytokines. In a pharmacokinetic study in rats after a single intravenous injection, AMP1 exhibited a low clearance showing a one-compartmental disposition. However, due to a low volume of distribution, AIMP1 had a short half-life of 0.1h. In a serum stability test, AIMP1 showed a half life of >60 min in human serum, 52 min in dog serum and 32 min in rat serum.

Phase I/II open-label study of the biologic effects of the interleukin-2 immunocytokine EMD 273063 (hu14.18-IL2) in patients with metastatic malignant melanoma.

BACKGROUND: To explore the biological activity of EMD 273063 (hu14.18-IL2), a humanized anti-GD2 monoclonal antibody fused to interleukin-2 (IL2), in patients with unresectable, stage IV cutaneous melanoma as measured by induction of immune activation at the tumor site and in peripheral blood. METHODS: Nine patients were treated with 4 mg/m2 per day of EMD 273063 given as a 4-h intravenous infusion on days 1, 2, and 3 every four weeks (one cycle). Peripheral blood was analyzed for T cell and natural killer cell phenotype and frequency, as well as levels of soluble IL2 receptor (sIL2R), IL10, IL6, tumor necrosis factor alpha and neopterin. Biopsies of tumor metastasis were performed prior to therapy and at day 10 of the first 2 cycles to study lymphocyte accumulation by immunohistochemistry. RESULTS: Treatment was generally well tolerated and there were no study drug-related grade 4 adverse events. Grade 3 events were mainly those associated with IL2, most commonly rigors (3 patients) and pyrexia (2 patients). Best response on therapy was stable disease in 2 patients. There were no objective tumor regressions by standard response criteria. Systemic immune activation was demonstrated by increases in serum levels of sIL2R, IL10, and neopterin. There was evidence of increased tumor infiltration by T cells, but not NK cells, in most post-dosing biopsies, suggesting recruitment of immune cells to the tumor site. CONCLUSION: EMD 273063 demonstrated biologic activity with increased immune-related cytokines and intratumoral changes in some patients consistent with the suspected mechanism of action of this immunocytokine.

Synergistic drug-cytokine induction of hepatocellular death as an in vitro approach for the study of inflammation-associated idiosyncratic drug hepatotoxicity.

Idiosyncratic drug hepatotoxicity represents a major problem in drug development due to inadequacy of current preclinical screening assays, but recently established rodent models utilizing bacterial LPS co-administration to induce an inflammatory background have successfully reproduced idiosyncratic hepatotoxicity signatures for certain drugs. However, the low-throughput nature of these models renders them problematic for employment as preclinical screening assays. Here, we present an analogous, but high-throughput, in vitro approach in which drugs are administered to a variety of cell types (primary human and rat hepatocytes and the human HepG2 cell line) across a landscape of inflammatory contexts containing LPS and cytokines TNF, IFN gamma, IL-1 alpha, and IL-6. Using this assay, we observed drug-cytokine hepatotoxicity synergies for multiple idiosyncratic hepatotoxicants (ranitidine, trovafloxacin, nefazodone, nimesulide, clarithromycin, and telithromycin) but not for their corresponding non-toxic control compounds (famotidine, levofloxacin, buspirone, and aspirin). A larger compendium of drug-cytokine mix hepatotoxicity data demonstrated that hepatotoxicity synergies were largely potentiated by TNF, IL-1 alpha, and LPS within the context of multi-cytokine mixes. Then, we screened 90 drugs for cytokine synergy in human hepatocytes and found that a significantly larger fraction of the idiosyncratic hepatotoxicants (19%) synergized with a single cytokine mix than did the non-hepatotoxic drugs (3%). Finally, we used an information theoretic approach to ascertain especially informative subsets of cytokine treatments for most highly effective construction of regression models for drug- and cytokine mix-induced hepatotoxicities across these cell systems. Our results suggest that this drug-cytokine co-treatment approach could provide a useful preclinical tool for investigating inflammation-associated idiosyncratic drug hepatotoxicity.

Opiate modulation of IL-1alpha, IL-2, and TNF-alpha transport across the blood-brain barrier.

Interleukin-1alpha (IL-1alpha), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-alpha) are proinflammatory cytokines with potent neuromodulatory effects and are implicated in the etiology and pathogenesis of various psychological and neurological disorders. The findings that chronic morphine treatment alters both blood-brain barrier (BBB) function and cytokine production raises the possibility that morphine can also modulate cytokine transport across the BBB. Here, we found that acute morphine treatment (12 mg/kg i.p.) did not alter blood-to-brain transport of IL-1alpha, IL-2 or TNF-alpha. Whereas chronic morphine treatment (48 h after implantation of 75 mg morphine pellets) and withdrawal from morphine (10-15 min after an i.p. injection of 1mg/kg of naltroxone 48 h after implantation of 75 mg morphine pellets) did not alter blood-to-brain transport of IL-1alpha or TNF-alpha, both the chronic morphine treatment and withdrawal from morphine groups had increased blood-to-brain transport of IL-2. Typically, the permeability of the BBB to IL-2 is dominated by brain-to-blood efflux, with only limited blood-to-brain transport. Here, we found that chronic morphine and withdrawal from morphine did not alter brain-to-blood efflux, but induced a novel saturable blood-to-brain transport system. Whereas IL-1alpha, IL-2, and TNF-alpha are all proinflammatory cytokines, morphine exposure has individualized effects on their blood-to-brain transport.

