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Bioequivalence of Two Intravenous Formulations of Antithrombin III: A Two-Way Crossover Study in Healthy Korean Subjects
Clinical Therapeutics/Volume 35, Number 11, 2013
Bioequivalence of Two Intravenous Formulations of Antithrombin III: A Two-Way Crossover Study in Healthy Korean Subjects Kyoung-Ah Kim, PhD1; Yoon-Young Lim, MS2; Sun-Ho Kim, MS2; and Ji-Young Park, MD, PhD1 1
Department of Clinical Pharmacology and Toxicology, Anam Hospital, Korea University College of Medicine, Seoul, Korea; and 2Clinical Research Biz, SK Chemicals Inc, Seongnam, Gyeonggi-do, Korea
ABSTRACT Background: Treatment with antithrombin (AT)-III is indicated for patients with sepsis or hereditary AT deficiency. Objective: The purpose of this study was to compare the pharmacokinetic and pharmacodynamic characteristics of 2 AT-III formulations in healthy Korean volunteers to satisfy the regulatory requirements for bioequivalence for marketing purposes. Methods: A single-center, single-dose, open-label, randomized, 2-period, 2-sequence crossover study was conducted in healthy Korean volunteers. Blood samples for the drug analysis were collected for up to 216 hours after drug administration. Participants received either the test or reference formulation of AT-III 100 U/kg IV for 20 minutes in the first period and the alternative formulation in the second period. Both the AT-III activity and antigen (Ag) were measured for the analysis of pharmacokinetic properties, and the prothrombin time and the activated partial thromboplastin time were assessed for the analysis of pharmacodynamic properties. Because AT-III is an endogenous compound, the analysis used data corrected from baseline values. The tolerability of the 2 formulations was also assessed based on physical examinations including vital sign measurements, laboratory tests, and 12-lead ECG. Results: Of the 20 subjects enrolled (mean [SD] age, height, and weight, 25.3 [2.3] years, 175.3 [4.5] cm, and 67.4 [6.3] kg, respectively), 19 completed both treatment periods; 1 subject withdrew consent for personal reasons. The observed mean (SD) Cmax, AUClast, and AUC0–1 of AT-III activity were, respectively, 279.24% (35.92), 14,364.10 (2325.25) % h, and 17,526.38 (3150.81) % h with the test formulation and 249.75% (31.96), 12,962.95 (1897.52) % h, and 15,957.67 (3189.21) % h with the reference formulation. The observed mean (SD) Cmax, AUClast, and
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AUC0–1 of AT-III Ag were 62.58 (5.66) mg/dL, 3051.94 (401.87) mg/dL h, and 3639.80 (726.01) mg/dL h, respectively, with the test formulation and 58.63 (5.27) mg/dL, 2805.08 (272.38) mg/dL h, and 3340.00 (428.46) mg/dL h with the reference formulation. The geometric mean ratios (90% CI) of the logtransformed data for AT-III activity between the 2 formulations were 1.11494 (1.08994–1.14053) for Cmax, 1.11305 (1.05435–1.17503) for AUClast, and 1.11527 (1.03754–1.19889) for AUC0–1; corresponding values for AT-III Ag were 1.08802 (1.06258– 1.11405), 1.10905 (1.05804–1.16242), and 1.11460 (1.02058–1.21726). During the study period, 8 adverse events were reported, and all were transient, mild, and resolved completely during the treatment period. Conclusion: The results of the present study showed that these 2 AT-III formulations met the regulatory criteria for pharmacokinetic bioequivalence with respect to AT-III activity and Ag in these healthy Korean subjects. ClinicalTrials.gov identifier: NCT00846274. (Clin Ther. 2013;35:1752–1761) & 2013 Elsevier HS Journals, Inc. All rights reserved. Key words: antithrombin III, bioequivalence, pharmacokinetic equivalence, pharmacokinetics.
INTRODUCTION Antithrombin (AT)-III is a 58,000-Da, vitamin K– dependent glycoprotein that inhibits coagulation factors and is synthesized in the liver.1,2 AT-III acts as a physiologic inhibitor of blood coagulation, and thus plays a key role in hemostasis.3 Accepted for publication August 31, 2013. http://dx.doi.org/10.1016/j.clinthera.2013.08.018 0149-2918/$ - see front matter & 2013 Elsevier HS Journals, Inc. All rights reserved.
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K.-A. Kim et al. Primarily AT-III treatment is indicated for sepsis or hereditary antithrombin deficiency.4 In addition, it has the potential to have antiangiogenic, antitumor, and antiviral activities.4–6 In the case of sepsis, the blood concentration of AT-III falls by 20% to 40% in septic patients, and the levels of AT-III correlate with disease severity and clinical outcomes.7,8 The reduction of ATIII levels occurs for various reasons, as follows: decreased production of AT-III in the liver; inactivation of the enzyme elastase, which is increased during inflammation; and the loss of AT-III from the circulation into tissues through inflamed and leaking capillary blood vessels.9 Patients with hereditary AT-III deficiency are at increased risk for venous thromboembolism, particularly in high-risk situations, such as surgery, trauma, and pregnancy. It has been reported that the risk for venous thromboembolism in these situations increases 10- to 50-fold compared with the general population.10,11 AT-III is used through the intravenous route for the management of acute thrombotic episodes and for prophylaxis of thrombotic episodes in patients. In healthy adults, the AT-III activity level ranges from 0.8 to 1.2 U/mL, with an average of 1 U/mL.12 Therefore, the goal of treatment is to maintain the antithrombin activity at 80% to 120% of normal to prevent thromboembolic episodes.4 The loading dose is based on targeting the 100% activity level in healthy patients, and is typically followed by subsequent doses to maintain the antithrombin activity between 80% and 120%, which is usually achieved with 60% of the loading dose.4 The most common and convenient laboratory method used for assessing AT-III is the detection of antithrombin activity.13 Antigenic testing to measure AT-III levels directly has been used in a limited number of studies; this test is primarily used for distinguishing qualitative from quantitative results.13 These antithrombin tests are performed after a thrombotic event has resolved and when a patient is suspected to have decreased AT-III levels clinically. Even though it has been reported that the elimination half-life of AT-III is 56.8 to 68.0 hours,4,14–16 little information is available concerning the pharmacokinetic characteristics of AT-III. Furthermore, the authors could not find any studies revealing the pharmacokinetic properties of AT-III in Asian populations, including Koreans. Like the conventional formulation of AT-III, the newly developed AT-III is isolated from the pooled plasma in humans. Preclinical data revealed that the new formulation has physicochemical characteristics
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similar to those of the conventional AT-III formulation, but with greater purity (99% vs 98%) (Physicochemical analysis test for AT-III formulations [Study no, INPAC06MS-203], Preclinical Data on Investigator’s Brochure, SK Chemicals, 2008). Additionally, the new formulation was shown to have excellent viral safety against hepatitis A, B, and C; HIV; and parvovirus B19, and efficacy similar to that of conventional AT-III in rats (validation of virus removal and inactivation [Study no, 2/06.159], AT-III Efficacy test in experimental DIC model [Study no, M07002-3], Preclinical Data on Investigator’s Brochure, SK Chemicals, 2008). A literature search revealed that studies on the pharmacokinetic properties of AT-III, including bioequivalence evaluations, are lacking. Different from the conventional formulation of AT-III, recombinant AT-III was developed to treat patients with antithrombin deficiency.4 In a comparison of the pharmacokinetic properties between recombinant AT-III and conventional AT-III, recombinant AT-III had greater clearance but a shorter half-life.17 The primary objectives of this study were to compare the pharmacokinetic characteristics between, and to determine the bioequivalence of, the newly developed and conventional formulations of AT-III in healthy Korean subjects for the purpose of registration approval of the test formulation in Korea.
