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Influence of Ethnic Origin and Sex on the Pharmacokinetics of Clazosentan

From Actelion Pharmaceuticals Ltd, Department of Clinical Pharmacology, Allschwil, Switzerland (Dr van Giersbergen, Dr Dingemanse), and Chiltern International Ltd, Slough, Berkshire, United Kingdom (Dr Gunawardena).

Address for correspondence: Paul L. M. van Giersbergen, PhD, Actelion Pharmaceuticals Ltd, Department of Clinical Pharmacology, Gewerbestrasse 16, 4123 Allschwil, Switzerland.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
This study investigated the influence of ethnic origin and, as a secondary objective, sex on the pharmacokinetics of the parenteral endothelin receptor antagonist clazosentan in healthy Caucasian and Japanese subjects. Twelve subjects of each ethnic origin (female/male ratio 1:1) were treated with sequential 4-hour infusions of 1, 5, and 15 mg/h. Blood samples were taken frequently to determine plasma levels of clazosentan. The exposure to clazosentan was approximately 16% higher in Japanese subjects compared with Caucasian subjects and 18% higher in females compared with males. These differences were mainly attributable to a difference in clearance. A 3-compartment model well described the plasma concentration-time profiles of clazosentan with disposition half-lives of approximately 6 minutes, 21 minutes, and 2.7 hours. The data suggest that Caucasian and Japanese patients can be treated with a similar dosing regimen of clazosentan. At the doses infused, administration of clazosentan was safe and well tolerated in both ethnic groups.

Key Words: Clazosentan • endothelin • ethnic origin • sex • pharmacokinetics

Clazosentan (Ro 61-1790; VML 588, AXV-034343) is an endothelin receptor antagonist formulated for parenteral use, which is currently in development for the treatment of SAH. The structure of clazosentan has been published, and in a canine model of SAH, this compound prevented and reversed cerebral vasospasm.⁵ In patients, clazosentan was shown to reduce the frequency and severity of vasospasm following severe aneurysmal SAH.⁶ Cerebral vasospasm, which typically occurs between 4 and 9 days after the insult, is thought to be 1 of the major causes for morbidity and mortality after SAH.⁷ The tolerability, safety, and pharmacokinetics of clazosentan in healthy male subjects have been described.⁸ Its pharmacokinetic profile can be described by a 2-compartment model. The volume of distribution at steady state (V_ss) and clearance (CL) were approximately 30 L and 40 L/h, respectively. The 2 disposition half-lives were approximately 9 minutes and 2 hours, with the first phase mainly determining the concentration-time profile after discontinuation of infusion. Metabolism of clazosentan is minimal, and this compound is mainly excreted as unchanged drug via the bile (Actelion Pharmaceuticals, data on file) with only 15% of an administered radioactively labeled dose recovered in urine. Dose-limiting adverse events were headache, nausea, and vomiting.

Thus far, the pharmacokinetics, tolerability, and safety of clazosentan have mainly been investigated in Caucasian male subjects. It is well known that ethnic origin and sex may affect the pharmacokinetics and pharmacodynamics of drugs, although the observed differences are rarely of clinical significance.^9-11 Female sex has been recognized as a risk factor for the occurrence of SAH. The female to male ratio is about 2.^12,13 Therefore, the pharmacokinetic characteristics of 3 different dosing regimens of clazosentan were investigated in both Caucasian and Japanese male and female subjects. The doses chosen were the same ones as investigated in a recently completed dose finding phase II study including more than 400 SAH patients.¹⁴ The study was performed to support the development of clazosentan in Japan.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Subjects
Twenty-four healthy male and female Caucasian and Japanese subjects were recruited in this study. Japanese and Caucasian subjects were matched for sex and body weight. For the latter, a 5% variation was allowed. A summary of their demographic characteristics is shown in Table I. Subjects were considered healthy based on the results of a screening examination, which included medical history, a physical examination, clinical laboratory tests, recording of vital signs, and electrocardiography (ECG). Because of the teratogenic properties of clazosentan, participating women of childbearing potential were required to use a reliable method of contraception (hormonal contraception or barrier method) and the partner had to use a condom. The Ravenscourt Ethics Committee (Ravenscourt Park, London, United Kingdom) approved the study protocol, and all subjects gave written informed consent before any screening procedures were performed. The study was conducted in full conformity with the principles of the Declaration of Helsinki.