Feasibility study of cytokine removal by hemoadsorption in brain-dead humans.

BACKGROUND: Inflammatory cytokines occur in the circulation and in the tissues after brain death and have been associated with dysfunction of donor organs before and after transplantation. OBJECTIVE: To determine the feasibility of removing cytokines using a hemoadsorption device. DESIGN: Two-center, randomized, open-label, feasibility study in which brain-dead subjects were randomized to two treatment groups. SETTING: Two U.S. academic hospitals. PARTICIPANTS: Eight brain-dead subjects deemed unsuitable for organ donation by respective organ procurement organizations. MAIN OUTCOME MEASURES: After obtaining consent from families, subjects were treated with hemoadsorption for 4 hrs using CytoSorb. Effects on cytokines (tumor necrosis factor, interleukin [IL]-6, and IL-10) were assessed both across the device and in the plasma over time. Feasibility for cytokine removal was assessed using objective criteria. RESULTS: Cytokine removal across the CytoSorb device ranged from 4% to 30% and was not significantly different from 1 hr to 4 hrs. Overall removal was greatest for IL-6, 28% (p = .006), and least for tumor necrosis factor, 8.5% (p = .13). Plasma concentrations of both IL-6 and tumor necrosis factor, but not IL-10, were significantly reduced after the first hour of therapy; mean differences were -13% +/- 7% for IL-6 (p = .039), -23% +/- 9% for tumor necrosis factor (p = .02), and -2% +/- 7% of IL-10 (p = 23). However, plasma concentrations for all three cytokines increased over time and were above baseline by the end of the intervention. No adverse effects of therapy were observed. However, removal of cortisol and triiodothyronine was similar to removal of cytokines. CONCLUSIONS: Hemoadsorption for removal of cytokines in brain-dead subjects is feasible. Evaluation of possible clinical benefit will require controlled trials in actual donors. However, the significant capacity for cytokine removal and absence of adverse events suggest that such trials are warranted.

Transcription, expression and tissue binding in vivo of INGAP and INGAP-related peptide in normal hamsters.

We studied islet neogenesis-associated protein (INGAP) transcription and its immunocytochemical presence in and binding in vivo of (125)I-tyrosylated INGAP pentadecapeptide ((125)I-T-INGAP-PP) to different normal male hamster tissues. (125)I-T-INGAP-PP was injected intraperitoneally with or without unlabeled T-INGAP-PP (0-1 mg/100 g bw), drawing blood samples at different times after injection; radioactivity was measured in serum, brain, skeletal muscle, dorsal root ganglia, liver, kidney, small intestine and pancreas samples, expressing results as organ:serum ratio. INGAP transcription (RT-PCR) and immunopositive cells were investigated in liver, kidney, brain, small intestine and pancreas. Total serum radioactivity increased progressively as a function of time; whereas 71% of this activity was displaced by unlabeled T-INGAP-PP at 5, 10 and 20 min, only 9% was at 60 min. Only liver, pancreas and small intestine specifically bound (125)I-T-INGAP-PP. The pancreas tissue dose-response curve showed a 50% displacement at 3.9x10(4) ng/100 g bw, suggesting a low binding affinity of its receptor. INGAP-mRNA was only identified in pancreatic islets and exocrine tissue. Our results suggest that INGAP transcription/expression is probably restricted to pancreas cells exerting its effect in a paracrine fashion. INGAP would be released and circulate bound to a serum protein from where it is bound and inactivated by the liver. Tissue binding could also explain INGAP's immunocytochemical presence in small intestine, where it could affect epithelial cell turnover.

Preclinical pharmacokinetics, tissue distribution, and antitumor activity of a folate-hapten conjugate-targeted immunotherapy in hapten-immunized mice.

Folic acid (pteroylglutamic acid) represents a useful ligand for targeted cancer therapies because it binds to a common epithelial tumor antigen known as the folate receptor. We previously devised an immunotherapy strategy that uses a bispecific ligand, a folate-hapten (FITC) conjugate, to redirect endogenously induced anti-FITC antibodies to folate receptor-positive tumor cells following parenteral administration. Here, we present results from preclinical pharmacokinetic and tissue biodistribution studies using a radioactive folate-FITC conjugate and results from dose optimization studies done in tumor-bearing animals. Folate-FITC was found to be rapidly eliminated in non-immunized mice; however, in immunized hosts, folate-FITC was shown to form immune complexes with FITC-specific antibodies, the consequence of which was a approximately 173-fold increase in drug exposure (i.e., area under the curve). Using a newly developed ELISA assay, the extent of circulating anti-FITC antibodies occupied by parenterally given folate-FITC was determined to be proportional to the given dose. Furthermore, high doses of folate-FITC were found to promote the cosaturation of tumor cell surface folate receptors and circulating FITC-specific antibodies, blocking the immune recognition of tumor cells and thereby reducing antitumor activity. Nonetheless, by extending the duration of treatment and administering subsaturating doses of folate-FITC, enhanced antitumor response was observed in mice bearing established folate receptor-positive M109 tumors. Overall, results from the present study may help to guide clinicians through on-going clinical investigations of folate-targeted immunotherapy.