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Dose, Serum Concentration, and Time Course of the Response to Acetazolamide

• Arve Dahl, MD; David Russell, MD, PhD, FRCPE; Kjell Rootwelt, MD, PhD; Rolf Nyberg-Hansen, MD, PhD; Emilia Kerty, MD

Abstract

Background and Purpose To improve the assessment of cerebral vasoreactivity using acetazolamide (ACZ), we studied the time course of the response and the relationship between dose, response, and serum concentration.

Methods Blood flow velocities were measured with the use of transcranial Doppler ultrasonography in one of the middle cerebral arteries of 48 healthy subjects after the intravenous administration of 1 to 1.6 g ACZ. In 34 subjects (group 1), velocities were measured every second minute to detect the maximum middle cerebral artery velocity increase. We also measured regional cerebral blood flow using single-photon emission computed tomography in 27 of the subjects in group 1 before and approximately 15 to 20 minutes after the ACZ injection. The serum concentration of ACZ was measured in 15 subjects. In the remaining 14 subjects (group 2), middle cerebral artery velocity measurements were made 10, 25, 30, and 45 minutes after ACZ administration to obtain information regarding the late time course of the response.

Results In group 1 the plateau phase of the velocity response was reached 8 to 15 minutes after ACZ administration. A large range of velocity increase was observed, and a significant correlation was found between the maximum velocity increase and the dose and serum concentration of ACZ. In group 2 subjects, maximum velocities were maintained 30 minutes after the injection, but after 45 minutes velocities had decreased to 68% of their highest level. No significant relationship was found between dose or serum concentration of ACZ and the regional cerebral blood flow increase. The velocity increase after ACZ was similar in both older and younger subjects.

Conclusions This study shows that cerebral vasoreactivity is best assessed 10 to 30 minutes after ACZ administration and that the dose should probably exceed 15 mg/kg if a maximum vasodilatory response in the cerebral circulation is to be obtained.

Information regarding the reserve capacity of the cerebral circulation has prognostic significance in patients with carotid artery occlusive disease.^{1 2} This information may be obtained by measuring changes in rCBF or blood flow velocities in the basal cerebral arteries after the administration of a potent vasodilatory stimulus such as ACZ or CO₂.^{3 4 5 6} ACZ, which is a carbonic anhydrase inhibitor, has been studied quite extensively,^{7 8} but some important practical information is still lacking when this substance is used in vasoreactivity studies. This includes information regarding the optimal dose and the time course of the vasodilatory response.

A standard dose of 1 g IV ACZ, usually administered in adult patients, is assumed to cause a high enough inhibition of carbonic anhydrase to allow a good estimation of cerebral vasodilatory reserve. However, some investigators have given considerably higher doses, up to 22 mg/kg.⁹ To our knowledge, no studies have been designed to determine the dose of ACZ required to cause a maximal vasodilatory response. This is important since we want to bring about maximal vasodilatation without causing unnecessary discomfort for the patient. Maximal cerebral vasodilatation is, on the other hand, probably important if the assessment of cerebral vasoreactivity is to have a high diagnostic sensitivity.

The first aim of this study was to obtain information regarding the relationship between the ACZ dose and the serum concentration required to bring about a maximum vasodilatory response when the latter was measured by rCBF with dynamic SPECT and ¹³³Xe inhalation. MCA blood flow velocities were measured with the use of TCD. The second aim was to record the time course of the response to obtain information regarding when rCBF and TCD measurements should be performed after ACZ administration.

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Subjects and Methods

Forty-eight healthy subjects (23 men and 25 women; age, 23 to 74 years; mean, 41.8 years) took part in the study after giving informed consent. They were divided into two groups. Group 1 included 34 subjects. Vasoreactivity was studied in these subjects with the use of SPECT, and maximum MCA velocity was measured, with particular attention paid to the early response after ACZ administration. In the 14 subjects of group 2, blood flow velocity measurements were performed between 10 and 45 minutes after ACZ administration to obtain information regarding the time course of the vasodilatory response beyond 15 minutes.

