Anesth Analg 2000;91:812-822
© 2000 International Anesthesia Research Society
CARDIOVASCULAR ANESTHESIA
Randomized Safety Studies of Intravenous Perflubron Emulsion. II. Effects on Immune Function in Healthy Volunteers
Robert J. Noveck, MD, PhD*,
E. J. Shannon, PhD
,
Phillip T. Leese, MD
,
Jolene S. Shorr, MA§,
Kathryn E. Flaim, PhD§,
Peter E. Keipert, PhD§, and
Catherine M. Woods, PhD§
*Clinical Research Center, New Orleans, Louisiana;
Louisiana State University, Baton Rouge, Louisiana;
Innovex Inc., Lenexa, Kansas; and
§Alliance Pharmaceutical Corp., San Diego, California
Address correspondence and reprint requests to Peter E. Keipert, PhD, Alliance Pharmaceutical Corp., 3040 Science Park Rd., San Diego, CA 92121.
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Abstract
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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; OxygentTM [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-
, interleukin-1 , and interleukin-1 ß. 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.
Implications: In major surgical procedures, perfluorocarbon-based temporary oxygen carriers are potentially important alternatives to blood transfusion. Early perfluorocarbon-based oxygen carriers were limited by side effects that have been overcome with the newer, second-generation oxygen carriers. This report summarizes Phase I clinical safety findings of an improved second-generation oxygen carrier, perflubron emulsion.
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Introduction
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Although the safety of the blood supply has markedly improved over the years with the introduction of more stringent donor screening practices and improved diagnostic testing, regional blood shortages and continued skepticism from the general public regarding the safety of the blood supply have led to the search for alternatives to allogeneic blood transfusion. A variety of products have been developed, ranging from hemoglobin-based blood substitutes to perfluorocarbon (PFC)-based temporary oxygen (O2) carriers (1,2).
PFCs exhibit a high solubility for O2 and carbon dioxide and readily release dissolved O2 to the tissues by simple diffusion because of a negligible O2-binding constant (3,4). Therefore, PFC emulsions provide an ideal vehicle for increasing tissue oxygenation in a variety of clinical settings, including the avoidance of allogeneic blood transfusion during large blood-loss surgical procedures, the protection of patients from cerebral hypoxia, and the prevention of myocardial damage during or after a period of low flow-induced ischemia (e.g., during cardiopulmonary bypass).
PFCs are inert, highly hydrophobic synthetic molecules that are immiscible with water. Therefore, they must be emulsified with a surfactant in an aqueous phase to make a biocompatible product for IV infusion. Some first-generation PFCs (e.g., Fluosol® [Green Cross Corp., Osaka, Japan], the only O2 carrier approved to date for marketing by the Food and Drug Administration), although effective O2 carriers, were limited by some of their physicochemical properties and were associated with clinical adverse events (5–7). In addition, certain emulsifiers, such as Pluronic-F68, the agent used to emulsify Fluosol®, was associated with complement activation accompanied by decreases in platelet count and adverse leukocyte effects (7–9). The first-generation perflubron-based emulsion formulations, although more concentrated (90% to 100% perflubron [wt/vol]) and stable than Fluosol® (2,7,10,11), were associated with marked flu-like symptoms and delayed febrile reactions (12) as well as a transient decrease in platelet count (13).
It has been shown that size is a major factor determining clearance rate of particles from the circulation, the degree of complement activation, and the site of primary clearance. Many studies with various liposomal formulations have indicated that particles 
0.3 µm in diameter are readily opsonized with complement and cleared more rapidly from the circulation than particles 
0.2 µm in diameter (14–18). Large particles appear to be cleared by the spleen, whereas small particles are cleared predominantly by the liver (17). Early studies with Fluosol® demonstrated a similar relationship of particle size with clearance from the circulation (19,20). The previous 90% perflubron emulsion formulations had a median particle diameter ranging from 0.25 to 0.35 µm. A systematic formulation effort led to the current perflubron-based emulsion (58% wt/vol perflubron, 2% wt/vol perfluorodecylbromide; OxygentTM [Alliance Pharmaceutical Corp., San Diego, CA]), which has a reduced particle size (median particle diameter of 0.16 to 0.18 µm) and increased particle stability (21). This reduction in particle size was shown to correlate with a reduced febrile response in rat (22).
