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Pharmacokinetics of Arundic Acid, an Astrocyte Modulating Agent, in Acute Ischemic Stroke

From Ono Pharma USA, Inc, Lawrenceville, New Jersey (H. Ishibashi, Y. Funakoshi); Stroke Program, Sanders-Brown Center on Aging, and Department of Neurology, University of Kentucky Medical Center, Lexington, Kentucky (L. C. Pettigrew); and Tsukuba Research Institute, Ono Pharmaceutical Co, Ltd, Tsukuba, Ibaraki, Japan (M. Hiramatsu).

Address for reprints: Hideyasu Ishibashi, Ono Pharma USA, Inc, 2000 Lenox Drive, Lawrenceville, NJ 08648; e-mail: hideyasu.ishibashi{at}ono-usa.com.

	ABSTRACT

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Arundic acid is an astrocyte modulating agent that improves neurological outcome in experimental acute stroke models. The pharmacokinetics of arundic acid in patients with acute ischemic stroke was investigated in a randomized, double-blind study. Six groups of 8 to 9 patients each received 2, 4, 6, 8, 10, or 12 mg/kg/h of arundic acid for a daily 1-hour infusion until completion of 7 doses. Maximum plasma concentrations of arundic acid increased with increasing dose; however, the systemic exposure was less than dose proportional at higher doses. The mean terminal half-life was approximately 2 to 3 hours. There was no excessive accumulation in plasma. Although systemic exposure in elderly patients was 30% greater than that in younger patients, the plasma concentration returned to nearly or below the limit of quantification prior to next administration. The pharmacokinetics of arundic acid in acute stroke patients assessed in this study were similar to that in healthy adults.

Key Words: Arundic acid • pharmacokinetics • cerebral ischemia • astrocyte

Arundic acid ((R)-(–)-2-propyloctanoic acid; ONO-2506) is an astrocyte modulating agent currently under development for the treatment of acute stroke. Arundic acid reduces growth of brain infarction by suppressing astrocyte-derived neurotoxic cascade following brain ischemia—that is, suppression of inflammation, enhancement of –aminobutyric acid A (GABA_A) responses, increase of glutamate transporters, and production of glutathione (antioxidant). In in vivo rat and primate ischemic stroke models, arundic acid has been shown to significantly reduce cerebral infarction volume as well as improve neurological deficits at a minimal plasma level of 7 to 15 µg/mL.⁵ Arundic acid is metabolized by hepatic oxidation and glucuronic acid conjugation and primarily excreted in urine as metabolites. The principal enzyme responsible is CYP2C9. When administered in healthy adults, systemic exposures increased in a dose-dependent manner, but not dose proportional at higher doses, with an apparent terminal half-life of approximately 2 hours. Repeated dosing had no effect on the pharmacokinetics. Plasma protein binding, primarily to albumin, was high (99%), and the volume of distribution was small. The primary metabolite in humans possessed few pharmacological activities (data on file).

The pharmacokinetics in patients with acute stroke may be significantly different from those in healthy adults due to several reasons, eg, greater incidence of stroke with advanced age, physiological changes secondary to brain damage, comorbid hepatic or renal impairment, and a number of concomitant medications for intensive care. To determine optimum doses of arundic acid in future studies, it is essential to understand the pharmacokinetic profile of arundic acid in the target population. Blood samples for pharmacokinetic assessment were collected during a phase I acute stroke study of arundic acid. The primary results, safety, and tolerability of arundic acid in a range of 2 to 12 mg/kg/h have been reported previously.⁶ In the present article, the results of the pharmacokinetic analysis in the phase I study are reported and compared to our previous experiences in healthy adults.

	METHODS

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Subjects and Study Design
We performed a randomized, double-blind, placebo-controlled, dose escalation phase I study at 18 medical centers in the United States. All research procedures using human subjects were approved by the institutional review board of each participating study center (see appendix), and all study patients provided written informed consent.

