Immunological evaluation of the new stable ultrasound contrast agent LK565: a phase one clinical trial

Echocardiography allows the analysis of ventricular motion and heart-valve morphology. The discovery that injection of small air bubbles dispensed in saline are capable of causing an opacification of the heart [1, 2] started an enormous effort to develop contrast agents to assist in diagnosing coronary artery disease and myocardial infarction [3]. Early developments led to stabilized bubbles in hyperosmolar solutions [4]. Albumin-stabilized bubbles were one of the first contrast agents to allow examination of both ventricles [5], but there are stability problems in patients with elevated pulmonary pressure [6]. Second-generation contrast agents improved the contrast especially in patients with subnormal echo signals [7–9]. LK565 and comparable substances represent a new concept in contrast agents as it consists of small, stable, air-filled particles [10] that provides a good contrast in both ventricles (figure 1, 2). Due to its high stability, LK565 improves examination of cardiac morphology and function as well as extracardiac vessel abnormalities for at least 15 minutes [11] (figure 3).

The particles consist of a synthetic polymer of aspartic acid. Via peptide bonds, the molecules form bold, stable beads filled with air. The average size of LK565 particles is 3 μm in diameter (figure 1). Because of their size and even polymeric surface the beads have similarities to microorganisms. This circumstance implies the induction of an immune response, which needs to be considered.

Results from previous studies indicate that phagocytes are responsible for eliminating the contrast agent from the blood stream [11]. In vitro, it has been shown that macrophages and neutrophils quickly devour the LK565 particles. Preliminary studies proved that the LK565 particles are phagocytosed by macrophages, resulting in slight activation with an increase in the intracellular calcium content. However, there were no symptoms of a respiratory burst [12].

The activation of macrophages can lead to different conditions: no activation, normal activation, and overreaction. In the last case, over-expression of tumor necrosis factor alpha (TNF-α) can lead to a septic shock by induction of intravascular blood clots apart from other effects, which may result in disseminated intravascular coagulation [13, 14]. Furthermore, macrophages act as antigen-presenting cells which are highly important to induce an adaptive immune response, i.e. specific antibody production. It is probable that the LK565 particles attract antigen-presenting cells to present LK565 fragments as antigens. This may induce the production of specific antibodies against the contrast agent, which in rare cases could result in pathogenic immune reactions. The aim of this study was to examine a possible immune response to LK565 considering phagocytosis, TNF-α secretion by macrophages, lymphocyte activation and specific antibody production.

We investigated 15 volunteers in a clinical phase one trial over a period of 14 days. The kinetics of activation surface markers CD69, CD25, CD71 and CD11b on leukocytes, the phagocytosis capacity, and TNF-α production in monocytes were analyzed, as was the specific antibody production after LK565 application.

The expression of CD69 [15], CD25 [16], CD71, HLA-DR [17], and CD11b [18] is increased after activation [19]. CD69 will be presented quickly after activation on almost all leukocytes normally after some hours but on neutrophils even after a few minutes due to mobilisation of intracellular storages [20]. The α-chain of the IL-2 receptor CD25, also known as the high-affinity receptor on lymphocytes, is developed after 6 hours at the earliest and up to 1–2 days. Transferrin receptor CD71 correlates directly with cell proliferation and a late marker which can be expected after 3–4 days similar to antigen presentation via MHC-II analyzed as HLA-DR. Integrin expression (CD11b) on monocytes and neutrophils is associated with phagocyte migration into tissue [21].

Independent of dosage or repeated contact, the contrast agent LK565 was well tolerated by all volunteers. After exposure to LK565 no erythema, no drop in blood pressure, neither significant change of the heart frequency nor fever occurred (data not shown). The volunteers did not complain of relevant clinical symptoms such as chest discomfort or asthenia. It exhibited good opacification of both ventricles a few seconds after application (figure 2). While the minimum dosage of 0.15 mg/kg LK565 was sufficient for just one echocardiogram, a suitable duration of echo contrast was obtained at a 0.4 mg/kg dose. A further increase in dosage provided no improvement in quality or contrast duration (data not shown).