Study Design
This was a single-center, open-label, parallel-group, ascending staggered dose study. Clazosentan was administered through an intravenous catheter to subjects in the supine position as sequential infusions of 1, 5, and 15 mg/h, each lasting for 4 hours. All subjects received the same treatment. Meals were standardized, and Japanese subjects consumed typical Japanese food whereas European food was served to the Caucasian subjects. Food is not expected to influence the pharmacokinetics of a parenteral drug such as clazosentan. Approximately 0.5 hours after consumption of breakfast, infusion of drug was initiated at an infusion rate of 10 mL/h. This rate was kept constant for the 3 consecutive infusions. Blood samples of 4 mL were collected into EDTA-containing tubes from an indwelling catheter immediately before and 0.5, 1, 2, 3, 4, 4.5, 5, 6, 7, 8, 8.5, 9, 10, 11, and 12 hours after start of the drug infusion as well as 2, 5, 10, 20, 30, and 45 minutes and 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 10, and 12 hours after discontinuation of the infusion. Plasma was separated and stored at -20°C pending analysis. Tolerability and safety were evaluated by monitoring premature withdrawals and adverse events, via clinical laboratory testing, vital sign (including ECG) recording, and physical examination.

No formal statistical hypothesis was set for this comparative pharmacokinetic study. The sample size was set on empirical considerations such as availability of subjects and expected recruitment rate. However, given that a previous study had shown that the mean (SD) of clearance is 39.2 (4.9) L/h,⁸ a sample size of 12 subjects in each group had a 90% power to detect a difference in means of 6.8 L/h assuming that the common standard deviation is 4.9 L/h.

Bioanalytical Method
Plasma concentrations of clazosentan were determined by means of a turbo ion spray liquid chromatography method coupled to tandem mass spectrometry (LC-MS/MS). The analyte and the internal standard, an analogue of clazosentan deuterated in 4 positions, were extracted from plasma by protein precipitation using a mixture of acetonitrile and trifluoroacetic acid (1:1, vol/vol). The chromatographic system consisted of 2 pumps used to elute the guard (Phenomenex Synergy MAX RP, 4-µm particle size, 80-Å pore size; 4 x 2.0 mm) and analytical column (Phenomenex Synergy Polar RP, 4-µm particle size, 80-Å pore size; 50 x 2.0 mm) (Phenomenex Synergy, Torrance, Calif) with a mixture of solvents A and B. Solvent A consisted of water-formic acid (99:1, vol/vol) and solvent B of acetonitrile-formic acid (99:1, vol/vol). After injection of the sample, the guard and analytical columns were eluted with a mixture of solvent A-solvent B (4:1, vol/vol) at a rate of 500 µL/min for 6 seconds and, for the next 24 seconds, at a rate of 300 µL/min. Then, a linear gradient was started, changing the mixture of solvents A and B over the course of 2 minutes from 4:1 to 1:20 at a flow rate of 300 µL/min. Elution with the 1:20 mixture was subsequently maintained for 1 minute at the same rate. Next, the rate was increased to 500 µL/min, and both columns were washed with a 4:1 (vol/vol) mixture of solvent A and solvent B. The retention time for both clazosentan and the internal standard was 3 minutes.

Mass spectrometric detection was performed with an API 4000^TM triple quadrupole mass spectrometer from MDS Sciex (Applied Biosystems, Rotkreuz, Switzerland) operating in electrospray ionization positive ion mode and equipped with a Turbolon^TM spray ion source (750°C). The collision energy and pressure were 30.0 eV and 3.4.10^-5 torr, respectively; dwell times for clazosentan and the internal standard were 300 and 50 milliseconds, respectively; and quadrupole resolution was 0.70 ± 0.15 amu at half peak intensity. Detection and quantification were performed using the standard software supplied in selected reaction monitoring mode. Precursor and product ion fragments used for quantification of clazosentan were at m/z 578 550 and for the internal standard at m/z 582 554. Samples were quantified using peak area ratios.

The performance of the chromatographic method was monitored by simultaneous analysis of independently prepared quality control samples of various concentrations. The assay was found to be linear in the concentration range 0.25 to 512 ng/mL. The interassay coefficient of variation and the inaccuracy were 7.7 and 9.6%, respectively.

Pharmacokinetic and Statistical Evaluations
Actual blood sampling times were used for the pharmacokinetic evaluation when they deviated by more than 5% from the scheduled ones. The pharmacokinetic variables were calculated with model-dependent techniques using WinNonlin (Pharsight Corporation, Mountain View, Calif). For this analysis, a 3-compartment model provided a more accurate description of the concentration-time profile than a 2-compartment model based on visual inspection of the fits, the Akaike information criterion, and the condition number. A weighting factor of 1/Y² was applied.