The present clinical studies described in this and the companion article (23) were designed to assess the safety profile of this 60% PFC wt/vol perflubron emulsion formulation in healthy volunteers. Both studies evaluated overall safety, hemostasis and coagulation responses, and blood cell differentials after perflubron emulsion dosing; both studies also evaluated blood perflubron pharmacokinetics. The study described in this report had special emphasis on assessment of immune function, and it is these aspects, together with overall safety results obtained from both studies, that are described in this article. The coagulation assessments and detailed pharmacokinetics are described in the companion report (23).
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Methods
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Twenty-four healthy volunteers (12 men, 12 women) were enrolled in the study between February and May 1995 after undergoing a medical history screening, a physical examination, and baseline laboratory testing 4 to 21 days before study entry to ensure that they fulfilled entry criteria. Eligibility criteria included healthy (as evidenced by lack of liver, renal, cardiovascular, or immune dysfunction) adults between 18 and 45 yr of age. Subjects were to show at least two positive reactions to a standard delayed hypersensitivity skin test (DHST 7-antigen kit of Multitest CMI; Connaught Laboratories, Toronto, Canada). Subjects were excluded from the studies if they had any signs of overt infection, had had any acute infection within the previous month, had abnormal bleeding times (defined as 2 x normal) or coagulation responses, or were taking chronic medications. Aspirin-containing medication was disallowed beginning 14 days before treatment (i.e., before Day 0), and medications other than contraceptives were excluded beginning 10 days before dosing. All women were required to be using an approved form of birth control and to exhibit a negative serum pregnancy test at baseline. Subjects were required to have a white blood cell (WBC) count and hemoglobin level within the normal clinical range and to have a platelet count above 200,000/µL at baseline before randomization.
This study was one of two randomized, double-blinded, parallel-group, placebo-controlled Phase I studies conducted in parallel at two investigational sites in the United States (Clinical Research Center, New Orleans, LA, and Innovex, Lenexa, KS). The primary focus of this study (OXHT-008) was to evaluate the effect of the 60% perflubron-based emulsion on immune function. The parallel study (OXHT-007) had particular emphasis on blood hemostasis and coagulation as well as perflubron pharmacokinetics and elimination in expired air. The immune responses to perflubron emulsion dosing and the safety profile are discussed in this report, and the coagulation and hemostasis data and pharmacokinetics are covered in the companion report (23).
The study was approved by the institutional review board and consisted of a screening visit, a 10-day inpatient period (from Day –3 to Day 7), with a follow-up visit on Day 14 (Table 1). Both this and the parallel study (OXHT-007) assessed bleeding times and coagulation responses with identical pre- and postdosing schedules and followed blood perflubron emulsion levels through 7 days after dosing, as well as adverse events, blood cell counts, and serum chemistries through Day 14. The primary endpoint for assessing the effect of perflubron emulsion on immune function was the DHST to recall antigens as a measure of cell-mediated immunity. Bleeding time served as the primary endpoint for assessing hemostasis and coagulation responses to dosing. Data from the companion study that were relevant to immune function (e.g., WBC counts) and safety profile have been included in this report.
After it was determined that they continued to fulfill entry criteria at baseline, subjects were randomized equally by sex to one of three treatment groups: saline Control (3 mL/kg), small-dose (P1.2) perflubron emulsion (1.2 g PFC/kg [2 mL/kg]), or large-dose (P1.8) perflubron emulsion (1.8 g PFC/kg [3 mL/kg]). The perflubron emulsion (OxygentTM) is a 60% wt/vol perflubron-based emulsion (formulation AF0144), primarily containing perflubron (perfluorooctyl bromide; C8F17Br) and a small quantity (2%) of perfluorodecylbromide (C10F21Br), emulsified with egg yolk lecithin in phosphate-buffered saline.