SUBJECTS AND METHODS Study Design This single-center, single-dose, randomized, openlabel, 2-sequence, 2-period, comparative crossover study was conducted at the Clinical Trial Center of Anam Hospital, Korea University College of Medicine, Seoul, Korea. The study protocol and informedconsent form were reviewed and approved by the institutional review board at Anam Hospital and by the Korea Food and Drug Administration (registration no. 693). The study was conducted in accordance with the principles of the Declaration of Helsinki, as outlined in the guideline for Good Clinical Practice.18,19 All data obtained from the study were monitored by the sponsor. Even though the present study was an openlabel study, the observer was neutral in relation to the participants and the investigational drugs. The purpose and procedures of the study were explained to the participants in detail before the study, and all subjects provided written, informed consent before participating in the study. Participants were supplied with written information that included the study purpose, methods,
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Clinical Therapeutics possible risks, and details of the honorarium that was paid to the participants.
Inclusion and Exclusion Criteria Screening occurred during a 3-week period before treatment administration. Healthy male and female Korean volunteers aged 19 to 40 years and within 20% of ideal body weight were recruited. Health was judged by physicians based on the results of a detailed physical examination including vital sign measurements, 12-lead ECG, and laboratory tests including blood chemistry, hematology, and urinalysis based on the inclusion and exclusion criteria. Participants were not enrolled if they had any of the following: a history or evidence of a hepatic, renal, gastrointestinal, or hematologic abnormality; prolonged prothrombin time (PT), activated partial thromboplastin time (aPTT), or bleeding time; hepatitis B, hepatitis C, or HIV infection revealed on laboratory testing; any other acute or chronic disease; a history of hypersensitivity to AT-III; a history of clinically significant allergic disease; a history of medical condition to influence drug (AT-III) absorption, distribution, metabolism, and elimination (cardiovascular, respiratory, renal, endocrine, hematologic, CNS, psychiatric disease, surgery, or malignant tumor); a history of or current hemorrhagic disease; hemolytic anemia or anemia due to blood loss (hemoglobin o14 g/dL and/or hematocrit o42%); a history of alcohol or drug abuse; repeated medication within 1 month from the day medicated through participation in a previous clinical trial; smoking habit (410 cigarettes daily); consumption of 45 glasses daily of beverages containing xanthine derivatives; or use of any medication having the potential to affect the study results (ie, estrogens, L-asparaginase, heparin) within 14 days of the start of the study.4
Study Procedures Study Drug Administration Subjects were randomly assigned to receive either the test* or the reference† formulation at the beginning of the study in accordance with randomization conducted using SAS version 9.13 (SAS Institute Inc, Cary, North Carolina). Subjects were admitted to the clinical trial *Antithrombin III injection containing 500 U per vial (batch no. CTG08001) (SK Chemicals Inc, Gyeonggi-do, Korea). † Antithrombin III injection containing 500 U per vial (batch no: 270B8159) (Green Cross Corporation, Gyeonggi-do, Korea).
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center on the evening before the day of drug administration. No food intake was allowed for at least 10 hours before dosing until 4 hours after dosing. The next morning, participants were given a single intravenous dose of either the test or the reference formulation, 100 IU/kg IV, under fasting conditions and under the supervision of physicians. Each drug was given over 20 minutes, with 100 IU/kg AT-III dissolved in normal saline using an infusion pump. After a washout period of 4 weeks, patients were switched to the alternate formulation. The administered dose was determined based on the results of the previous AT-III pharmacokinetic studies.17,18 The authors chose the dose of AT-III to achieve 200% of the AT-III activity from 100% of normal using the following equation: AT-III amount required (U) = [(Desired AT-III level – Baseline AT-III level) Weight (kg)]/1.4].20,21 The intake of beverages containing xanthine derivatives or alcohol, extreme physical activity, and smoking were not allowed over the course of the study. The subjects were under continuous medical supervision. Physical examinations, including vital sign measurements (blood pressure, heart rate, and body temperature), 12-lead ECG, and laboratory tests including chemistry, hematology, and urinalysis were conducted before drug administration and at the end of each study period. Vital signs were measured in each subject by a nurse after the subject had rested for at least 5 minutes, with the arm supported at heart level. All laboratory tests were performed at an accredited central laboratory (Department of Laboratory Medicine, Anam Hospital, Korea University College of Medicine, Seoul, Korea).
Blood Sampling Ten-milliliter blood samples were collected into an indwelling catheter placed into the forearm vein. Blood samples were collected at –2, –1, and immediately before study drug administration (0 hour) and then at 10, 20, 30, and 45 minutes and at 1, 2, 4, 6, 8, and 12 hours after the start of drug administration through the indwelling catheter kept by normal saline. Blood samples were also collected at 24, 48, 72, 96, 120, 168, and 216 hours after the start of drug administration by direct venipuncture. Blood samples were collected into a plain tube (for pharmacokinetic analysis) and into citrated tubes (for pharmacodynamic analysis) (Vacutainer, Becton, Dickinson, and
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K.-A. Kim et al. Company, Franklin Lakes, New Jersey). Tubes for pharmacokinetic analyses were centrifuged for the isolation of plasma at 41C in a refrigerated benchtop centrifuge at 1500g for 15 minutes, and tubes for pharmacodynamic analyses were sent to the central laboratory at Anam Hospital for the measurements of PT and aPTT. Separated samples were stored at –701C until assayed. After a 4-week washout period, assessments were repeated in the same manner in the second period to complete the crossover design.
The area under the time-versus-effect curve from time 0 to the last time measured (AUEClast) was calculated using the linear trapezoidal rule.
Tolerability Assessment Adverse events were assessed throughout each study period based on direct questioning and spontaneous reports, and were recorded regardless of their suspected relationship to the study drugs.
Statistical Analysis Assessments of AT-III Activity and Antigen Concentrations AT-III activity was measured by a chromogenic method using a commercial AT-III activity kit (Berichrome AT-III, Siemens Healthcare Diagnostics, Inc, Tarrytown, New York) with a Sysmex CA-540 Coagulation Analyzer (Siemens Healthcare Diagnostics). AT-III antigen (Ag) was measured using a turbidity immunoassay method with a Hitachi 7600110 Clinical Chemistry Analyzer (Hitachi HighTechnologies Corporation, Tokyo, Japan).