Major inclusion criteria were as follows: (1) men or nonpregnant women, aged 18 to 85 years; (2) clinical diagnosis of acute ischemic stroke and initiation of study medication within 24 hours of symptom onset; (3) computed tomography (CT) brain scan and/or conventional magnetic resonance imaging (MRI) compatible with the diagnosis of ischemic stroke; and (4) baseline National Institutes of Health Stroke Scale (NIHSS) of 7 to 22. The NIHSS is a validated scale to evaluate neurological deficits in acute stroke with a score range of 0 to 42, where a score of 0 and 42 indicates no neurological symptom and worst neurological deficits, respectively.⁷ A range of 7 to 22 of NIHSS scores represents moderate to moderately severe strokes. In addition, patients with significant hepatic or renal disease or dysfunction were excluded: total bilirubin >2.0 mg/dL, alanine aminotransferase (ALT) or aspartate aminotransferase (AST) >2 times upper limit of normal, or serum creatinine >2.0 mg/dL. Concomitant administration of tissue plasminogen activator (t-PA) was prohibited.

Patients were randomized to receive 2, 4, 6, 8, 10, or 12 mg/kg/h arundic acid (8-9 subjects/group) for a 1-hour infusion until completion of 7 doses. The interval between each dosing was at least 12 hours but no longer than 25 hours for the first 3 infusions to adjust the dosing schedule to regular working hours, and 24 ± 1 hours for the remaining 4 infusions. The study medication was given as a 300-mL intravenous infusion. A complete description of the methods is included in a previous report.⁶

Pharmacokinetic Evaluation
Blood samples were collected at preinfusion, 0.25, 1, 1.25, 3, 7, and 12 hours poststart infusion for the first and third infusions; and only at preinfusion for the remaining 5 infusions. Seven mL of blood samples was drawn via a cannula or by repeated venipuncture into lithium heparin tubes. Plasma was separated by centrifugation and stored at –20°C.

Plasma concentrations of arundic acid were determined by a validated high-performance liquid chromatography (HPLC) with fluorescence detection. After addition of the internal standard (2-undecenoic acid) and hydrochloric acid, 0.5 mL of plasma was extracted with chloroform. The organic layer was evaporated and reconstituted in solution of acetonitrile/methanol/0.2% acetic acid (40/20/40, v/v/v) before the injection of an aliquot onto the HPLC system. The chromatographic separation was performed using Inertsil ODS-2 column (5 µm, 4.6 x 150 mm; GL Science, Tokyo, Japan). The mobile phase was composed of acetonitrile/methanol/water (40/20/40, v/v/v) (phase A) and acetonitrile (phase B), and the following gradient program was run in a sequential manner: phase A for 30 minutes, phase B for 9 minutes, and then switching back to phase A for 6 minutes. The wavelengths for excitation and emission were set at 470 nm and 580 nm, respectively. An assay range was 200 to 8000 ng/mL, and the overall imprecision and inaccuracy of the assay ranged from 1.9% to 8.1% and 1.1% to 8.7%, respectively.

Pharmacokinetic and Statistical Analysis
The pharmacokinetic parameters were calculated by standard noncompartmental methods using WinNonlin Version 3.0 (Pharsight, Inc, Mountain View, Calif). These parameters included maximum plasma concentration (C_max), time to C_max (t_max), apparent terminal half-life (t_1/2; calculated from ln2/_Z, where _Z is the terminal rate constant calculated by log-linear regression of the terminal phase of the plasma concentration-time curve), area under the plasma concentration-time curve from time 0 to 12 hours (AUC_0-12h; calculated by the linear trapezoidal rule and, if below the limit of quantification at 12 hours, extrapolated to a value of 0 at 12 hours) and to infinity (AUC_0-; calculated using _Z), plasma clearance of arundic acid (CL; calculated from dose/AUC_0-), and apparent volume of distribution at steady state (V_ss; calculated from (Dose/AUC_0-) x (AUMC/AUC_0-) – T/2, where AUMC is the area under the first moment curve to infinity calculated by the linear trapezoidal rule, and T is the length of the infusion). Actual sampling times were used for individual concentration-time profiles.

Statistical analyses were performed using SAS Version 8 (SAS Institute, Inc, Cary, NC). All tests were 2-sided, and statistical significance was considered at the 5% level. Patients who deviated from the scheduled infusion duration (60 minutes) or infusion volume (300 mL) by more than 10% were excluded from the analyses. Dose proportionality was assessed by a power model approach.⁸ A mean exponent of slope with a 95% confidence interval (CI) was presented, and if the 95% CI excludes 1, the relationship between dose and a pharmacokinetic parameter was considered to be not dose proportional. Also, dose-normalized C_max and AUC_0- values were log-transformed and tested in a 1-way analysis of variance (ANOVA) with a factor of dose.

