Edward J. Shannon

Randomized safety studies of intravenous perflubron emulsion. II. Effects on immune function in healthy volunteers.

Particle size distribution is a major determinant of particle clearance by the mononuclear phagocytic system and the potential for concomitant activation of resident macrophages. To test the safety of a second-generation perflubron-based emulsion (60% perfluorocarbon [PFC] wt/vol; Oxygent [Alliance Pharmaceutical Corp., San Diego, CA]) with a small mean particle size, two parallel, randomized, double-blinded, placebo-controlled studies were conducted in 48 healthy volunteers (n = 24 per study). The study described herein focuses on safety concerning immune function. The primary endpoint was defined prospectively as delayed hypersensitivity skin test responses with lymphocyte proliferative responses to mitogenic stimulation providing a secondary measure for changes in cell-mediated immunity. Subjects received either perflubron emulsion IV (1.2 g PFC/kg or 1.8 g PFC/kg) or saline (3 mL/kg) control. Perflubron emulsion had no effect on delayed hypersensitivity skin reactions, lymphocyte proliferative potential, circulating immunoglobulins, complement activation, or plasma levels of the inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1 alpha, and interleukin-1 beta. Perflubron emulsion was generally well tolerated, although there was a dose-dependent increase in minor flu-like symptoms in the perflubron treatment groups at 24 h after dosing. Increased serum levels of interleukin-6 were observed in those subjects exhibiting febrile responses. The clinical safety profile of perflubron emulsion supports its continued investigation as a temporary oxygen carrier in surgical patients to reduce exposure to allogeneic blood transfusion.Previous perfluorocarbon (PFC) emulsions have been associated with transient adverse events (i.e., platelet activation, decreased platelet count, febrile responses, changes in hemodynamic function). The Phase I studies described in this report were parallel, randomized, double-blinded, placebo-controlled studies conducted in 48 healthy volunteers (n = 24 per study) with perflubron emulsion (Oxygent™; Alliance Pharmaceutical Corp., San Diego, CA). Because of the decreased platelet counts observed with previous PFC emulsions and the intended use of perflubron emulsion in surgical patients, these studies assessed postdosing coagulation responses and hemostasis. PFC pharmacokinetic variables were also evaluated. The primary endpoint for examination of coagulation effects was prospectively defined as bleeding time. Subjects received either saline (3 mL/kg) control, or perflubron emulsion at 1.2 g PFC/kg or 1.8 g PFC/kg, and were evaluated for a 14-day period. No postinfusion changes in bleeding time or differences in ex vivo agonist-induced platelet aggregation were observed. A 17% reduction in platelet count was observed 3 days after dosing in the 1.8-g PFC/kg group; levels recovered to baseline by Day 7. The intravascular half-life of perflubron for the first 24 h was dose dependent: 9.4 ± 2.2 h and 6.1 ± 1.9 h in the 1.8- and 1.2-g PFC/kg groups, respectively. Results indicate that this perflubron emulsion did not affect coagulation function in healthy volunteers. Implications In major surgical procedures, perfluorocarbon-based temporary oxygen carriers are potentially important alternatives to blood transfusion. Newer, second-generation perfluorocarbon-based oxygen carriers have been developed to improve on the short-term side effects observed with earlier formulations. This report summarizes Phase I clinical safety data in healthy volunteers receiving the oxygen carrier, perflubron emulsion.

Thalidomide Can Be Either Agonistic or Antagonistic to LPS Evoked Synthesis of Tnf-α by Mononuclear Cells

The effect if thalidomide on tumor necrosis factor alpha (TNF-alpha) produced in vitro by lipopolysaccharide (LPS) stimulated human cells was investigated. In cultures of LPS stimulated human mononuclear cells enriched for adherent cells and in cultures of LPS stimulated human monocytes of the cell line-THP-1, thalidomide enhanced the synthesis of TNF-alpha. When cultures of unfractionated peripheral blood mononuclear cells were stimulated with LPS, thalidomide decreased the synthesis of TNF-alpha. Depending on the type of cells stimulated with LPS in vitro, thalidomide, at concentrations achieved in vivo, can either enhance or suppress the synthesis of TNF-alpha.

Immunomodulatory assays to study structure-activity relationships of thalidomide.