Baseline variables of immune system function and WBC counts were assessed from a sample of blood collected 1 h before dosing. Study drug or saline was administered by the pharmacist by IV infusion for a period of 5 to 20 min (infusion rate, approximately 15 mL/min) on Day 0. All other study personnel, who were responsible for subject monitoring and study assessments, were blinded to treatment. Blood samples were drawn at 20 min after the start of infusion and at selected intervals thereafter for assessment of immune responses and perflubron blood levels as outlined in Table 1. The primary endpoint for assessing whether there were any changes in immune response after dosing was defined prospectively as DHST responses on Day 3 (72 h) compared with baseline responses. Lymphocyte ex vivo proliferative responses to mitogenic stimulation provided secondary functional assessments for cell-mediated immunity: these were performed on Days 1, 3 and 7. In addition, complete blood cell counts and WBC differentials (neutrophils, lymphocytes, monocytes, eosinophils, and basophils), lymphocyte subsets (CD3+, CD4+, CD8+, CD14+, CD16+, CD19+), immunoglobulin classes (IgG, IgA, IgM, IgD, IgE), and immune complex levels were measured at selected postdosing intervals (Table 1). To assess whether perflubron emulsion had triggered any inflammatory reaction, serum levels of tumor necrosis factor- (TNF-), interleukin-1 (IL-1) and ß (IL-1ß), IL-2, IL-6, and C-reactive protein (CRP) were measured. Complement components, C3, C3a, C4, and total serum hemolytic complement (CH50) were measured through the first 24 h of dosing to monitor for evidence of complement activation.
Body temperature and vital signs, including heart rate, systolic and diastolic arterial blood pressures, and respiratory rate, were measured at baseline, at 15 and 30 min after infusion, at 1, 2, 4, 6, 8, 12, 16, and 24 h after infusion, and daily through Day 7. Routine clinical chemistries to assess hepatic and renal function were measured at baseline, daily through Day 7, and on Day 14. Hepatic function was assessed by measuring aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, and bilirubin. Renal function was assessed by measuring serum creatinine, blood urea nitrogen, and uric acid. Adverse events were monitored throughout the study period and classified as defined by World Health Organization Adverse Reaction Terminology.
Cell-mediated immune responses were measured using a 7-antigen hypersensitivity skin test comprising Corynebacterium diphtheriae, Clostridium tetani, Mycobacterium tuberculosis, Streptococcus, Candida, Trichophyton, and Proteus antigens. A positive response to a given recall antigen was defined as an induration of 10 mm 2 days after administration. Ex vivo lymphocyte proliferation was measured by [3H]-thymidine incorporation after exposure of mononuclear cells to phytohemagglutinin (PHA) and pokeweed mitogen (PWM). Mononuclear cells were isolated from peripheral blood collected in sodium heparin treated vacuum tubes, the plasma removed by centrifugation and the cells resuspended in 0.85% saline. The cell suspension was then added to Ficoll-Paque (Pharmacia, Uppsala, Sweden) to harvest the mononuclear cells. The cells were washed twice, then resuspended to 2 x 106 viable lymphocytes (trypan-blue and peroxidase negative)/mL in RPMI-1680 culture medium containing 10% calf serum. Aliquots (150 µL) were added to each well of 96-well plates and 4 µg of PHA or 4 µg of PWM in 50 µL RPMI or medium alone was added per well. After 48 h of culture at 37°C in a humidified atmosphere with 5% CO2, 0.5 µCi of methyl-{H3}-thymidine (7 Ci/nmole; ICN Radiochemicals, Irvine CA) was added to each well. Twenty-four hours later, cells were harvested, and the 3H-thymidine content was measured in a Beckman LS 6000IC liquid scintillation counter (Beckman, Fullerton, CA). Cytokine levels were measured with enzyme-linked immunoadsorbent assay kits for TNF- and IL-6 (BioSource International, Camarillo, CA), and for IL-1 and IL-2 (Endogen, Inc., Woburn, MA). The CRP was quantified by radioimmunoprecipitation (The Binding Site, Inc., San Diego, CA), and C3 and C4 complement levels were quantified by radial immunodiffusion. C3a, which is generated during activation of the complement system by both the classical and alternate pathway, and circulating immune complexes were measured by enzyme immunoassay (QUIDEL, San Diego CA). CH50 titers were measured by using lysis of hemolysin-sensitized sheep erythrocytes (DiaMedix, Miami, FL). Lymphocyte subsets were quantified by fluorescence-activated cell-sorter analysis (FACScan; Beckton Dickinson, San Jose, CA). For this, erythrocyte-lysed whole blood was incubated with Becton-Dickinson-conjugated monoclonal antibodies and the percent CD3+, CD4+, CD8+, CD14+, CD16+, and CD19+ cells determined by flow cytometry. A radial immunodiffusion method was used to measure immunoglobulins IgG, IgM, IgA, IgD, and IgE (The Binding Site, Inc.), and WBC count and differentials were quantitated by using standard clinical laboratory methods.