TCD Examinations

Blood flow velocities were measured in the left or right MCA with the use of a TCD instrument (TC 2-64B, Eden/Nicolet Inc). The instrument and the procedure for artery identification have been described elsewhere.¹⁰ A permanently fixed 2-MHz probe was used in 26 subjects and a hand-held probe in 22. Time-mean velocities from the spectral outline were recorded.

We calculated mean velocity values from velocity samples using the mean of at least 10 to 15 heart cycles. In group 1, measurements were made at the end of the ACZ injection and repeated 2, 4, 6, 8, 10, 12, and 15 minutes later or until no further increase in velocity was observed. Measurements were therefore not usually performed after 15 minutes if there was no increase or a drop in velocities was observed from 10 to 12 minutes. In group 2, MCA velocities were measured before ACZ injection and again 10, 15, 20, 25, and 45 minutes after.

rCBF Measurements

rCBF measurements were performed in 27 subjects from group 1 before and from 15 to 20 minutes after ACZ administration. This was carried out with the use of SPECT and ¹³³Xe inhalation (Tomomatic-64, Medimatic Inc). This instrument and method have been described in detail elsewhere.^{11 12} rCBF was calculated in milliliters per 100 g per minute in standardized regions of interest corresponding approximately to the estimated perfusion territories of the MCAs. The measurements were carried out in a 2-cm-thick slice located 6 cm above the orbitomeatal line. A software package for automatic calculation of flow values in regions of interest was used for this purpose.¹²

ACZ Administration

Twenty-eight subjects in group 1 received 1 g ACZ. The remaining 6 received doses from 1.0 to 1.6 g so that the vasodilatory effect of higher ACZ doses could be studied. All subjects in group 2 were given 1 g ACZ. ACZ was injected intravenously during a 3-minute period after the subject had been resting for at least 5 minutes in the supine position.

Dosage and Serum Concentration of ACZ Measurements

Blood samples were taken from an antecubital vein contralateral to the side of the injection in 15 of the subjects in group 1 who received a standard dose of 1 g ACZ. The first sample was taken 5 minutes after the injection and the second immediately after the second rCBF measurement. This was approximately 20 minutes later in 9 subjects, 25 minutes later in 5, and 30 minutes later in 1. The whole blood was centrifuged within 1 hour and the serum frozen for later analysis. The drug concentration in serum was analyzed with the use of reverse-phase high-performance liquid chromatography.¹³

Statistical Evaluation

All values are given as mean±SD. All statistical tests were two-tailed, and differences were considered statistically significant at P≤.05. Correlations between variables were analyzed with the Pearson correlation model. Linear regression analyses were also performed.

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Results

Mean blood flow velocities before the ACZ injection in group 1 were 63.5±13.5 cm/s; the maximum increase in velocity was 43.4±10.5%. The maximum velocity increase in group 1 (n=34) was reached 6 minutes after the ACZ injection in 3 subjects, 8 minutes after in 7 subjects, 10 minutes after in 15 subjects, and 12 minutes after in 9 subjects.

Fig 1⇓ shows the time course of the ACZ response in group 1. The velocity increases as a percentage of baseline values are used in the plot. In 29 of the 34 subjects a velocity increase was observed before completion of the 3-minute ACZ injection, with a rapid velocity increase during the next 6 to 10 minutes. Values then remained stable until the rCBF study.

Figure 1.

Graph shows time course of MCA velocity increases after ACZ in 34 healthy subjects. The basal levels are set to zero, and the increases are percentages. Error bars indicate SDs.

In group 2 the MCA velocities remained stable for approximately 30 minutes. However, 45 minutes after ACZ administration the mean response had decreased to 68% of the maximum level, a response that was significantly different from the values at 30 minutes (P<.01).

In 27 subjects studied with SPECT, basal rCBF (means of both sides) was 54.5±7.2 mL/100 g per minute, and the increase in flow was 14.8±5.0 mL/100 g per minute, which was an increase of 27.0±8.6%.