Assessments of Pharmacokinetic and Pharmacodynamic Properties A noncompartmental method was used to determine the pharmacokinetic and pharmacodynamic properties of AT-III using WinNonlin version 5.21 (Pharsight Corporation, Mountain View, California).22 For pharmacokinetic analysis, Cmax and Tmax were obtained using direct inspection of individual plasma concentration–time data. AUClast was calculated using the linear trapezoidal rule. AUC0–1 was calculated as AUClast þ Ct/ke, where Ct is the last plasma concentration measured and ke is the elimination rate constant, determined by linear regression analysis of the linear portion of the log plasma concentration–time curve. The t½ values were calculated as ln2/ke, and the clearance (CL) of AT-III was calculated as Dose/ AUC0–1. Because AT-III is an endogenous compound, data corrected from baseline values were analyzed. Corrected values were calculated by subtracting the baseline values from the values obtained at each time point after the start of infusion. We used the means of the values measured at –2, –1, and 0 hours before treatment as the baseline values. For pharmacodynamic analysis, the peak effect (Emax) and time to Emax (Tmax) were obtained by direct inspection of individual effect-versus-time data.
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The sample size for this study was estimated using a power calculation conducted on the basis of the data derived from previous pharmacokinetic studies.18 At least 14 participants were required to detect a 20% difference in the pharmacokinetic parameters (Cmax and AUC) between the test and reference formulations with 80% statistical power at a 0.05 level of significance.23 To determine whether the 2 AT-III formulations exhibited pharmacokinetic equivalence for the respective AT-III activity and Ag, we compared individual Cmax and AUC values and their ratios (test/reference) using log-transformed data; means and 90% CIs were analyzed using parametric variance analysis. Two-way ANOVA was used to assess the effects of formulation, period, and sequence on the pharmacokinetic parameters as fixed effects and of subjects nested within the sequence as a random effect. Other main effects were tested at the 5% level of significance against the residual error (mean square error) from the ANOVA model as the error term. Due to the nature of normaltheory CIs, this was equivalent to carrying out two 1sided t tests at the 5% level of significance.23,24 To satisfy the requirement for bioequivalence of AT-III in Korea, the geometric mean ratios and 90% CIs of the test formulation to the reference formulation for Cmax and AUC were to be within the range of 80% to 125% for log-transformed data. All statistical analyses were performed using SAS version 9.13 (SAS Institute Inc).
RESULTS Study Population Twenty participants who met the inclusion and exclusion criteria were enrolled in this study; mean (SD) age, height, and weight were 25.3 (2.3) years, 175.3 (4.5) cm, and 67.4 (6.3) kg, respectively. One subject withdrew consent during the first period for
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Table I. Baseline demographic and clinical characteristics of the healthy subjects in this study of the bioequivalence of 2 intravenous formulations of antithrombin III (N ¼ 20). Characteristic Age, y Mean (SD) Range %CV Height, cm Mean (SD) Range %CV Weight, kg Mean (SD) Range %CV
RT
TR
All Patients
25.3 (2.1) 23–30 8.1
25.0 (2.7) 22–31 10.7
25.3 (2.3) 22–31 8.9
177.9 (3.8) 173–184 2.2
172.6 (3.6) 167–181 2.1
175.3 (4.5) 167–184 2.6
69.8 (4.5) 62.7–75.0 6.4
64.9 (7.0) 55.3–73.6 10.8
67.4 (6.3) 55.3–75.0 9.3
RT ¼ reference–test sequence; TR ¼ test–reference sequence.
(0.12) L/h with the test and reference formulations. Summarized pharmacokinetic properties of AT-III activity with both formulations are presented in Table II.
AT-III Antigen The mean plasma concentration–time profiles of AT-III Ag after single-dose administration the 2 ATIII formulations are shown in Figure 2. The baseline AT-III Ag levels before treatment were 24.64 (1.71) mg/dL (range, 21.6–27.8 mg/dL) with the test formulation and 25.05 (1.50) mg/dL (range, 21.1–27.0 mg/ dL) with the reference formulation (P ¼ 0.284). Similar to the AT-III activity, all pharmacokinetic properties of AT-III Ag were comparable between the 2 formulations. The mean (SD) Cmax values with the test and reference formulations were 62.58 (5.66) and 58.63 (5.27) mg/dL, respectively; Tmax values were 0.66 and 0.64 hours. The mean (SD) AUC0–1 values were 3639.80 (726.01) mg/dL h with the test formulation and 3340.00 (428.46) mg/dL h with the reference formulation. CL values of AT-III Ag were 1.91 (0.37) and 2.07 (0.33) L/h and for the test and reference
personal reasons. Therefore, 19 subjects completed both treatment periods and were included in the pharmacokinetic and pharmacodynamic analyses. The participants’ demographic characteristics are summarized in Table I.
350 Reference Test
300
The mean plasma concentration–time AT-III activity profiles of the 2 AT-III formulations after the administration of a single intravenous dose of 100 IU/ kg of each are shown in Figure 1. The baseline activities before treatment were 93.6% (7.4) (range, 77.4%–106.3%) with the test formulation and 95.6% (6.6) (range, 82.4%–107.2%) with the reference (P ¼ 0.150). All pharmacokinetic properties of AT-III activity between the 2 formulations were comparable. The mean (SD) Cmax values of the test and reference formulations were 279.24% (35.92) and 249.75% (31.96), respectively; Tmax values were 0.75 and 0.79 hours. The mean (SD) AUC0–1 values were 17,526.38 (3150.81) and 15,957.67 (3189.21) % h with the test and reference formulation (P ¼ NS). CL values of AT-III activity were 0.40 (0.09) and 0.44
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AT-III Activity (%)
250
Pharmacokinetic Properties AT-III
200 150 100 50 0 0
24
48
72
96 120 144 168 192 216 Time (h)
Figure 1. Plasma concentration profiles of 2 intravenous formulations of antithrombin (AT)-III 100 IU/kg after single-dose administration in healthy Korean subjects (n ¼ 19).
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Table II. Pharmacokinetic properties* of 2 intravenous formulations of antithrombin III 100 IU/kg after single-dose administration in healthy Korean subjects (n ¼ 19). Parameter Baseline activity level, % ke, h–1 t½, h Tmax, h Cmax, % AUClast, % h AUC0–1, % h Cmax/dose, % AUC0–1/dose, % h CL, L/h
Test Formulation, Mean (SD) 93.6 0.008 92.80 0.75 279.24 14,364.1 17,526.38 0.042 2.627 0.40
Reference Formulation, Mean (SD)
(7.4) (0.001) (15.14) (0.49) (35.92) (2325.25) (3150.81) (0.007) (0.526) (0.09)
95.6 0.008 93.46 0.79 249.75 12,962.95 15,957.67 0.037 2.408 0.44
(6.6) (0.004) (29.97) (0.57) (31.96) (1897.52) (3189.21) (0.005) (0.606) (0.12)
Geometric Mean Ratio 0.978 0.964 1.039 0.991 1.118 1.106 1.104 1.118 1.104 0.906
CL ¼ clearance; ke ¼ elimination rate constant. *Corrected from baseline activity.
formulations. Summarized pharmacokinetic parameters of AT-III Ag with both formulations are presented in Table III. 80
Pharmacodynamic Properties of AT-III
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Reference Test
60 AT-III Ag (mg/dL)
For the pharmacokinetic analysis of AT-III, we assessed PT and aPTT as previously reported.1,2 The baseline PT values before treatment were 11.18 (0.50) seconds with the test formulation and 11.02 (0.53) seconds and with the reference formulation (P = 0.162), and the baseline aPTT values before treatment were 26.85 (1.99) seconds with the test formulation and 27.04 (2.52) seconds with the reference formulation (P = 0.210) (Figure 3). The mean (SD) Emax values of PT of the test and reference formulations were 11.59 (0.53) and 11.65 (0.51) seconds, respectively; Tmax was 6 hours with both. The mean (SD) AUEC values of PT were 2363.95 (106.70) and 2372.61 (86.69) sec h with the test and reference formulations. In the case of aPTT, Tmax for both was 1 hour, and the mean (SD) Emax values of aPTT of the test and reference formulations were 29.40 (2.25) and 29.92 (2.58) seconds, respectively. The mean (SD) AUEC values of aPTT were 5608.88 (407.08) and 5666.21 (461.59) sec h with the test and reference formulations. Summarized pharmacodynamic parameters for both formulations are presented in Table IV.