Differences between Caucasian and Japanese subjects and between female and male subjects, with respect to pharmacokinetic variables of clazosentan, were investigated using analysis of variance (ANOVA) including the factors race and sex and an interaction term between these 2 dichotomous variables based on logarithmically transformed values. A P value of <.05 was taken to indicate a statistically significant difference. No adjustment to account for multiple comparisons was made. To test for an effect of sex on the pharmacokinetics of clazosentan, uncorrected and body weight-corrected CL and V values were used. Because Caucasian and Japanese subjects were matched for body weight (Table I), correction for body weight was not performed to test for an effect of ethnic origin. To explore dose-proportionality of clazosentan pharmacokinetics, the area under the curve values of the 3 infusion intervals were subjected to a power model as described by Gough et al.¹⁵ This test was performed by ethnic group, by sex, and by ethnic group and sex.

The tolerability findings were evaluated descriptively. Mean values for vital signs and ECG variables obtained predose and clinical laboratory variables at screening were compared with those obtained during and after the end of the drug infusion to detect drug-related changes.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Pharmacokinetics
The mean plasma concentration-time curves of clazosentan in the 2 ethnic groups during and following infusion of clazosentan are shown in Figure 1. Steady-state conditions were attained within 2 hours after start of the respective infusion. Following termination of the infusion, the plasma concentrations of clazosentan quickly decreased following a triphasic profile. Derived and calculated pharmacokinetic variables obtained by 3-compartmental analysis are given in Table II. The ANOVA did not indicate any significant interaction between race and sex for any of the pharmacokinetic variables compared. No statistically significant differences were detected for any of the pharmacokinetic variables determined by 3-compartmental analysis except for a significantly lower CL in Japanese subjects (Table II).

View larger version (13K): [in this window] [in a new window]   Figure 1. Arithmetic mean ± SD plasma concentration versus time profiles both on a linear and a semilogarithmic (insert) scale of clazosentan in Caucasian (n = 12) and Japanese subjects (n = 12) after infusion of 1, 5, and 15 mg/h, each for a period of 4 hours.

View larger version (13K): [in this window] [in a new window]   Figure 2. Arithmetic mean ± SD plasma concentration versus time profiles both on a linear and a semilogarithmic (insert) scale of clazosentan in male (n = 12) and female subjects (n = 12) after infusion of 1, 5, and 15 mg/h, each for a period of 4 hours.

The pharmacokinetics of clazosentan were dose proportional in both Caucasian and Japanese subjects as evidenced by values for ß (95% confidence interval) of 0.99 (0.93, 1.06) and 0.95 (0.90, 1.00), respectively. They were also dose proportional by sex and by ethnic group and sex (data not shown).

Tolerability and Safety
All subjects completed the study according to protocol. The adverse events reported in the study are presented in Table IV. Headache was the most frequent adverse event, and its incidence was similar in both ethnic groups. Both groups also reported dizziness and feeling hot with similar frequency. In addition to these 3 adverse events, Caucasian subjects incidentally reported other adverse events whereas no other adverse events were reported by Japanese subjects. Three of the 42 reported adverse events were of moderate intensity (all in Caucasian subjects), whereas all others were rated as mild by the investigator. The type of adverse events reported as similar between male and female subjects within each ethnic group, but females reported more adverse events than males (25 vs 17, respectively, data not shown). Most subjects reporting headache were administered acetaminophen (International Nonproprietary Name, paracetamol) for pain relief. In addition to receiving acetaminophen to treat a headache, 1 Caucasian male subject was administered a single 10-mg dose of metoclopramide to treat nausea. All adverse events resolved without sequelae.

No treatment-related pattern was detected that indicated an effect of clazosentan on clinical laboratory or ECG variables. When compared with the values obtained before clazosentan infusion, decreases in supine systolic and diastolic blood pressure were observed with the mean maximum decrease (approximately 10 mm Hg for both supine systolic and diastolic blood pressure) approximately 3 to 7 hours after start of the infusion. At 12 hours after termination of infusion, blood pressure values had nearly returned to baseline. These decreases were similar in Caucasian and Japanese subjects, and no influence of sex was apparent (data not shown).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Ethnic differences and effects of sex in drug disposition and/or tolerability and safety exist but are difficult to predict because many genetic and environmental factors may play a role.^9-11 Usually these differences are not of clinical relevance, that is, do not lead to dose adaptation. However, for some drugs, especially those with a narrow therapeutic index like warfarin, ethnic origin and sex do affect the dose regimen.^16,17 The design of stepwise infusions in individual subjects allowed comparison of pharmacokinetic variables, including dose proportionality, in a low number of subjects.

