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(Stroke. 1995;26:2048-2052.)
© 1995 American Heart Association, Inc.

Articles

Phase I: Transcranial Echo Contrast Studies in Healthy Volunteers

Manfred Kaps, MD; Peter Schaffer, MD; Klaus-Dieter Beller, MD; Günther Seidel, MD; Harald Bliesath, MD, MSc Wilhelm Wurst, MD, MSc

From the Department of Neurology, University at Lübeck (M.K., G.S.), and Byk Gulden Pharmaceuticals, Clinical Pharmacology, Konstanz (P.S., K.-D.B., H.B., W.W.), Germany.

Correspondence to Prof Dr M. Kaps, Klinik für Neurologie, Universität zu Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Background and Purpose Transcranial ultrasound diagnostics are particularly hindered by insufficient ultrasound penetration through the temporal bone. The use of ultrasonic contrast media to enhance the Doppler signal is an important step toward the solution of this problem. In the present study we investigated the tolerability and the diagnostic value of a new intravenous transpulmonary ultrasonic contrast medium, BY963.

Methods In two phase I studies, 8 healthy volunteers received a spherosome suspension containing a phospholipid as the active ingredient. The intravenous injection was performed in three doses (2.5, 5, and 10 mL) at four different injection rates (0.25, 0.5, and 1 mL/s and bolus). The duration and degree of the signal enhancement were measured by two transcranial ultrasonic procedures presently used in clinical practice: transcranial Doppler sonography (TCD) and transcranial color-coded sonography (TCCS). The assessment of tolerability was based on chemical laboratory parameters and hemodynamic data (heart rate, blood pressure, electrocardiogram) and on questionnaires relating to general well-being.

Results BY963 was tolerated without complications. All 38 administrations of the echo contrast medium produced a marked increase in the TCD signal (>30 dB) in the intracranial basal cerebral arteries. To obtain the optimum time window for diagnostic use, higher doses with slower injection rates are advantageous. The duration of optimal contrasting was 42 to 68 seconds (TCD) and 12 to 132 seconds (TCCS), depending on the method and mode of administration. Bolus injections gave rise to an increased incidence of color artifacts.

Conclusions BY963 significantly improves intracranial Doppler imaging while being well tolerated. The signal enhancement lasts long enough for TCCS to display all basal cerebral arteries after just one injection.

Key Words: cerebrovascular disorders • diagnostic imaging • transcranial Doppler • ultrasonics

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Transcranial examination of the cerebral arteries has proved itself in clinical practice during the last decade.¹ The list of applications of this method has grown ever longer since 1982 and currently includes the diagnostics of occlusive cerebrovascular processes and a broad spectrum of hemodynamic studies that can be performed under both physiological and pathological conditions. As an imaging technique, the recently developed TCCS also enables a representation of the course of the basal cerebral arteries and an assessment of the spatial relationships of the vessels to one another and relative to the parenchymatous structures.

The most serious problem of transcranial ultrasound diagnostics, however, is still the acoustic "window failure."² Depending on age, sex, and race, approximately 18% of stroke patients cannot be examined in this way because of insufficient ultrasound transmission through the temporal region of the skull, and the percentage of failures can be as high as 50% among nonwhites.³

High hopes are therefore pinned on the use of echo contrast media, which allow a significant signal enhancement.^{4 5 6 7 8} An ideal echo contrast medium must be well tolerated, capable of intravenous administration, and sufficiently stable to pass through the pulmonary circulation and the heart into cerebral circulation. There should also be a standardized microbubble size, and the course of image degradation should be calculable as a function of time.

Studies with a galactose microbubble suspension (SHU 508 A) have shown that by appropriate enhancement of the signal, the distal brain arteries and the veins can also be imaged, even though as a rule they escape noncontrast examination.^{6 8} According to recent experience, the time course, duration, and extent of the Doppler signal enhancement depend to some extent on the echo contrast medium used. In the present study we have investigated for the first time the cerebral circulation using a new standardized substance (BY963) that contains a phospholipid derived from soybean lecithins as its active ingredient (Table 1).

View this table: [in this window] [in a new window]   Table 1. Properties of BY963

The aim of the study was to test the clinical tolerability of BY963 and to clarify how its diagnostic usefulness is influenced by the dose and mode of administration. For this purpose the signal enhancement was measured with two different transcranial ultrasonic techniques (TCD and TCCS).