In 15 subjects the serum concentration of ACZ after 5 minutes was 107.6±17.7 mg/L; concentration at the second measurement (after 20 to 25 minutes) was 71.0±14.7 mg/L. In each subject the two values were closely related, with a correlation coefficient of .86. In 5 subjects the second blood sample was taken after 25 minutes and in 1 after 30 minutes. In these subjects we assumed that the serum concentration fell at the same rate between 20 and 25 minutes as it did in the previous 15 minutes. We therefore interpolated the assumed value for 20 minutes after injection in these 6 subjects and plotted these values against the 5-minute value. The correlation coefficient between the two serum concentrations then increased to .97. This strongly suggests that the relative differences in serum concentrations between the 15 subjects were preserved throughout the study period.

The relationship between the dose in milligrams per kilogram and the serum concentration measured in the samples taken 5 minutes after ACZ administration was close and the correlation coefficient (r=.835) statistically significant (P<.001) (Fig 2⇓).

Figure 2.

Dose (x axis) is plotted against serum concentration (y axis) measured 5 minutes after ACZ administration (conc 1) (n=15).

Fig 3⇓ shows the relationship between the dose in milligrams per kilogram and the maximal MCA velocity response measured with TCD. A significant linear correlation was found, and the plot shows that within the small dose range assessed in this study the highest response was found in the subjects who received the highest doses. The plot suggests that a standard dose of 1 g to subjects who weigh more than 70 kg (14.3 mg/kg) is insufficient to provoke a maximal vasodilatation.

Figure 3.

Scatterplot shows the relationship between the dose of ACZ (x axis) and the maximal MCA velocity increase (y axis) in 34 healthy subjects.

A significant relationship was also found between maximum velocity increase and the serum concentration measured 5 minutes after ACZ injection (Fig 4⇓).

 Figure 4.

Scatterplot shows the relationship between serum concentration of ACZ (x axis) measured 5 minutes after injection (conc 1) and the maximum MCA velocity increase (y axis) (n=15).

No significant correlation was found for rCBF increase in the MCA perfusion territory, in absolute values or in percentage, between the dose of ACZ or serum concentration.

There was no significant correlation between the percent velocity increase after ACZ administration and the age of the subjects (r=.09, P=NS). A significant negative correlation with age was found for rCBF increases (r=−.62, P<.001).

In all but 3 subjects the side effects of ACZ were slight and consisted of slight paresthesia in the extremities and perioral region. In 3 subjects the adverse effects were more severe and consisted of a diffuse headache, which required treatment with acetaminophen. These 3 subjects received more than 18 mg/kg of ACZ.

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Discussion

ACZ inhibits carbonic anhydrase, which reversibly catalyses the conversion of CO2+H₂O to H₂CO₃. The effects of ACZ on the brain have been studied extensively.^{14 15 16 17 18 19 20 21} It increases the H⁺ and CO₂ concentration in the extracellular fluid of the brain, which is assumed to be the stimuli for the increase in flow.²² Carbonic anhydrase is found inside the glial cells and in the plexus choroideus but is especially richly distributed in the endothelial cells of the brain capillaries, probably at the luminal side of the cell membrane.²³ ACZ penetrates slowly through the blood-brain barrier, and the almost immediate increase in flow velocity observed in this study suggests that inhibition at the capillary endothelium is important when decreasing pH in the extracellular fluid of the brain.^{24 25}

The dose-response curve suggests that in many subjects a dose below 15 mg/kg is insufficient to give maximum cerebral vasodilatation. Our study was not designed to determine where the dose-response curve levels off, but doses above 18 mg/kg seemed to increase the risk of unpleasant side effects. The dose should therefore preferably be at the level of 15 to 18 mg/kg. The relationship between dose and response found in our study indicates that a higher dose is needed to obtain maximal vasodilatation of the brain capillaries than that needed to obtain maximal effect on intraocular pressure, where 5 mg/kg as a single injection seems to give a near-maximal effect.²⁶