The pharmacokinetics of clazosentan as assessed in this study are in good agreement with results from a previous study with the exception of the variable V_ss.⁸ Using noncompartmental analysis, a value of about 30 L, irrespective of the dose administered, was found in the entry-into-man study.⁸ Across several studies in healthy subjects, 2- or 3-compartmental modeling consistently yielded a value of about 15 L for V_ss, whereas noncompartmental analysis yielded negative values in some subjects, or mean values ranging from 10 to 30 L (Actelion Pharmaceuticals, data on file). In our experience, noncompartmental assessment of V_ss is very sensitive to the quality of the data, and the duration of the infusion and should be interpreted with caution.

This study showed that the pharmacokinetics of clazosentan are similar in Caucasian and Japanese subjects. A slightly higher exposure to clazosentan was observed in Japanese subjects compared with Caucasian subjects and in females compared with males. After we corrected for body weight, the observed sex difference disappeared. However, differences in body weight cannot explain the lower clearance of clazosentan in Japanese subjects compared with Caucasian subjects because the subjects were matched for body weight. Clazosentan is excreted essentially unchanged by the liver into bile. In analogy with the structurally related endothelin receptor antagonist tezosentan,¹⁸ it is expected that the drug transporter protein MRP2 (also called cMOAT) plays a role in the elimination of clazosentan. Polymorphisms of MRP2 in healthy Japanese subjects have been identified,¹⁹ but the influence of these polymorphisms on drug disposition in general and clazosentan in particular is unknown. The influence of ethnic origin on drug transporter function has been poorly investigated,¹¹ and more research is necessary to unravel the mechanisms explaining observed differences in drug disposition in subjects from different ethnic origins.

In a previous study,⁸ the plasma concentration-time profile of clazosentan could be described by a 2-compartment model, but in the present study a 3-compartment model described the concentration-time data more accurately. This is probably related to the more frequent blood sampling after discontinuation of the infusion in the present study. In the entry-into-humans study, in which dosing regimens of 3 to 60 mg/h for 3 hours were given to healthy subjects, the pharmacokinetics of clazosentan were shown to be dose proportional.⁸ The data obtained in this study confirm that observation. Neither ethnic group nor sex had an effect on dose-proportionality.

Intravenous administration of clazosentan was safe and well tolerated in both study groups, and there were no serious or severe adverse events. Within the limitations of the study (ie, low subject numbers and the absence of a placebo group), the adverse event profile was similar between Caucasian and Japanese subjects and between males and females, although Caucasian subjects reported more adverse events than Japanese subjects and females more than males. In line with a previous study in healthy subjects, the most frequently reported adverse event was headache.⁸ This adverse event was not dose-limiting in this study, most likely attributable to the doses chosen, the relative short duration of infusion,⁸ and the administration of acetaminophen.

Clazosentan reduced blood pressure by approximately 10 mm Hg to a similar extent in both groups. This observation is in line with a previous study with clazosentan⁸ and is consistent with the pharmacological action of clazosentan in that it blocks the actions of one of the most potent vasoconstrictors known. No dose relationship could be discerned for the clazosentan-induced decrease in blood pressure, suggesting that for this effect, a dose of 1 mg/h is at the top of the dose-response curve. One important aspect of the treatment of SAH patients is maintaining their blood pressure.²⁰ In this respect, it is reassuring to note that the incidence of arterial hypotension was not greater in the clazosentan group compared with the placebo group in the proof-of-concept study including 32 SAH patients.⁶ This, however, needs to be confirmed in larger clinical trials.

	CONCLUSIONS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The pharmacokinetic, tolerability, and safety profile of clazosentan in Caucasian and Japanese subjects is similar. The results obtained in a healthy population indicate that males and females from both ethnic groups can be treated with a similar dose regimen. However, a study in patients is needed to confirm this conclusion.

	ACKNOWLEDGEMENTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Financial disclosure: Dr van Giersbergen and Dr Dingemanse are full-time employees of Actelion Pharmaceuticals Ltd. Dr Gunawardena is the principal investigator of the clinical trial and received financial compensation for the clinical costs associated with conducting the study.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 

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This Article Abstract Full Text (PDF) All Versions of this Article: 0091270007307337v1 47/11/1374    most recent 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 ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Request Reprints Citing Articles Citing Articles via Google Scholar Google Scholar Articles by van Giersbergen, P. L. M. Articles by Dingemanse, J. Search for Related Content PubMed PubMed Citation Articles by van Giersbergen, P. L. M. Articles by Dingemanse, J. Social Bookmarking                   What's this?