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
This phase I study on healthy volunteers was approved by an independent ethics committee and performed in accordance with the guidelines of the Declaration of Helsinki (1964) with the supplements of the Tokyo (1975), Venice (1983), and Hong Kong (1989) revisions. Eight subjects took part in this open, nonrandomized study. The inclusion criteria were age 18 to 40 years, normal weight, and absence of any general medical abnormalities. The exclusion criteria were a history of allergy, heart rhythm disturbances, previous psychiatric illness, serological signs of hepatitis pathology, and a positive result on drug screening. A total of 38 injections of doses ranging from 2.5 to 10 mL were administered on 4 study days. All injections were through the antecubital vein and were followed by a 10-mL flush of saline.

The spherosome suspension of the echo contrast medium was prepared immediately before the injection by agitating 5 mL of the lyophilizate with 0.18 mL of air in a special mixing chamber. The system comprises two syringes connected by an adapter, and the carrier solution is prepared for use by repeated agitation of first one side and then the other to obtain a homogeneous suspension of air-filled microbubbles with a standardized bubble size of approximately 3.8 mm (Table 1).

Tolerability and Drug Safety
The tolerability of the echo contrast medium injections was assessed on the basis of 12-channel electrocardiographic recordings, blood pressure and pulse measurements, questionnaires relating to changes in general well-being, clinical laboratory parameters, and urinalyses. The tolerability data were collected 30 minutes before and 3, 5, and 24 hours after the injections and 14 days after the end of the study. During the injection an electrocardiogram was recorded continuously in one lead.

TCD Studies
The TCD recordings were made with the use of a pulsed ultrasound system (Multi-Dop X, DWL) following the usual guidelines.⁹ The sound signals were recorded continuously with the use of a 2-MHz head fixed over the temporal acoustic window with a spectacle-frame holder. The signal enhancement was quantified with the use of an on-line fast Fourier transformation and displayed on the screen in flash color in 3-dB steps.

At the start of the study the Doppler sample volume was centered on the main stem of the right MCA, and the signal amplification was then reduced until the frequency spectrum was no longer discernible on the screen. The contrast medium was administered with the instrument kept at this constant setting.

BY963 (Table 1) was administered into the right cubital vein in three healthy volunteers (age, 23 to 27 years; height, 1.79 to 1.89 m; weight, 70 to 76 kg) in two doses (2.5, 5 mL) and at three different speeds (0.25, 0.5, 1.0 mL/s). After each of the six injections, 0.9% saline was used to rinse any residues of the contrast medium out of the arm veins. The data were recorded on diskettes and on videotape.

The evaluation was performed off-line by two experienced investigators who determined the time to the appearance of the echo contrast medium in the MCA main stem after the start of injection (Fig 1). This time to contrast appearance is designated TA. In addition, the maximum signal enhancement was recorded on the basis of the color-coded image, as was the duration of homogeneous representation of the Doppler frequency spectrum (as a parameter of diagnostic value). The decline of the effect of the echo contrast medium was recognizable acoustically by a characteristic "splashing" noise and optically by interruptions in the envelope curve of the Doppler frequency spectrum (Fig 1). The following parameters were measured after each contrast medium injection: (1) the latency period (TA) from the start of injection to the detection of echo contrast medium in the MCA (in seconds) and (2) maximum signal enhancement (in decibels) and the duration of the optimum/homogeneous enhancement without overcontrasting or fragmentation of the Doppler envelope curve (in seconds).

View larger version (0K): [in this window] [in a new window]   Figure 1. TCD recording; echo contrast enhancement in the MCA with the use of BY963. A, After antecubital intravenous injection, the echo contrast agent reaches the MCA after a latency period (TA). B, Optimal signal enhancement by more than 30 dB with homogeneous imaging of the Doppler frequency spectrum and a continuous envelope curve. C, Fading of the enhancement effect, showing inhomogeneous frequency spectrum and fragmented envelope curve. The color decibel scale for signal enhancement is on the right margin; time axis is in seconds.

TCCS Studies
For the color imaging studies we used a standard 64-channel phased-array system with a 2.7-MHz ultrasonic head (HP Sonos 1500, Hewlett-Packard Co). The imaging of the basal cerebral arteries was performed transtemporally with an overall view in the axial plane.^{10 11} Before administration of the echo contrast medium, the color amplification was reduced so that parenchymatous structures were still visible but not the color coding of the cerebral arteries.

Five more healthy volunteers (age, 26 to 36 years; height, 1.66 to 1.89 m; weight, 63 to 99 kg) were injected through a cubital vein with three different doses (2.5, 5.0, 10 mL) of BY963 at an injection rate of 0.25 or 1.00 mL/s or as a bolus (3 mL/s). An interval of 1 hour was allowed between injections to ensure that the previous dose of echo contrast medium had been completely washed out.