All data were presented as the mean ± SD or SEM. Twenty-four subjects from the parallel study (OXHT-007) were included in the analyses of body temperature, incidence of adverse events, and WBC counts and differentials. All other analyses included only the 24 subjects from this study (OXHT-008). Data were entered into a clinical database by subject, and the treatment code and group assignment blind were broken only once all the data had been entered. Differences between treatment groups were determined by analysis of variance (analysis of variance, with Fisher’s least significant difference method) by using StatView IITM software (Abacus Concepts, Inc., Berkeley, CA). For all statistical comparisons, a P value < 0.05 was considered significant.
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Results
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Twenty-four healthy subjects were screened and enrolled in this study (OXHT-008): 8 subjects were randomized to each of the three treatment groups (P1.2, P1.8, Control). All subjects completed the study through Day 14. Subject demographic and baseline clinical characteristics are summarized in Table 2 by treatment group. Between-group demographics and baseline immunologic variables were similar at study entry. This study cohort showed similar demographics to the parallel study described by Leese et al. (23) with respect to age, weight, and height.
Cell-Mediated Immune Responses
The principal test for assessing the integrity of cell-mediated immune function was the DHST response at 3 days after infusion compared with that at baseline. Anergy was not observed in any of the perflubron emulsion-treated subjects as assessed by loss of response on Day 3 to any of the seven antigens that had elicited a positive response (defined as appearance of an induration 10 mm) at baseline. Furthermore, as shown in Table 3, there were no between-group differences with regard to the number of positive reactions before and after infusion. The most frequently observed positive skin reactions were to Corynebacterium diphtheriae, Clostridium tetani, and M. tuberculosis antigens. Only 2 of 24 (8.3%) volunteers, 1 from each of the two perflubron emulsion dose groups, exhibited a different response pattern to the seven antigens after infusion. However, these differences were attributed by the investigator (before the treatment code was known) to technical difficulties in applying antigens to the skin and not to any PFC-induced effects because subsequent tests showed shifting responses rather than a continuing loss of response to those antigens that had elicited a positive reaction at baseline.
Ex vivo responses of T and B lymphocytes to PWM and PHA stimulation were assessed in blood samples drawn at baseline, 24 h, 72 h, 7 days, and 14 days after dosing. The results, expressed in terms of stimulation index (fold increase in 3H-thymidine incorporation relative to control, nonmitogen-stimulated levels) are shown by treatment group in Figure 1. Although baseline values were highly variable, responses to PWM and PHA were similar between treatment groups, and no significant between-group differences in stimulation index were observed after the administration of perflubron emulsion throughout the duration of the study.
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Figure 1. Proliferation potential of T and B lymphocytes in response to stimulation with pokeweed mitogen (PWM) (A) and phytohemagglutinin (PHA) (B). P1.2 = 1.2 g PFC/kg (closed circles); P1.8 = 1.8 g PFC/kg (closed triangles); Control (open circles). Data are mean ± SEM (n = 8 per group). PFC = perfluorocarbon.
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Hematology
Mean WBC counts were similar between treatment groups at baseline (data were pooled from both studies for this analysis, n = 48). A transient reduction in total WBC count was observed in both perflubron emulsion-treated groups (36%, P1.2 group; 21%, P1.8 group) at 20 min after infusion (Figure 2). However, this effect was short lived: total WBC counts returned to baseline values at the next time point (24 h after infusion) and remained stable thereafter. Furthermore, mean values remained within the normal range (3.8 to 10.8 x 109 cells/L) at all time points. Changes in WBC differential counts for each treatment group are also shown in Figure 2. Transient decreases in neutrophil, monocyte, and eosinophil counts and, to a lesser extent, basophil counts (data not shown) were observed at 20 min after infusion in both the P1.2 and P1.8 groups. Similar to overall WBC counts, neutrophil counts recovered rapidly to above baseline values at 24 h after infusion, whereas monocyte and eosinophil counts recovered more slowly, returning to normal levels by 48 h after infusion. Five PFC-treated subjects experienced transient decreases in their neutrophil counts to <40% of baseline values, and one subject exhibited a severe decrease in neutrophil count to 477 x 106 cells/L at 20 min after infusion. However, in all subjects, neutrophil counts returned to normal levels at the next time point, 24 h, and no infectious complications were observed. Lymphocyte counts decreased at a slower rate than other cell types, reaching a nadir at 24 h and recovering to baseline by 48 h.