40
20
0 0
24
48
72
96 120 144 168 192 216 Time (h)
Figure 2. Plasma concentration profiles of antithrombin (AT)-III antigen (Ag) after single-dose administration of 2 intravenous formulations of antithrombin III 100 IU/kg in healthy Korean subjects (n ¼ 19).
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Table III. Pharmacokinetic properties* of antithrombin III antigen after single-dose administration 2 intravenous formulations of antithrombin III 100 IU/kg in healthy Korean subjects (n ¼ 19). Parameter
Test Formulation, Mean (SD)
Baseline Ag level, mg/dL ke, h–1 t½, h Tmax, h Cmax, mg/dL AUClast, mg/dL h AUC0–1, mg/dL h Cmax/dose, mg/dL AUC0–1/dose, mg/dL h CL, L/h
24.64 0.009 84.46 0.66 62.58 3051.94 3639.80 0.009 0.542 1.91
Reference Formulation, Mean (SD)
(1.71) (0.003) (23.14) (0.39) (5.66) (401.87) (726.01) (0.001) (0.098) (0.37)
25.05 0.009 84.00 0.64 58.63 2805.08 3340.00 0.009 0.498 2.07
(1.50) (0.002) (14.90) (0.41) (5.27) (272.38) (428.46) (0.001) (0.076) (0.33)
T/R Ratio 0.983 0.988 1.013 1.045 1.088 1.109 1.114 1.088 1.111 0.900
CL ¼ clearance; ke ¼ elimination rate constant. *Corrected from baseline activity.
Pharmacokinetic Equivalence The equivalence statistics of the pharmacokinetic parameters of AT-III, including the Cmax, AUClast, and AUC0–1, for the 2 formulations are summarized in Table V. Considering that AT-III is an endogenous compound and that there are baseline AT-III activities and Ag levels in the body, we assessed the pharmacokinetic equivalence of these 2 formulations. The mean log-transformed ratios of these parameters and their 90% CIs for AT-III activity and Ag levels all fell
within the predefined pharmacokinetic equivalence range of 80% to 125%.
Tolerability No serious or unexpected adverse events were observed in this study. Eight subjects experienced 8 adverse events during the study periods (3 and 5 with the test and reference formulations, respectively). Among them, 2 adverse events were suspected to be unrelated to the formulation (1 case of erythema and 1
12.0
32 Reference Test
Reference Test 30
PT (sec)
aPTT (sec)
11.5
11.0
28
26
10.5 Time (h)
72 96 12 0 14 4 16 8 19 2 21 6
24 48
18
12
6
0
72 96 12 0 14 4 16 8 19 2 21 6
24 48
18
12
6
0
24 Time (h)
Figure 3. Change of (A) PT and (B) aPTT after single-dose administration of 2 intravenous formulations of antithrombin III 100 IU/kg in healthy Korean subjects (n ¼ 19).
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Table IV. Pharmacodynamic properties of 2 intravenous formulations of antithrombin III 100 IU/kg after single-dose administration in healthy Korean subjects (n ¼ 19). Parameter PT Basal, sec Tmax, h* Emax, sec AUEC, sec h aPTT Basal, sec Tmax, h* Emax, sec AUEC, sec h
Test Formulation, Mean (SD)
Reference Formulation, Mean (SD)
Geometric Mean Ratio
11.18 6 11.59 2363.95
(0.50) (0.17–216) (0.53) (106.70)
11.02 6 11.65 2372.61
(0.53) (0.17–120) (0.51) (86.69)
1.014 — 0.995 0.996
26.85 1 29.40 5608.88
(1.99) (0.33–216) (2.25) (407.08)
27.04 1 29.92 5666.21
(2.52) (0.33–8) (2.58) (461.59)
0.995 — 0.984 0.991
AUEC ¼ area under the peak effect curve; Emax ¼ peak effect. *Median (range).
case of sore throat), while 6 adverse events were considered to be related to the formulation tested (3 per formulation). These adverse events included 2 cases of fatigue, 2 cases of headache, and 1 case each of itching sensation, and ecchymosis at the injection site.
DISCUSSION This study revealed the pharmacokinetic characteristics of AT-III in detail in healthy Korean subjects, and that a newly developed AT-III formulation showed similar pharmacokinetic and pharmacodynamic characteristics to those of the currently used AT-III formulation. We demonstrated that the 2 AT-III formulations met the regulatory requirements for pharmacokinetic equivalence. The mean ratios and the 90% CIs for the log-transformed pharmacokinetic parameters of AT-III, including the Cmax and AUC, met the criteria for pharmacokinetic equivalence with respect to AT-III activity and Ag levels. In this study, the average activity values of the Cmax for the test and reference formulations after correction from baseline values were 279.24% and 249.75%, respectively, and those of AUClast for the test and reference formulations were 14,364.10 and 12,962.95 % h. Marzo et al18 reported that the administration of 100 IU/kg of AT-III achieved a Cmax of 262.03% and an AUClast of 10,452 % h in
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12 healthy subjects.18 This finding suggests a 24% to 38% greater AUC of AT-III in our study despite the comparable Cmax after the identical dosing regimen. Interestingly, the study done by Marzo et al showed that the elimination half-life of AT-III activity was 116.9 hours, but our study showed a half-life of 93 hours, which is a 23-hour difference. Based on a literature review, the results of our study and those of
Table V. Comparison of point estimates for logtransformed pharmacokinetic parameters of 2 intravenous formulations of antithrombin III 100 IU/kg after singledose administration in healthy Korean subjects (n ¼ 19). Parameter Activity Cmax AUClast AUC0–1 Antigen Cmax AUClast AUC0–1
Point Estimate
90% CI
1.11494 1.11305 1.11527
1.08994–1.14053 1.05435–1.17503 1.03754–1.19889
1.08802 1.10905 1.11460
1.06258–1.11405 1.05804–1.16242 1.02058–1.21726
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Clinical Therapeutics the Marzo group demonstrated an almost 2-fold longer half-life than other previous findings.16,25,26 Unfortunately, we could not determine which factor determined the half-life of AT-III. However, the presence of heparin decreased the half-life of AT-III from 56.8 to 33.7 hours.16 This means that endogenous compounds, such as heparin, might modulate the levels of AT-III. However, the notion that heparin modulates AT-III levels has a limitation to explain the pharmacokinetic discrepancies with other populations. We thus speculate the plausible mechanisms as follows. First, considering that thrombin is irreversibly bound by AT-III and forms thrombin–AT-III complex,1,2 one may speculate that the extent of thrombin–AT-III complex formation may influence the life of AT-III. Second, study design might be a factor to cause the variability of AT-III half-life. Because AT-III is an endogenous compound, the time interval of assessment of AT-III could make difference in pharmacokinetics. For instance, we collected the blood samples until 216 hours after the start of drug administration, but a previous study revealing the detailed AT-III pharmacokinetics collected samples for 168 hours after treatment.18 Additionally, it has been reported that serum albumin levels anticipate AT-III activity in patients with disseminated intravascular coagulation.26 The test and reference formulations showed similar baseline activity (93.6% vs 95.6%) and Ag (24.64 vs 25.05 mg/dL) values in this study. Because AT-III is an endogenous compound, the difference in baseline activity or Ag can influence the overall exposure of AT-III. However, the data we analyzed here were corrected from baseline values in order to overcome the difference in baseline activity or Ag, so the basal difference would influence the results of the present study. The Cmax and AUC of a test formulation should lie within the 20% deviation of the reference formulation for the acceptance of bioequivalence. The 90% CIs of the log-transformed ratio of Cmax and AUC for AT-III activity of the 2 AT-III formulations (test/ reference) were within the stipulated pharmacokinetic equivalence range of 80% to 125%.22 Furthermore, those of the AT-III Ag levels were also within the stipulated range. The results of this study indicate that the test AT-III formulation exhibits a pharmacokinetic profile, with regard to the extent and rate of absorption, comparable to that of the reference formulation.