The echo contrast effect was recorded on videotape and evaluated off-line. Four different phases were defined for the purposes of this evaluation (Fig 2): phase I, latency period from the beginning of injection of BY963 to the first color image of the cerebral arteries (Fig 2, top left panel); phase II, excessive enhancement of the signal, with color artifacts ("blooming") and limited diagnostic usefulness; phase III, optimum imaging of the basal cerebral arteries (Fig 2, top right panel); and phase IV, "fragmentation" of the color signal, again with limited diagnostic usefulness (Fig 2, bottom left panel). Recording was stopped after 3 minutes even if some contrast effects were still discernible in individual artery sections (Fig 2, bottom right panel).

View larger version (0K): [in this window] [in a new window]   Figure 2. TCCS recordings; axial section in overall view. Top left, Color enhancement reduced until only parenchymatous structures are visible before appearance of echo contrast agent (phase I). Top right, After injection of 2.5 mL BY963, the anterior, middle, and posterior cerebral arteries are all visible together ipsilaterally (phase II). Bottom left, Fading echo contrast effect such that individual arterial segments are no longer connected (phase IV). Bottom right, Approximately 3 minutes after the start of injection, fragmented images of some peripheral MCA branches are still evident.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Tolerability and Drug Safety
No adverse events were observed during the study. Evaluation of the laboratory data obtained and the scales of subjective health rating revealed no clinically relevant changes compared with the baseline situation.

TCD Studies
Table 2 shows that the TA varied from 12 to 28 seconds. The TA depends on the heart rate and on the speed of injection. Individually (ie, at a constant heart rate) the TA varied only slightly (1.7±2.4 seconds; maximum, 4 seconds), independent of the infusion velocity. Higher injection rates decreased the TA. The signal amplitude was in all cases increased by more than 30 dB.

View this table: [in this window] [in a new window]   Table 2. Time to Appearance of Echo Contrast Effect and Optimal Diagnostic Window in TCD

The duration of optimum contrasting was dose dependent and lasted from 42 to 68 seconds (Table 2). A higher dose of BY963 increased the optimum diagnostic time window. Slow speed of injection is preferable because it results in a shorter phase of overcontrasting.

TCCS Studies
After all 20 injections of BY963, we noted a distinct enhancement of the color signal, which permitted differentiation of the large-caliber basal cerebral arteries on both ipsilateral and contralateral sides; moreover, on the ipsilateral side the distal MCA branches, which otherwise escape detection, were imaged (Fig 2).

Evaluations of the TCCS images showed that the TA depended on the mode of injection and not on the dose (Fig 3; Table 3). Bolus injections resulted in a prolonged blooming phase at the expense of the period of optimum diagnostic usefulness. The length of phase III was dose dependent (minimum, 12 seconds; maximum, 132 seconds). In all tests, phase IV lasted more than 3 minutes after the start of injection.

View larger version (39K): [in this window] [in a new window]   Figure 3. Bar graph shows that the diagnostic benefit of echo contrast imaging depends on both the quantity and the rate at which the medium is administered. After injection there is an initial phase of excessive signal reinforcement ("blooming"), following which the basal arteries are well evaluable for 18 to 100 seconds. Partial effects persist longer than 180 seconds.

View this table: [in this window] [in a new window]   Table 3. Time to Appearance of Echo Contrast Effect and Optimal Diagnostic Window in TCCS

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
General Aspects
Echo contrast media permit the use of transcranial ultrasound diagnostics in patients who do not yield useful results on conventional ultrasonographic examination. From the perspective of neuroangiology, an echo contrast medium must be tolerated without side effects and provide a diagnostic window of sufficient duration and image quality to allow an evaluation of the cerebral vessels.

No relevant adverse events were observed in this study, as in all previous studies with BY963.¹² The tolerability even in high doses of up to 30 mL as a bolus injection appears to be good (M.K. et al, unpublished data). In particular, there were no clinical central nervous symptoms. Further studies of drug safety using more differentiated cerebral function tests and electroencephalographic monitoring are currently under way.

Since all of the healthy volunteers provided good transcranial ultrasound images even without echo contrast media, after adjusting the TCD measurement volume in the main stem of the MCA (in the TCCS projection plane), we reduced the Doppler gain until no Doppler signal was detectable. This adjustment was not altered during the course of the study and served as the reference for evaluating the echo contrast effect. As the next step we envisage studies under clinical conditions in patients with the problem of poor ultrasound penetration.