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Figure 2. Total white blood cell (WBC) counts and differentials after the administration of perflubron emulsion or placebo on Day 0 combined from both the OXHT-007 and OXHT-008 studies: WBC (A); neutrophils (B); lymphocytes (C); monocytes (D); and eosinophils (E). P1.2 = 1.2 g PFC/kg (n = 16; closed circles); P1.8 = 1.8 g PFC/kg (n = 16; closed triangles); Control (n = 16; open circles). Data are mean ± SEM (n = 16 per group). WBC analysis at 20 min after dosing was performed in the OXHT-008 study only. PFC = perfluorocarbon, ULN = upper limit of normal, LLN = lower limit of normal.
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Different lymphocyte subset and monocyte counts were analyzed by using flow cytometry to determine if infusion of perflubron emulsion selectively affected any particular cell types. For all treatment groups, no changes were observed in the percent of CD3+ (T), CD19+ (B), or CD16+ (natural killer) cells in the WBC population on Days 1, 3, and 7 after administration of perflubron emulsion (Table 4). In addition, no between-group differences were observed in the proportions of CD4+ and CD8+ T cells. However, a small decrease in the CD14+ monocyte fraction was observed at 24 h after infusion in both perflubron emulsion-treated groups, similar to the small decrease in monocytes observed by standard laboratory measurements (Figure 2).
Complement and Immunoglobulin Levels
The effect of perflubron emulsion on activation of the complement system was investigated by measuring levels of C3, C3a, C4, and CH50 activity. Levels of C3 and C4 did not change significantly during the 24-h postinfusion period. C3a levels were transiently elevated to a similar extent in both the saline control and perflubron emulsion treatment groups (about four-fold) at 20 min after the start of infusion, returning toward baseline values between 16 and 24 h after infusion (Figure 3). Activity of CH50 was inversely correlated with C3a level, decreasing at 20 min and returning to normal between 16 and 24 h (Figure 3).
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Figure 3. C3a concentration and CH50 U after the administration of perflubron emulsion or saline placebo (solid line) on Day 0. P1.2 = 1.2 g PFC/kg (solid circles); P1.8 = 1.8 g PFC/kg (solid triangles). Data are mean ± SEM (n = 8 per group). PFC = perfluorocarbon.
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Infusion of perflubron emulsion had no effect on serum levels of any Ig subclasses on postinfusion Days 1, 3, 7, and 14 (data not shown). No consistent differences between placebo- and perflubron emulsion-treated subjects were observed. Likewise, levels of circulating immune complexes were unaffected by perflubron emulsion.
Cytokine Levels
Serum levels of the inflammatory cytokines, IL-1, IL-1ß, and TNF-, remained below or near the limit of detection of the assay in all treatment groups after dosing. Infusion of perflubron emulsion was associated with a small dose-dependent increase in serum concentrations of IL-6 and CRP (Table 5), both of which are associated with the acute phase response. All 24 volunteers (i.e., both saline control- as well as perflubron emulsion-treated subjects) exhibited a transient increase in IL-6 serum levels above baseline by 8 h after infusion, but these levels returned to baseline by 16 h. A larger increase was observed in the P1.8 group resulting from the higher peak IL-6 serum levels (142 and 190 pg/mL) in two female subjects who also exhibited the most pronounced febrile response. Similarly, a dose-related increase in serum CRP levels was observed from 16 to 24 h in both treatment groups, returning toward baseline by Day 3. No change from baseline was observed for CRP levels in the Control group in the 3-day period after dosing. Mean serum IL-2 levels increased in all three groups after treatment with peak levels noted at 24 h after infusion. However, the increase was similar for both treatment groups as well as saline controls.