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In this study, we evaluated the effects of 2 AT-III formulations on the PT and aPTT prolongation as a pharmacodynamic assessment. AT-III inhibits coagulation serine proteases, factor IX, factor XI, and factor IIa (thrombin).1,2 Therefore, the administration of AT-III might prolong aPTT and PT through the inhibition of the coagulation pathway. In this study, PT was prolonged maximally to 0.41 seconds with the test formulation and 0.63 seconds with the reference formulation at about 6 hours after treatment. In addition, aPTT also showed a similar maximal prolongation at 1 hour after treatment (2.55 and 2.88 seconds with the test and reference formulations, respectively). However, there were no statistical differences in PT and aPTT prolongation between the 2 formulations. This study had some potential limitations. First, it was conducted using an open-label design, and thus there may have been limitations in objectively addressing the effectiveness and safety profiles of the formulations tested. Second, we recruited healthy subjects into this study; because the pharmacokinetics of AT-III may differ in patients, there remains a theoretical uncertainty about generalizing the results to a patient group.
CONCLUSION The results of this study reveal that the test and reference formulations of AT-III tested met the regulatory criteria for bioequivalence in these healthy Korean volunteers.
ACKNOWLEDGMENTS The study was designed, conducted, and analyzed by the Department of Clinical Pharmacology and Toxicology, Anam Hospital, Korea University College of Medicine, Seoul, Korea. Drs. K.-A. Kim and Park contributed to the literature search, figures, study design, writing, data collection, interpretation, and analysis. Ms. Lim and Kim contributed to the writing, interpretation, and analysis.
CONFLICTS OF INTEREST This study was sponsored by SK Chemicals Inc, Gyeonggi-do, Korea. The authors have no other conflicts of interest with regard to the content of this article.
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REFERENCES 1. Menache D. Antithrombin III: Introduction. Semin Hematol. 1991;28:1–2. 2. Menache D, Grossman BJ, Jackson CM. Antithrombin III: physiology, deficiency, and replacement therapy. Transfusion. 1992;32:580–588. 3. Pratt CW, Church FC. Antithrombin: structure and function. Semin Hematol. 1991;28:3–9. 4. Pal N, Kertai MD, Lakshminarasimhachar A, Avidan MS. Pharmacology and clinical applications of human recombinant antithrombin. Exp Opin Biol Ther. 2010;10:1155–1168. 5. Onizuka S, Kawakami S, Taniguchi K, et al. Pancreatic carcinogenesis: apoptosis and angiogenesis. Pancreas. 2004;28:317–319. 6. Saif MW, Bona R, Greenberg B. Aids and thrombosis: retrospective study of 131 HIV-infected patients. AIDS Patient Care and STDs. 2001;15:311–320. 7. Opal SM. The clinical relevance of endotoxin in human sepsis: a critical analysis. J Endotoxin Res. 2002;8: 473–476. 8. Wiedermann ChJ, Romisch J. The anti-inflammatory actions of antithrombin—a review. Acta Medica Austriaca. 2002;29:89–92. 9. Afshari A, Wetterslev J, Brok J, Moller AM. Antithrombin III for critically ill patients. Cochrane Database Syst Rev. 2008:CD005370 10. Buchanan GS, Rodgers GM, Ware Branch D. The inherited thrombophilias: genetics, epidemiology, and laboratory evaluation. Best Practice Res Clin Obstet Gynaecol. 2003;17:397–411. 11. Walker ID, Walker JJ, Colvin BT, et al. Investigation and management of haemorrhagic disorders in pregnancy. Haemostasis and thrombosis task force. J Clin Pathol. 1994;47:100–108. 12. Maclean PS, Tait RC. Hereditary and acquired antithrombin deficiency: epidemiology, pathogenesis and treatment options. Drugs. 2007;67:1429– 1440. 13. Roshal M. Antithrombin testing. In: Transfusion Medicine and Hemostasis:
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14.
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Clinical and Laboratory Aspects. San Diego, Calif: Elsevier; 2013:893–897. Knot EA, de Jong E, ten Cate JW, et al. Antithrombin III: biodistribution in healthy volunteers. Thromb Haemost. 1987;58:1008–1011. Collen D, Schetz J, de Cock F, et al. Metabolism of antithrombin III (heparin cofactor) in man: effects of venous thrombosis and of heparin administration. Eur J Clin Invest. 1977;7:27–35. de Swart CA, Nijmeyer B, Andersson LO, et al. Elimination of intravenously administered radiolabelled antithrombin iii and heparin in humans. Thromb Haemost. 1984;52:66–70. Lu W, Mant T, Levy JH, Bailey JM. Pharmacokinetics of recombinant transgenic antithrombin in volunteers. Anesth Analg. 2000;90:531–534. Marzo A, Ceppi-Monti N, Giusti A, et al. Pharmacokinetic behaviour of antithrombin III following intravenous infusion in healthy volunteers. Arzneimittel-Forschung. 2002;52:187– 193. Korea Food and Drug Administration. Korea good clinical practice (KGCP) guidelines [in Korean]. http://clinic altrials.Kfda.Go.Kr/guide/laws/board_ list.Jsp?Category_seq=12. Accessed February 12, 2013. Schwartz RS, Bauer KA, Rosenberg RD, et al, for the Antithrombin Iii Study Group. Clinical experience with antithrombin iii concentrate in treat-
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ment of congenital and acquired deficiency of antithrombin. Am J Med. 1989;87:53S–60S. European Medicines Agency. Note for guidance on the clinicgal investigation of plasma derived antithrombin products. http://www.Emea.Europa.Eu/ pdfs/human/bpwg/222099en.Pdf. Accessed 2013. Chow SC, Liu JP. Design and analysis of bioavailability and bioequivalence studies. New York, NY: Marcel Dekker; 2000:1–30. Chow SC, Wang H. On sample size calculation in bioequivalence trials. J Pharmacokinet Pharmacodyn. 2001;28: 155–169. Schuirmann DJ. A comparison of the two one-sided tests procedure and the power approach for assessing the equivalence of average bioavailability. J Pharmacokinet Biopharm. 1987;15:657–680. Aibiki M, Fukuoka N, Nishiyama T, et al. Differences in antithrombin III activities by administration method in critical patients with disseminated intravascular coagulation: a pharmacokinetic study. Shock. 2007;28:141–147. Aibiki M, Fukuoka N, Umakoshi K, et al. Serum albumin levels anticipate antithrombin III activities before and after antithrombin III agent in critical patients with disseminated intravascular coagulation. Shock. 2007;27:139–144.