The effect of multiple dosing on accumulation was evaluated by comparing AUC_0-12h and C_max between dose 1 and dose 3, and the linearity of the kinetics from dose 1 to dose 3 was evaluated by comparing terminal t_1/2. The log-transformed parameters were analyzed by a paired t test, and the ratios (dose 3/dose 1) of least squares geometric means with the 95% CI are presented.

Effect of gender and age was assessed by 2-way ANOVA. Dose-normalized C_max and AUC_0-, t_1/2, CL, and V_ss values on day 1 were log-transformed. To assess the effect of age, the older population was predefined as those aged 65 years or older and compared to those aged less than 65 years, according to the International Conference on Harmonization (ICH) guideline.⁹ The interaction (Dose x Gender, Dose x Age) was not found to be significant and dropped from the model.

	RESULTS

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Plasma samples were collected from all 49 patients who received arundic acid, but 6 and 4 patients were excluded from the statistical analysis for the first and third infusions, respectively, because of the following reasons: deviation by more than 10% from the scheduled dose or duration of infusion (6), interruption of infusion (1), missing blood sampling at 15 minutes and 1 hour postinfusion (1), blood sampling time unknown (1), and only 1 blood sample available (1). In addition, 2 patients were withdrawn from the study before receiving the third infusion. The demographic and baseline characteristics of the pharmacokinetic analysis population (n = 43) are shown in Table I.

Pharmacokinetic profiles of arundic acid are illustrated in Figure 1. Pharmacokinetic parameters are summarized by treatment group in Table II. Mean C_max was reached at the end of the 1-hour infusion period, and plasma concentrations of arundic acid subsequently declined with a mean t_1/2 of approximately 2 to 3 hours. Mean C_max and AUC_0- increased with increasing dose levels; however, at higher doses, the increase tended to be less than dose proportional. An estimate of slope from the dose-proportionality power model for C_max was 0.73 (95% CI: 0.54, 0.92), indicating that the observed non–dose proportionality was statistically significant (Table III). An estimate of slope for AUC_0- was 0.85 (0.67, 1.02). The finding is consistent with the results of the 1-way ANOVA in dose-normalized C_max and AUC_0- (Table III, P = .007 and .314, respectively).

View larger version (11K): [in this window] [in a new window]   Figure 1. Mean plasma arundic acid concentration-time profiles in acute ischemic stroke after (A) first dose and (B) third dose. Once-daily 1-hour intravenous infusion: 2 mg/kg/h (filled diamonds); 4 mg/kg/h (open squares); 6 mg/kg/h (filled triangles); 8 mg/kg/h (open circles); 10 mg/kg/h (filled circles); 12 mg/kg/h (filled squares).

View this table: [in this window] [in a new window]   Table III Dose Proportionality on Cmax and AUC0- at Dose 1

The effect of multiple dosing was assessed by comparing the pharmacokinetic parameter values at dose 3 with corresponding values at dose 1. The C_max and AUC_0-12h values at dose 3 were similar to those at dose 1 across a dose range of 2 to 12 mg/kg/h (Table IV). The ratio (dose 3/dose 1) for the log-transformed C_max tended to be greater in the 10- and 12-mg/kg/h groups compared to lower dose groups, but the magnitude of increase was not statistically significant, and trough plasma concentrations of arundic acid were generally below the limit of quantification throughout the 7-day drug administration period. In addition, the geometric mean t_1/2 values at dose 3 were similar to those at dose 1 over the entire dose range, and there were no consistent findings for an alteration in half-life on repeated dosing.

The effects of gender and age were examined by 2-way ANOVA, and the results are summarized in Tables V and VI. The pharmacokinetic parameters in women were similar to those in men. The dose-normalized AUC_0- in the elderly patients was approximately 30% greater than that in younger patients (Figure 2), and this age-related difference was statistically significant. There was a correspondingly lower systemic clearance and a tendency toward a larger volume of distribution in elderly patients. The lower clearance and larger volume of distribution were manifested as a statistically significant longer t_1/2 in elderly patients. Dose-normalized C_max in elderly patients was similar to that in younger patients. Exclusion of the 8- and 10-mg/kg/h dose groups, in which there were no and one younger patient(s), respectively, resulted in similar results. There were no correlations between dose-normalized AUC_0- values at dose 1 and serum albumin, ALT, AST, alkaline phosphatase, total bilirubin, or creatinine clearance values measured on day 1 prior to study medication (r < 0.3, Pearson's correlation method).