The results of TCD and TCCS are in agreement regarding the typical course of the echo contrast effect after BY963. After intravenous injection into a cubital vein (TCD studies), 12 to 28 seconds pass (varying between individuals) before an enhancement of the Doppler signals from the MCA becomes apparent. A phase of overcontrasting follows, which eventually (depending on the mode of administration) passes into a phase of diagnostic interest, during which optimum imaging of the cerebral arteries is obtained, free from unwanted signal artifacts. Both in TCD and in TCCS the injection rate has a particular significance for diagnostic usefulness. Bolus injections shorten TA and prolong overenhancement (blooming), but they do not prolong the diagnostically useful time. For the same injection rate the TAs measured in TCD studies agree well with the values from TCCS (20 to 22 seconds for an injection rate of 0.25 mL/s). Comparable measurements have been reported by other working groups using galactose particles as the contrast substance.^{5 6 8}

In subjects with healthy hearts the TA, which we have expressed in seconds, depends on the heart rate: the echo contrast medium enters the cerebral circulation sooner when the heart rate is faster. In individuals (ie, for the same heart rate) the TAs remained constant regardless of the amount of substance injected.

TCD Studies
The time for which the envelope curve of the Doppler frequency spectrum remained uninterrupted and free from artifacts was used to quantify the duration of the diagnostic effect. During this time quantitative analysis of the spectrum delivers relevant and useful data.¹³ In the phase that follows quantification is more problematic, but the positions of the brain vessels can still be identified. Thus, the meaning of "diagnostically useful" is to some extent a question of definition.

The signal enhancement was quantified with the use of a color scale for relative decibel values. Since in the later phases the Doppler frequency spectrum becomes inhomogeneous (Fig 1C) but individual particles of contrast medium continue to amplify the signal by more than 30 dB even after several minutes have elapsed, we have not constructed a dose-effect curve in this study. BY963 can also be detected in the jugular vein in healthy volunteers, ie, it passes through the capillaries and into the venous system (M.K. et al, unpublished data, 1994) and undergoes recirculation.

TCCS Studies
Because of the different assessment criteria, the duration of the diagnostically relevant window cannot be compared with the values from TCD. Experience shows that after a slow injection of 10 mL BY963 (0.25 mL/s), all basal cerebral arteries may be imaged simultaneously for a period of approximately 1.5 minutes, even when unfavorable technical parameters have been selected (eg, axial view plane of the entire brain, 2.7-MHz sector probe, insensitive color parameters, and gain reduction). In the case of acute stroke, it would therefore be possible to identify patients with a main trunk occlusion in the middle or posterior cerebral arteries on arrival at the hospital in a reliable manner and without difficulty or any great expense. Diagnosis would thus be considerably hastened, and better planning of the next steps in treatment would be possible. Recent clinical echo contrast studies in patients with cerebrovascular disease have demonstrated that the change in diagnosis from uncertain to firmly established was possible in the majority of the cases.⁸

Distal arterial sections were also imaged, but they were not analyzed in greater detail because the study protocol specified a fixed projection plane. More detailed studies on this topic have been performed with the use of galactose microparticles, which revealed otherwise invisible intracranial arterial and venous segments by color duplex sonography.^{6 8}

In TCCS studies superimposed artifacts have a special impact; individual arterial sections run together or become round and swollen, and clouds of color and intense reflections can complicate an assessment considerably. TCCS seems to be more sensitive to overcontrasting than TCD. Even after phase III (optimum investigation conditions), the cerebral arteries can still be localized by TCCS for some time, although the images of arterial segments are no longer connected.

While all transpulmonary echo contrast media used thus far in neuroangiology depend for their action on encapsulated microbubbles of air, there are significant differences between carriers. The diagnostic properties will also be different. Because of methodological differences, a comparison of our results with other echo contrast media is difficult, since we used healthy volunteers rather than patients with insufficient temporal acoustic transparency. Moreover, the intervals measured depend on how the echo contrast effect is defined, and the technical parameters of the study (eg, signal amplification) play a special part. In routine examinations the initial overcontrasting phase would be reduced simply by turning down the amplification on the instrument. Similarly, the diagnostic window could be considerably prolonged in the later phase simply by increasing amplification.

It must be emphasized that the data on administration methods are applicable only to the imaging of basal cerebral arteries. Other regions of the vascular system (eg, coronary or peripheral arteries) may require a different mode of administration.

	Selected Abbreviations and Acronyms

 

MCA = middle cerebral artery TA = time to appearance (latency period from start of intravenous injection to first appearance of echo contrast effect in MCA) TCCS = transcranial color-coded sonography TCD = transcranial Doppler sonography

	Acknowledgments

 
We are indebted to P. Willmes of DWL Elektronische Systeme for his help and technical advice in the TCD studies.

Received May 2, 1995; revision received July 5, 1995; accepted July 7, 1995.

	References

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kaps, M. Articles by Wurst, W. Search for Related Content PubMed PubMed Citation Articles by Kaps, M. Articles by Wurst, W.