Address correspondence to: Ji-Young Park, MD, PhD, Department of Clinical Pharmacology and Toxicology, Anam Hospital, Korea University College of Medicine, 126-1, 5-Ga, Anam-dong, Sungbuk-Gu, Seoul 136705, Korea. E-mail: [email protected]
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Bioequivalence of Two Intravenous Formulations of Antithrombin III: A Two-Way Crossover Study in Healthy Korean Subjects Kyoung-Ah Kim, PhD1; Yoon-Young Lim, MS2; Sun-Ho Kim, MS2; and Ji-Young Park, MD, PhD1 1
Department of Clinical Pharmacology and Toxicology, Anam Hospital, Korea University College of Medicine, Seoul, Korea; and 2Clinical Research Biz, SK Chemicals Inc, Seongnam, Gyeonggi-do, Korea
ABSTRACT Background: Treatment with antithrombin (AT)-III is indicated for patients with sepsis or hereditary AT deficiency. Objective: The purpose of this study was to compare the pharmacokinetic and pharmacodynamic characteristics of 2 AT-III formulations in healthy Korean volunteers to satisfy the regulatory requirements for bioequivalence for marketing purposes. Methods: A single-center, single-dose, open-label, randomized, 2-period, 2-sequence crossover study was conducted in healthy Korean volunteers. Blood samples for the drug analysis were collected for up to 216 hours after drug administration. Participants received either the test or reference formulation of AT-III 100 U/kg IV for 20 minutes in the first period and the alternative formulation in the second period. Both the AT-III activity and antigen (Ag) were measured for the analysis of pharmacokinetic properties, and the prothrombin time and the activated partial thromboplastin time were assessed for the analysis of pharmacodynamic properties. Because AT-III is an endogenous compound, the analysis used data corrected from baseline values. The tolerability of the 2 formulations was also assessed based on physical examinations including vital sign measurements, laboratory tests, and 12-lead ECG. Results: Of the 20 subjects enrolled (mean [SD] age, height, and weight, 25.3 [2.3] years, 175.3 [4.5] cm, and 67.4 [6.3] kg, respectively), 19 completed both treatment periods; 1 subject withdrew consent for personal reasons. The observed mean (SD) Cmax, AUClast, and AUC0–1 of AT-III activity were, respectively, 279.24% (35.92), 14,364.10 (2325.25) % h, and 17,526.38 (3150.81) % h with the test formulation and 249.75% (31.96), 12,962.95 (1897.52) % h, and 15,957.67 (3189.21) % h with the reference formulation. The observed mean (SD) Cmax, AUClast, and
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AUC0–1 of AT-III Ag were 62.58 (5.66) mg/dL, 3051.94 (401.87) mg/dL h, and 3639.80 (726.01) mg/dL h, respectively, with the test formulation and 58.63 (5.27) mg/dL, 2805.08 (272.38) mg/dL h, and 3340.00 (428.46) mg/dL h with the reference formulation. The geometric mean ratios (90% CI) of the logtransformed data for AT-III activity between the 2 formulations were 1.11494 (1.08994–1.14053) for Cmax, 1.11305 (1.05435–1.17503) for AUClast, and 1.11527 (1.03754–1.19889) for AUC0–1; corresponding values for AT-III Ag were 1.08802 (1.06258– 1.11405), 1.10905 (1.05804–1.16242), and 1.11460 (1.02058–1.21726). During the study period, 8 adverse events were reported, and all were transient, mild, and resolved completely during the treatment period. Conclusion: The results of the present study showed that these 2 AT-III formulations met the regulatory criteria for pharmacokinetic bioequivalence with respect to AT-III activity and Ag in these healthy Korean subjects. ClinicalTrials.gov identifier: NCT00846274. (Clin Ther. 2013;35:1752–1761) & 2013 Elsevier HS Journals, Inc. All rights reserved. Key words: antithrombin III, bioequivalence, pharmacokinetic equivalence, pharmacokinetics.
INTRODUCTION Antithrombin (AT)-III is a 58,000-Da, vitamin K– dependent glycoprotein that inhibits coagulation factors and is synthesized in the liver.1,2 AT-III acts as a physiologic inhibitor of blood coagulation, and thus plays a key role in hemostasis.3 Accepted for publication August 31, 2013. http://dx.doi.org/10.1016/j.clinthera.2013.08.018 0149-2918/$ - see front matter & 2013 Elsevier HS Journals, Inc. All rights reserved.