Body Temperature
Febrile responses were not observed in the Control and P1.2 groups. Delayed febrile responses occurring approximately 8 h after dosing were observed in 5 of 16 subjects treated with P1.8 across the two studies (3 subjects from OXHT-008 and 2 from OXHT-007). However, in all cases, the fever was mild and had resolved by 9 to 12 h. The most pronounced temperature increases (2°C increase) were observed in 2 female subjects in the P1.8 group within the OXHT-008 study. These same 2 subjects exhibited substantially increased levels of IL-6 at 8 h and increase IL-2 and CRP levels at 24 h after infusion. Overall, the mean body temperature for each treatment group was normal throughout the first 24-h postinfusion (Figure 4).
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Figure 4. Body temperature (mean ± SEM) after the administration of perflubron emulsion or saline placebo (open circles) on Day 0. P1.2 = 1.2 g PFC/kg (solid circles); P1.8 = 1.8 g PFC/kg (solid triangles). Data combined from both the OXHT-007 and OXHT-008 studies (n = 16 per group). PFC = perfluorocarbon.
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Adverse Events
As shown in Table 6, adverse events were more prevalent among the treated groups, with all subjects experiencing at least one adverse event. Further, 9 of 16 (56.3%) Control subjects reported at least one adverse event during the study. The majority of events occurred within the first 48 h after dosing and were in two broad categories: (1) early onset (within minutes to 1 h), transient events such as facial flushing, taste perversion ("oily taste"), back and chest pain, dyspnea, and dizziness; and (2) delayed onset (4 to 8 h) symptoms of headache, malaise, myalgia, fever, that could be broadly categorized as "flu-like." These flu-like symptoms typically resolved within 12 to 24 h. The most commonly reported events for the perflubron emulsion-treated groups included headache (81.3%), dizziness (71.9%), nausea (62.5%), vomiting (43.8%), back pain (34.4%), and chest pain (31.3%). Back pain (18.8%) and dizziness (25%) were also observed in the Control group. The majority of adverse events were mild (79.8% of the total of 233 separate events reported by the 48 subjects throughout the 14-day posttreatment period) or moderate (18.5%) in severity. Three cases of headache and one of transient back pain were reported as severe.
No changes of note were observed throughout the study in arterial systolic and diastolic blood pressure, heart rate, or respiratory rate. No significant changes were observed in either hepatic or renal function (data not shown).
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Discussion
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PFC emulsions have potential use in a variety of clinical indications as temporary O2 carriers to prevent tissue hypoxia, provided they have an acceptable safety profile. Factors such as the surfactant used to emulsify the PFC, emulsion particle size, O2-carrying capacity, and side effects can affect the clinical safety and use of PFCs. First-generation PFC emulsions were often associated with a variety of adverse events, including flu-like symptoms, complement activation, decreased platelet counts, WBC changes, and transient febrile responses (8,9,10,24,25).
Previous Phase I studies with 90% to 100% PFC wt/vol perflubron emulsion formulations (doses from 0.68 to 1.35 g/kg) indicated that most treated subjects experienced marked febrile and flu-like responses. In some cases, there was evidence of increased levels of serum TNF-. These data indicated that this first-generation perflubron emulsion triggered a transient inflammatory response. Subsequent formulation development indicated that the addition of small levels of perfluorodecylbromide reduced mean particle size and improved emulsion particle stability (21). Formulations with smaller mean particle size reduced the febrile response and improved intravascular retention times in the rat (22) similar to earlier observations on particle size effects of Fluosol® (19,20) and liposomes (16,17,19).
These Phase I studies were designed to assess whether the 60% PFC formulation (OxygentTM), which has a median particle diameter of 0.16 to 0.18 µm, exhibits an improved safety profile in humans. In this study, the impact of perflubron emulsion dosing on subsequent cell-mediated immune response was evaluated. With respect to the primary (DHST) and secondary functional assessments (lymphocyte proliferative responses), the data clearly indicated that there was no change in either set of responses after perflubron dosing. The impact on humoral immunity was assessed by monitoring levels of circulating immunoglobulins, but none of the subclasses showed any changes on dosing. The only change observed was a decrease in WBC in the circulation at 20 minutes after the start of dosing. This appeared to be a relatively nonspecific, transient margination effect as all WBC cell type levels were affected to a similar extent and levels had recovered by the next time point (24 hours). Because the protocol only specified WBC and differential measurements at 20 minutes and 24 hours during the first day after dosing, it was not possible to determine the duration of this effect. However, there were no reports of infection during the first week after dosing, suggesting that this transient decrease had no clinical sequelae in this study population.