Thalidomide, which has a long history of tragedy because of its ability to cause severe birth defects, is very effective in alleviating erythema nodosum leprosum in leprosy patients and aphthous ulcers in AIDS patients. The causes of these inflammatory diseases and the mechanism by which thalidomide diminishes them are unknown. It has been suggested that modulation of the immune response plays an important role. We found that thalidomide exerts immunomodulatory activity in three bioassays. It suppresses an IgM plaque forming cell response in mice injected with sheep erythrocytes: it inhibits TNF-alpha production by LPS stimulated human mononuclear cells: and it enhances IL-2 production by Con-A stimulated human mononuclear cells. We employed these bioassays to compare the activity of 15 analogs of thalidomide with thalidomide itself. Eight of the compounds were derivatives of the glutarimide moiety of thalidomide and the others were phthalimide or derivatives of the phthalimide moiety of thalidomide. N-hydroxyphthalimide, a simple derivative of phthalimide, was more effective than thalidomide and was also the most effective of the compounds assayed in suppressing the IgM plaque and TNF-alpha responses, but it did not enhance the IL-2 response, instead, it significantly suppressed it.

Thalidomide Suppressed IL-1β While Enhancing TNF-α and IL-10, When Cells in Whole Blood were Stimulated with Lipopolysaccharide

Thalidomide is used to treat erythema nodosum leprosum (ENL). The events that precipitate this inflammatory reaction, which may occur in multibacillary leprosy patients, and the mechanism by which thalidomide arrest ENL, are not known. Thalidomides ability to inhibit tumor necrosis factor alpha (TNF-α) in vitro has been proposed as a partial explanation of its effective treatment of ENL. In in vitro assays, thalidomide can enhance or suppress TNF-α. This is dependent on the stimulant used to evoke TNF-α; the procedure used to isolate the mononuclear cells from blood, and the predominant mononuclear cell type in the culture. To avoid artifacts that may occur during isolation of mononuclear cells from blood, we stimulated normal human blood with LPS and evaluated the effect of thalidomide and dexamethasone on TNF-α, and other inflammatory cytokines and biomarkers. Thalidomide suppressed interleukin 1 β (IL-1β) (p = 0.007), and it enhanced TNF-α (p = 0.007) and interleukin 10 (IL-10) (p = 0.031). Dexamethasone enhanced IL-10 (p = 0.013) and suppressed IL-1β, TNF-α, interleukin 6 (IL-6), and interleukin 8 (IL-8) (p = 0.013). The two drugs did not suppress: C-reactive protein (CRP), Ig-superfamily cell-adhesion molecule 1 (ICAM 1), tumor necrosis factor receptor 1 (TNFR1), tumor necrosis factor receptor 2 (TNFR2), or amyloid A. In vitro and in vivo evidence is accumulating that TNF-α is not the primary cytokine targeted by thalidomide in ENL and other inflammatory conditions.

THALIDOMIDE DOES NOT PERTURB CD2, CD4, CD5, CD8, HLA-DR, OR HLA-A, B, C MOLECULES IN VITRO ON THE MEMBRANES OF CELLS WITH IMMUNE POTENTIAL

Thalidomide dramatically relieves the signs and symptoms of erythema nodosum leprosum (ENL). ENL is an acute inflammatory complication of lepromatous leprosy. The cause(s) of ENL as well as the mechanism of action of thalidomide in arresting ENL are unknowns. It has been suggested that ENL is the consequence of a transient activation of a cell-mediated-immune (CMI) response to Mycobacterium leprae. To initiate a CMI response, an interaction between adhesion and/or signal transducing molecules on T-cells and molecules on antigen presenting cells would occur. An alteration, induced by thalidomide, of one or more of the molecules on T-cells or antigen presenting cells that are essential to maintaining the reactive state of ENL, could explain Thalidomides ability to attenuate ENL. Thalidomide did not modify: (a) adhesion and/or signal transducing molecules such as CD2, CD4, CD5 and CD8, or (b) molecules that facilitate antigen presentation such as HLA-DR, HLA-A, HLA-B, or HLA-C.

Thalidomide delayed the ability of 4T1 cells to amass into tumors in Balb/c mice.

Thalidomide (Thal) can suppress the growth of established, as well as explanted tumors in mice. We wanted to determine if it could suppress the ability of tumor cells to assemble and establish a primary tumor at the injection site. Using the mouse 4T1 mammary tumor model, we fed Thal to mice for 4 days, then injected 105 4T1 cells into the interscapular region of Balb/c mice. After 20 days on treatment with Thal, all seven control mice, fed with meal had tumors ranging from 3 to 93 mm3 (median 20). Two of the eight mice fed with meal + Thal had no tumors, and the remaining mice had tumors ranging from 2 to 22 mm3 (median 5). The median volume of the tumors in the control group was significantly more than that of mice treated with Thal (p = 0.03, Mann–Whitney test). In vitro treatment of the 4T1cells with Thal did not inhibit their ability to proliferate, to adhere to plastic, or to bind to Concanavalin-A. Thal caused a marked reduction in the ability of the 4T1 cells to assemble into palpable tumors.