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K.-A. Kim et al. Primarily AT-III treatment is indicated for sepsis or hereditary antithrombin deficiency.4 In addition, it has the potential to have antiangiogenic, antitumor, and antiviral activities.4–6 In the case of sepsis, the blood concentration of AT-III falls by 20% to 40% in septic patients, and the levels of AT-III correlate with disease severity and clinical outcomes.7,8 The reduction of ATIII levels occurs for various reasons, as follows: decreased production of AT-III in the liver; inactivation of the enzyme elastase, which is increased during inflammation; and the loss of AT-III from the circulation into tissues through inflamed and leaking capillary blood vessels.9 Patients with hereditary AT-III deficiency are at increased risk for venous thromboembolism, particularly in high-risk situations, such as surgery, trauma, and pregnancy. It has been reported that the risk for venous thromboembolism in these situations increases 10- to 50-fold compared with the general population.10,11 AT-III is used through the intravenous route for the management of acute thrombotic episodes and for prophylaxis of thrombotic episodes in patients. In healthy adults, the AT-III activity level ranges from 0.8 to 1.2 U/mL, with an average of 1 U/mL.12 Therefore, the goal of treatment is to maintain the antithrombin activity at 80% to 120% of normal to prevent thromboembolic episodes.4 The loading dose is based on targeting the 100% activity level in healthy patients, and is typically followed by subsequent doses to maintain the antithrombin activity between 80% and 120%, which is usually achieved with 60% of the loading dose.4 The most common and convenient laboratory method used for assessing AT-III is the detection of antithrombin activity.13 Antigenic testing to measure AT-III levels directly has been used in a limited number of studies; this test is primarily used for distinguishing qualitative from quantitative results.13 These antithrombin tests are performed after a thrombotic event has resolved and when a patient is suspected to have decreased AT-III levels clinically. Even though it has been reported that the elimination half-life of AT-III is 56.8 to 68.0 hours,4,14–16 little information is available concerning the pharmacokinetic characteristics of AT-III. Furthermore, the authors could not find any studies revealing the pharmacokinetic properties of AT-III in Asian populations, including Koreans. Like the conventional formulation of AT-III, the newly developed AT-III is isolated from the pooled plasma in humans. Preclinical data revealed that the new formulation has physicochemical characteristics
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similar to those of the conventional AT-III formulation, but with greater purity (99% vs 98%) (Physicochemical analysis test for AT-III formulations [Study no, INPAC06MS-203], Preclinical Data on Investigator’s Brochure, SK Chemicals, 2008). Additionally, the new formulation was shown to have excellent viral safety against hepatitis A, B, and C; HIV; and parvovirus B19, and efficacy similar to that of conventional AT-III in rats (validation of virus removal and inactivation [Study no, 2/06.159], AT-III Efficacy test in experimental DIC model [Study no, M07002-3], Preclinical Data on Investigator’s Brochure, SK Chemicals, 2008). A literature search revealed that studies on the pharmacokinetic properties of AT-III, including bioequivalence evaluations, are lacking. Different from the conventional formulation of AT-III, recombinant AT-III was developed to treat patients with antithrombin deficiency.4 In a comparison of the pharmacokinetic properties between recombinant AT-III and conventional AT-III, recombinant AT-III had greater clearance but a shorter half-life.17 The primary objectives of this study were to compare the pharmacokinetic characteristics between, and to determine the bioequivalence of, the newly developed and conventional formulations of AT-III in healthy Korean subjects for the purpose of registration approval of the test formulation in Korea.
SUBJECTS AND METHODS Study Design This single-center, single-dose, randomized, openlabel, 2-sequence, 2-period, comparative crossover study was conducted at the Clinical Trial Center of Anam Hospital, Korea University College of Medicine, Seoul, Korea. The study protocol and informedconsent form were reviewed and approved by the institutional review board at Anam Hospital and by the Korea Food and Drug Administration (registration no. 693). The study was conducted in accordance with the principles of the Declaration of Helsinki, as outlined in the guideline for Good Clinical Practice.18,19 All data obtained from the study were monitored by the sponsor. Even though the present study was an openlabel study, the observer was neutral in relation to the participants and the investigational drugs. The purpose and procedures of the study were explained to the participants in detail before the study, and all subjects provided written, informed consent before participating in the study. Participants were supplied with written information that included the study purpose, methods,
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Clinical Therapeutics possible risks, and details of the honorarium that was paid to the participants.
Inclusion and Exclusion Criteria Screening occurred during a 3-week period before treatment administration. Healthy male and female Korean volunteers aged 19 to 40 years and within 20% of ideal body weight were recruited. Health was judged by physicians based on the results of a detailed physical examination including vital sign measurements, 12-lead ECG, and laboratory tests including blood chemistry, hematology, and urinalysis based on the inclusion and exclusion criteria. Participants were not enrolled if they had any of the following: a history or evidence of a hepatic, renal, gastrointestinal, or hematologic abnormality; prolonged prothrombin time (PT), activated partial thromboplastin time (aPTT), or bleeding time; hepatitis B, hepatitis C, or HIV infection revealed on laboratory testing; any other acute or chronic disease; a history of hypersensitivity to AT-III; a history of clinically significant allergic disease; a history of medical condition to influence drug (AT-III) absorption, distribution, metabolism, and elimination (cardiovascular, respiratory, renal, endocrine, hematologic, CNS, psychiatric disease, surgery, or malignant tumor); a history of or current hemorrhagic disease; hemolytic anemia or anemia due to blood loss (hemoglobin o14 g/dL and/or hematocrit o42%); a history of alcohol or drug abuse; repeated medication within 1 month from the day medicated through participation in a previous clinical trial; smoking habit (410 cigarettes daily); consumption of 45 glasses daily of beverages containing xanthine derivatives; or use of any medication having the potential to affect the study results (ie, estrogens, L-asparaginase, heparin) within 14 days of the start of the study.4
Study Procedures Study Drug Administration Subjects were randomly assigned to receive either the test* or the reference† formulation at the beginning of the study in accordance with randomization conducted using SAS version 9.13 (SAS Institute Inc, Cary, North Carolina). Subjects were admitted to the clinical trial *Antithrombin III injection containing 500 U per vial (batch no. CTG08001) (SK Chemicals Inc, Gyeonggi-do, Korea). † Antithrombin III injection containing 500 U per vial (batch no: 270B8159) (Green Cross Corporation, Gyeonggi-do, Korea).
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center on the evening before the day of drug administration. No food intake was allowed for at least 10 hours before dosing until 4 hours after dosing. The next morning, participants were given a single intravenous dose of either the test or the reference formulation, 100 IU/kg IV, under fasting conditions and under the supervision of physicians. Each drug was given over 20 minutes, with 100 IU/kg AT-III dissolved in normal saline using an infusion pump. After a washout period of 4 weeks, patients were switched to the alternate formulation. The administered dose was determined based on the results of the previous AT-III pharmacokinetic studies.17,18 The authors chose the dose of AT-III to achieve 200% of the AT-III activity from 100% of normal using the following equation: AT-III amount required (U) = [(Desired AT-III level – Baseline AT-III level) Weight (kg)]/1.4].20,21 The intake of beverages containing xanthine derivatives or alcohol, extreme physical activity, and smoking were not allowed over the course of the study. The subjects were under continuous medical supervision. Physical examinations, including vital sign measurements (blood pressure, heart rate, and body temperature), 12-lead ECG, and laboratory tests including chemistry, hematology, and urinalysis were conducted before drug administration and at the end of each study period. Vital signs were measured in each subject by a nurse after the subject had rested for at least 5 minutes, with the arm supported at heart level. All laboratory tests were performed at an accredited central laboratory (Department of Laboratory Medicine, Anam Hospital, Korea University College of Medicine, Seoul, Korea).
Blood Sampling Ten-milliliter blood samples were collected into an indwelling catheter placed into the forearm vein. Blood samples were collected at –2, –1, and immediately before study drug administration (0 hour) and then at 10, 20, 30, and 45 minutes and at 1, 2, 4, 6, 8, and 12 hours after the start of drug administration through the indwelling catheter kept by normal saline. Blood samples were also collected at 24, 48, 72, 96, 120, 168, and 216 hours after the start of drug administration by direct venipuncture. Blood samples were collected into a plain tube (for pharmacokinetic analysis) and into citrated tubes (for pharmacodynamic analysis) (Vacutainer, Becton, Dickinson, and
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K.-A. Kim et al. Company, Franklin Lakes, New Jersey). Tubes for pharmacokinetic analyses were centrifuged for the isolation of plasma at 41C in a refrigerated benchtop centrifuge at 1500g for 15 minutes, and tubes for pharmacodynamic analyses were sent to the central laboratory at Anam Hospital for the measurements of PT and aPTT. Separated samples were stored at –701C until assayed. After a 4-week washout period, assessments were repeated in the same manner in the second period to complete the crossover design.