Levels of the proinflammatory cytokines, TNF-, IL-1, and IL-1ß remained at baseline values, indicating that perflubron emulsion dosing was not associated with any systemic inflammatory response. The only change observed was a minor increase in the acute phase proteins, IL-6 and CRP, in those patients experiencing the most marked fever responses (Table 5). The increase (maximal response in the 100–200 pg/mL range) was three orders of magnitude less than has been observed during systemic inflammatory disease such as sepsis (26). Circulating immune complexes provide another measure of immune mediated inflammation but no differences were observed after dosing. Complement effects were assessed by measuring C3, C4, and C3a, the short-lived cleavage product of the activated complement classical and alternate pathways, but again there were no differences between perflubron emulsion-treated and Control groups. These results confirmed that the egg yolk phospholipid-coated perflubron emulsion particles were not associated with the activation of the complement pathway that had been observed with pluronic-based formulations such as Fluosol® (20).
Studies with Fluosol® had indicated that the infusion was associated with adverse hemodynamic effects which were attributed to complement activation (3,8,9). No evidence for hemodynamic changes were observed after perflubron emulsion infusion in either of the two studies presented here, providing further evidence that perflubron emulsion was not associated with complement activation.
Although the febrile responses were not entirely abolished with this 60% PFC formulation, no changes were observed compared with baseline in the Control or P1.2-treated subjects, and the P1.8 group showed only a slight increase of 0.6°C to a mean value of 37.4°C, well within the normal range. Only 5 of the 16 subjects in the P1.8 group experienced a febrile response, defined in this study as a temperature increase to 38°C. However, in three cases, only a minor increase (1°C) was observed, with the maximum temperature observed (single individual with a 2°C increase) was 39.0°C. This contrasts with the more marked increase in body temperature observed between 6 and 8 hours of dosing with the earlier 100% and 90% perflubron formulations (27). These data suggest that the reformulated perflubron-based emulsion with smaller sized particles was indeed associated with a minimal febrile response. Flu-like symptoms were observed in both PFC treatment groups, but these were typically mild with onset approximately 4 to 8 hours and resolution within 12 to 24 hours.
The clearance of perflubron from the circulation appeared to consist of two phases. The half-life over the first 24 hours was 6.1 ± 1.9 hours and 9.4 ± 2.2 hours for the 1.2 and 1.8 g/kg doses, respectively (23). This first rapid phase is presumed to reflect the initial clearance of perflubron particles from the circulation by the mononuclear phagocyte system (21), similar to the reduction in "fluorocrit" measured in Fluosol® studies (19,20). In the second phase, there appeared to be a much slower terminal half-life, which most likely reflects perflubron that has redistributed after phagocytosis to lipid compartments within the blood and released via expiration from the lungs (21). The timing of the observed transient flu-like responses correlates with the first phase of elimination from the circulation: mononuclear phagocyte system-mediated uptake in the liver, spleen, and bone marrow. There was no evidence in these studies of any adverse events associated with the subsequent phase of slow terminal perflubron clearance from the circulation.
The short-lived responses previously discussed will likely be clinically insignificant for surgical patients. First, the results of several studies indicate that WBC and macrophage responses are attenuated during anesthesia (28,29). Therefore, flu-like responses associated with perflubron emulsion clearance are likely to be attenuated in anesthetized patients. Second, early onset febrile responses have not been reported in surgical patients treated with perflubron emulsion. A previous pilot study in surgical patients with the 90% perflubron emulsion formulation, showed no differences in postoperative adverse events in control and treated patients (30). A recently completed, controlled study of 147 orthopedic surgical patients showed little difference in postoperative febrile responses between control and perflubron emulsion-treated (0.9 or 1.8 g/kg of the current 60% perflubron emulsion) patients (31). These findings further support the conclusion that the minor febrile, flu-like responses observed in conscious, healthy adults are unlikely to be of any significance in anesthetized surgical patients.
In conclusion, the results of this and the companion study by Leese et al. (23) indicate that the safety profile of the 60% PFC perflubron emulsion (OxygentTM) support its continued clinical investigation as a temporary O2 carrier to avoid allogeneic blood transfusion and potentially to treat or prevent myocardial and cerebral hypoxia during ischemia.
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Accepted for publication June 5, 2000.
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Abstract
Introduction