View larger version (6K): [in this window] [in a new window]   Figure 2. Multiplot showing age and dose-normalized AUC0- at dose 1 in individual patients with acute ischemic stroke.

	DISCUSSION

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The purpose of this investigation was to characterize the pharmacokinetics of arundic acid in acute ischemic stroke. Following intravenous infusion of arundic acid, mean maximum plasma drug levels were reached at the end of the 1-hour infusion period; thereafter, plasma concentrations declined with a mean apparent terminal half-life of approximately 2 to 3 hours after single and repeated doses. Systemic exposure increased, less than proportionally, with increasing dose levels. There was no excessive accumulation of arundic acid in plasma, consistent with the short half-life relative to the dosing interval. Mean systemic clearance of arundic acid was approximately 40 to 60 mL/h/kg, which is low relative to estimated hepatic blood flow (1200 mL/h/kg). Apparent volume of distribution was 90 to 200 mL/kg, which approximates the nominal extracellular fluid volume (300 mL/kg) and was greater than plasma volume (50 mL/kg).

The pharmacokinetic parameters of arundic acid in acute ischemic stroke were similar to those measured in our previous phase I study with healthy adult male subjects (Table VII, data on file). It is noted that nonlinear systemic exposure at higher doses was observed in the study with healthy adults as well.

The reason for the nonlinear systemic exposure is likely due to a high protein-binding property of arundic acid in plasma (99%; primarily binding to albumin), resulting in saturation of protein-binding sites at higher doses. In an in vitro study, the saturation of protein binding in human serum is observed at 40 µg/mL of arundic acid or higher, similar to concentrations at which the nonlinear systemic exposure was observed in our study. V_ss and CL values were increased as the nonlinear drug exposure became apparent at 10 to 12 mg/kg/h, suggesting that a proportion of the unbound drug was increased. Saturation of renal reabsorption after intravenous administration might be another possibility of the nonlinear systemic exposure. However, in the previous healthy adult study, no differences in urine excretion of arundic acid and its metabolites were found regardless of doses of arundic acid, indicating little contribution of this mechanism. Although less than a proportional increase was observed in the total drug concentration, an increase in the unbound concentration is expected to remain dose proportional in the dose range investigated in this study, ie, drug effect could increase linearly with increasing dose, and thus careful monitoring is needed. However, no safety concerns were identified in this study.⁶

While pharmacokinetic variables of arundic acid in men were not appreciably different from those in women, systemic exposure in elderly patients was greater than that in younger patients, associated with a lower systemic clearance and prolonged terminal half-life. We explored a possible relationship with hepatic or renal parameters or plasma albumin levels, but no association was identified. There are several reports that age alone can affect hepatic oxidation pathways.¹⁰ Because arundic acid is metabolized by hepatic oxidation, aging itself may be the direct cause of the higher systemic exposure. Although systemic exposure was 30% greater in elderly patients, the plasma concentration after each infusion fell below or approximated the lower limit of quantification prior to next administration, and no particular safety findings were observed. This result indicates that dose adjustment would not be required to prevent systemic drug intoxication in elderly individuals within the dose range investigated in this study.

Currently available treatment for acute ischemic stroke is aimed at resupplying blood flow by dissolving or mechanically removing occlusive thrombi in cerebral arteries. These therapies can dramatically improve neurological deficits but are only effective when initiated within the first few hours after onset of stroke symptoms, and thus only a limited number of patients can benefit from these therapies.^2,3 An alternative approach is neuroprotection, ie, minimizing growth of the brain infarct by suppressing neurotoxic cascades, which may extend the therapeutic time window. Thus far, a number of neuroprotective agents have failed to detect clinical benefits despite promising results in preclinical experiments.¹¹ One possible explanation for the failure of previously examined neuroprotective agents is that those clinical trials did not follow the design features of preclinical studies showing benefit in animal models. Differences between actual drug concentrations achieved in humans and effective drug concentrations obtained in preclinical experiments would be an important source of this error.^11,12 The present study confirmed that 1-hour intravenous infusion of arundic acid ranging from 2 to 12 mg/kg/h reached or exceeded minimal plasma drug levels, 7 to 15 µg/mL, shown to be efficacious in stroke models in both rats and primate animals, without any safety concerns.⁶