The uptake of LK565 led to the saturation of phagocytes. The uptake capacity of the phagocytes from blood samples 6 h after injection was exhausted (figure 4). Neither macrophages nor neutrophils phagocytosed any more labeled bacteria after LK565 uptake, the observed effect was reversible. After 24 h the phagocytic capacity reached almost the starting level.

During the first 24 h an increase in intracellular TNF-α production was detected in monocytes and macrophages (figure 5a). However, in relation to the positive control after 2 h of LPS stimulation, the slight increase in intracellular TNF-α was negligible. No connection between repeated exposure and dosage was found. The amount of intracellular TNF-α decreased to starting levels after 24 h.

An increase in integrin expression CD11b was detected on monocytes and macrophages 6 hours after application. Compared to the positive control, the upregulation of the integrin was only poor. In all cases, the upregulation of the integrin was only of brief duration and returned to normal levels after 24 h (figure 6d). Only a slight and short increase in CD69 after 6 h was determined in neutrophils in a few cases. Most volunteers exhibited no CD69 increase in neutrophils. Even less CD69 expression was found on monocytes and macrophages where CD69 stimulation is associated with the production of prostaglandines, leukotriens and TNF-α. Only minor CD69 expression was observed on T-cells and on B-cells (figure 6c). No increase of CD25 and CD71 (also on monocytes) was detected (figure 6a,6b).

Discrete MHC class II (HLA-DR) upregulation on macrophages and lymphocytes was observed (figure 7a). We found no further evidence of an adaptive immune response via antibody production. None of the serum samples during our clinical trial exhibited any development of specific antibodies (IgM, IgG) versus the contrast agent (figure 5b).

The results show that there is only a slight activation of leukocytes esp. of phagocytes after application of LK565, a particular contrast agent based on a polymer of the naturally occurring amino aspartic acid. No specific antibody development against the contrast agent was detected. Thus, the risk of a major immunological activation cascade after repeated dosage can be regarded as minor. Since LK565 does not lead to an adaptive immune activation in this study, the possibility of an overexpression of IgE, which plays a key role in allergic diseases, also becomes more unlikely. Although none of the 15 volunteers enrolled in this phase I study developed an allergic-type reaction, such reactions could, however, occur in further phases of clinical testing or application. An allergic reaction against a pharmaceutical drug is a rare event which is impossible to exclude even in large clinical studies.

An increase in CD11b/CD18 is connected with phagocytosis and the diapedesis readiness of phagocytes. LK565 is eliminated from the blood stream via phagocytosis, therefore neutrophils exhibited an increase in CD11b expression and phagocytosis activity. This is also connected to a temporary higher intracellular TNF-α level in monocytes.

On lymphocytes, especially T-cells, the cascade of CD69, CD25 and CD71 is closely related to an immune response. After expression of CD69, the interlinkage of the receptor leads to proliferation and secretion of IL-2, IFN-γ and TNF-α. After effective activation, the T-cells secrete IL-2 which activates the development of the high-affinity IL-2 receptor CD25 and therefore launches cell proliferation with an expression of CD71 after 3 – 4 days. During our study after LK565 application no major activation cascade was detected on either T-cells or B-cells.

As mentioned above, the best contrast performance for echocardiography was obtained at a LK565 dosage of 0.4 mg/kg. Opacification of both ventricles was not enhanced by a further increase in dosage. Bearing in mind the fact that the contrast agent is phagocytosed and uptake capacity is limited, it is recommended that the daily dosage does not exceed 90 mg, which is equivalent up to three echocardiograms per day.

To minimize the risk of an undesirable adverse event such as an anaphylactoid reaction, immunological studies should be included in clinical trials for new UCAs. The use of LK565 as another new ultrasound contrast agent (UCA) with a comfortable duration of signal enhancement esp. in echocardiography should be encouraged as a means to provide additional diagnostic information without causing a major activation cascade or triggering an adaptive immune response. This can be an advantage for the "difficult-to-image" patient with adverse reactions related to other UCAs.