At the time of the present study, information was incomplete regarding the time course of the response after ACZ administration. In one study in which Doppler measurements were taken of the carotid artery on the neck, a maximum response was observed after 25 minutes. Studies in which rCBF measurements with ¹³³Xe have been used conflict regarding the rate of flow increase.^{14 15 16} We therefore studied the relationship between the serum concentration and the response in proximity to the beginning of the suspected flow/velocity increase and again close to the second rCBF study. We found that ACZ has a similar rate of elimination from serum in the first 20 minutes after an intravenous injection in all subjects. The most likely explanation for the observed fall in serum concentration is distribution of the drug from the blood to other body fluids (different compartments). Although the elimination of ACZ from serum follows a nonlinear model, animal studies have demonstrated that the drug disappears very quickly from plasma in the first 60 minutes before the plasma concentration levels out.^{25 27} The elimination constant was also very similar in different animal species. The relationship between the two measurements in our study was so close that the relative differences in drug concentrations between subjects seem to be preserved throughout the 20-minute period. These findings suggest that the concentration-dose responses may also be calculated for the 5- to 20-minute interval after administration.

TCD provides the opportunity to continuously follow the time course of a pharmacological stimulus on the cerebral circulation. This information is impossible to obtain with rCBF measurements with radioactive xenon. The effect of ACZ was seen very soon after the start of injection. In this study the maximum effect was reached earlier than reported by Hauge et al,¹⁶ who administrated ACZ 0.5 g IV and measured the velocity increase in the internal carotid artery in the neck. Slowness in achieving a high enough ACZ concentration in the target cells when a lower dose is used may explain their somewhat delayed response. The time course of the response observed in the present study is similar to that observed regarding the pressure-reducing effect in the eye.²⁶ This suggests that vasoreactivity may first be assessed 10 minutes after ACZ administration and that it should be performed during the next 20 minutes. In the next 15 minutes, asymmetries may possibly be evaluated, but the response is no longer maximal.

In our study we did not find any significant relationship between dose and response when performing rCBF measurements with SPECT. One possible explanation for this is the precision of the method used. Assessing vasoreactivity with the use of SPECT and xenon inhalation is dependent on two rCBF measurements of absolute flow in milliliters per 100 g per minute. However, it is difficult to achieve precise measurements of absolute flow values because of methodological problems.^{28 29} Measurement of the increase in flow velocities during a 15-minute period, with subjects supine and with the use of fixed Doppler probes or repeated hand-held measurements, seems to be more precise than two rCBF measurements.^{28 30} It may therefore be easier to discover a relationship between vasoreactivity and dose with TCD rather than rCBF measurements with the use of SPECT and xenon inhalation.

This study also demonstrated that the increase in velocity after ACZ is similar in both older and younger subjects. Although the number of older subjects was small, this finding supports the use of ACZ when vasoreactivity is assessed in this age group. Højer-Pedersen,³¹ using ACZ and measuring rCBF with stationary detectors, found that the vasodilatory response appeared unchanged with advancing age. However, others have found a reduced response with age when using CO₂ as the vasodilatory stimulus.^{32 33} We have no good explanation for the discrepancy between the TCD and rCBF findings regarding the ACZ response in older subjects. Both methodological and physiological explanations are possible. The TCD method measures blood flow velocities that are dependent on volume flow in the distribution territory of the vessel under study and the diameter of the vessel. A possible explanation is that the increase in flow after ACZ measured with SPECT may be influenced by factors that differ in older and younger subjects (for example, possible shunting of blood in the microcirculation after ACZ). In addition, since the measurements were not performed simultaneously in the present study, physiological factors may have differed during the two study situations.

In conclusion, the results from this study suggest that an ACZ dose of 15 to 18 mg/kg IV should be given when cerebral vasoreactivity is evaluated and that measurements of the vasoreactive response may be performed between 10 and 30 minutes after ACZ administration.