There are several limitations in this study. First, the study was designed on the basis of practical considerations in the emergency treatment setting. Although plasma samples were obtained from all of the study participants, several patients were excluded from the analysis because of protocol deviations. However, despite the suboptimal design, the present study reasonably characterized the pharmacokinetic profile of arundic acid in acute stroke for further clinical development and confirmed that it was generally comparable with that in healthy adults. Second, the impact of severe hepatic or renal impairment on the exposure of arundic acid was not a part of this investigation, and these patients were excluded from this study. In particular, severe hepatic impairment could significantly affect the pharmacokinetics of arundic acid, and further investigation is needed. Lastly, because of the nature of intensive treatment for acute stroke, all of the patients evaluated in this study received a broad range of medications, and it was not possible to evaluate the effects of concomitant medications. Most frequent concomitant medications were heparin and acetylsalicylic acid in this study. Although the pharmacokinetic comparison with the results of the previous phase I study in the drug-free, healthy adults suggests that the impact of concomitant medications may be relatively less, further investigation is required in the future.

In summary, the pharmacokinetics of arundic acid were similar to those in healthy adult subjects, systemic exposure appeared to be less than dose proportional at higher doses, and there was no accumulation in plasma after repeated dosing. Systemic exposure in elderly patients was greater than that in younger patients, associated with a lower systemic clearance and manifested as a longer terminal half-life.

	ACKNOWLEDGEMENTS

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Financial disclosure: Mr H. Ishibashi, Mr Y. Funakoshi, and Mr M. Hiramatsu are employees of Ono Pharmaceutical Co, Ltd or its subsidiary. Dr L. Creed Pettigrew received financial support from Ono Pharmaceutical Co, Ltd, as a grant awarded to the participating study center.

	REFERENCES

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1. Mackay J, Mensah GA. The Atlas of Heart Disease and Stroke. Geneva, Switzerland: World Health Organization; 2004.

2. National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. Tissue plasminogen activator for acute ischemic stroke. N Engl J Med. 1995;333: 1581-1587.[Abstract/Free Full Text]

3. Smith WS, Sung G, Starkman S, et al. Safety and efficacy of mechanical embolectomy in acute ischemic stroke: Results of the MERCI trial. Stroke. 2005;36: 1432-1438.[Abstract/Free Full Text]

4. Heiss WD, Thiel A, Grond M, Graf R. Which targets are relevant for therapy of acute ischemic stroke? Stroke. 1999;30: 1486-1489.[Abstract/Free Full Text]

5. Asano T, Mori T, Shimoda T, et al. Arundic acid (ONO-2506) ameliorates delayed ischemic brain damage by preventing astrocytic overproduction of S100B. Curr Drug Targets. 2005;4: 127-142.

6. Pettigrew LC, Kasner SE, Albers GW, et al. Safety and tolerability of arundic acid in acute ischemic stroke. J Neurol Sci. 2006;251: 50-56.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

7. Brott T, Adams HP, Olinger CP, et al. Measurements of acute cerebral infarction: a clinical examination scale. Stroke. 1989;20: 864-870.[Abstract/Free Full Text]

8. Gough K, Hutchison M, Keene O, et al. Assessment of dose proportionality: report from the statisticians in the pharmaceutical industry/pharmacokinetics UK joint working party. Drug Information J. 1995;29: 1039-1048.

9. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. Studies in support of special populations: Geriatrics E7. Current Step 4 version; 24 June 1993.

10. Schmucker DL. Liver function and phase I drug metabolism in the elderly: a paradox. Drugs Aging. 2001;18: 837-851.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

11. Gladstone DJ, Black SE, Hakim AM. Toward wisdom from failure: lessons from neuroprotective stroke trials and new therapeutic directions. Stroke. 2002;33: 2123-2136.[Abstract/Free Full Text]

12. Fisher M, Brott TG. Emerging therapies for acute ischemic stroke: new therapies on trial. Stroke. 2003;34: 359-361.[Free Full Text]
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This Article Abstract Full Text (PDF) 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 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 Ishibashi, H. Articles by Hiramatsu, M. Search for Related Content PubMed Articles by Ishibashi, H. Articles by Hiramatsu, M. Social Bookmarking                   What's this?