The area under the time-versus-effect curve from time 0 to the last time measured (AUEClast) was calculated using the linear trapezoidal rule.
Tolerability Assessment Adverse events were assessed throughout each study period based on direct questioning and spontaneous reports, and were recorded regardless of their suspected relationship to the study drugs.
Statistical Analysis Assessments of AT-III Activity and Antigen Concentrations AT-III activity was measured by a chromogenic method using a commercial AT-III activity kit (Berichrome AT-III, Siemens Healthcare Diagnostics, Inc, Tarrytown, New York) with a Sysmex CA-540 Coagulation Analyzer (Siemens Healthcare Diagnostics). AT-III antigen (Ag) was measured using a turbidity immunoassay method with a Hitachi 7600110 Clinical Chemistry Analyzer (Hitachi HighTechnologies Corporation, Tokyo, Japan).
Assessments of Pharmacokinetic and Pharmacodynamic Properties A noncompartmental method was used to determine the pharmacokinetic and pharmacodynamic properties of AT-III using WinNonlin version 5.21 (Pharsight Corporation, Mountain View, California).22 For pharmacokinetic analysis, Cmax and Tmax were obtained using direct inspection of individual plasma concentration–time data. AUClast was calculated using the linear trapezoidal rule. AUC0–1 was calculated as AUClast þ Ct/ke, where Ct is the last plasma concentration measured and ke is the elimination rate constant, determined by linear regression analysis of the linear portion of the log plasma concentration–time curve. The t½ values were calculated as ln2/ke, and the clearance (CL) of AT-III was calculated as Dose/ AUC0–1. Because AT-III is an endogenous compound, data corrected from baseline values were analyzed. Corrected values were calculated by subtracting the baseline values from the values obtained at each time point after the start of infusion. We used the means of the values measured at –2, –1, and 0 hours before treatment as the baseline values. For pharmacodynamic analysis, the peak effect (Emax) and time to Emax (Tmax) were obtained by direct inspection of individual effect-versus-time data.
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The sample size for this study was estimated using a power calculation conducted on the basis of the data derived from previous pharmacokinetic studies.18 At least 14 participants were required to detect a 20% difference in the pharmacokinetic parameters (Cmax and AUC) between the test and reference formulations with 80% statistical power at a 0.05 level of significance.23 To determine whether the 2 AT-III formulations exhibited pharmacokinetic equivalence for the respective AT-III activity and Ag, we compared individual Cmax and AUC values and their ratios (test/reference) using log-transformed data; means and 90% CIs were analyzed using parametric variance analysis. Two-way ANOVA was used to assess the effects of formulation, period, and sequence on the pharmacokinetic parameters as fixed effects and of subjects nested within the sequence as a random effect. Other main effects were tested at the 5% level of significance against the residual error (mean square error) from the ANOVA model as the error term. Due to the nature of normaltheory CIs, this was equivalent to carrying out two 1sided t tests at the 5% level of significance.23,24 To satisfy the requirement for bioequivalence of AT-III in Korea, the geometric mean ratios and 90% CIs of the test formulation to the reference formulation for Cmax and AUC were to be within the range of 80% to 125% for log-transformed data. All statistical analyses were performed using SAS version 9.13 (SAS Institute Inc).
RESULTS Study Population Twenty participants who met the inclusion and exclusion criteria were enrolled in this study; mean (SD) age, height, and weight were 25.3 (2.3) years, 175.3 (4.5) cm, and 67.4 (6.3) kg, respectively. One subject withdrew consent during the first period for
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Table I. Baseline demographic and clinical characteristics of the healthy subjects in this study of the bioequivalence of 2 intravenous formulations of antithrombin III (N ¼ 20). Characteristic Age, y Mean (SD) Range %CV Height, cm Mean (SD) Range %CV Weight, kg Mean (SD) Range %CV
RT
TR
All Patients
25.3 (2.1) 23–30 8.1
25.0 (2.7) 22–31 10.7
25.3 (2.3) 22–31 8.9
177.9 (3.8) 173–184 2.2
172.6 (3.6) 167–181 2.1
175.3 (4.5) 167–184 2.6
69.8 (4.5) 62.7–75.0 6.4
64.9 (7.0) 55.3–73.6 10.8
67.4 (6.3) 55.3–75.0 9.3
RT ¼ reference–test sequence; TR ¼ test–reference sequence.
(0.12) L/h with the test and reference formulations. Summarized pharmacokinetic properties of AT-III activity with both formulations are presented in Table II.
AT-III Antigen The mean plasma concentration–time profiles of AT-III Ag after single-dose administration the 2 ATIII formulations are shown in Figure 2. The baseline AT-III Ag levels before treatment were 24.64 (1.71) mg/dL (range, 21.6–27.8 mg/dL) with the test formulation and 25.05 (1.50) mg/dL (range, 21.1–27.0 mg/ dL) with the reference formulation (P ¼ 0.284). Similar to the AT-III activity, all pharmacokinetic properties of AT-III Ag were comparable between the 2 formulations. The mean (SD) Cmax values with the test and reference formulations were 62.58 (5.66) and 58.63 (5.27) mg/dL, respectively; Tmax values were 0.66 and 0.64 hours. The mean (SD) AUC0–1 values were 3639.80 (726.01) mg/dL h with the test formulation and 3340.00 (428.46) mg/dL h with the reference formulation. CL values of AT-III Ag were 1.91 (0.37) and 2.07 (0.33) L/h and for the test and reference
personal reasons. Therefore, 19 subjects completed both treatment periods and were included in the pharmacokinetic and pharmacodynamic analyses. The participants’ demographic characteristics are summarized in Table I.
350 Reference Test
300
The mean plasma concentration–time AT-III activity profiles of the 2 AT-III formulations after the administration of a single intravenous dose of 100 IU/ kg of each are shown in Figure 1. The baseline activities before treatment were 93.6% (7.4) (range, 77.4%–106.3%) with the test formulation and 95.6% (6.6) (range, 82.4%–107.2%) with the reference (P ¼ 0.150). All pharmacokinetic properties of AT-III activity between the 2 formulations were comparable. The mean (SD) Cmax values of the test and reference formulations were 279.24% (35.92) and 249.75% (31.96), respectively; Tmax values were 0.75 and 0.79 hours. The mean (SD) AUC0–1 values were 17,526.38 (3150.81) and 15,957.67 (3189.21) % h with the test and reference formulation (P ¼ NS). CL values of AT-III activity were 0.40 (0.09) and 0.44
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AT-III Activity (%)
250
Pharmacokinetic Properties AT-III
200 150 100 50 0 0
24
48
72
96 120 144 168 192 216 Time (h)
Figure 1. Plasma concentration profiles of 2 intravenous formulations of antithrombin (AT)-III 100 IU/kg after single-dose administration in healthy Korean subjects (n ¼ 19).
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Table II. Pharmacokinetic properties* of 2 intravenous formulations of antithrombin III 100 IU/kg after single-dose administration in healthy Korean subjects (n ¼ 19). Parameter Baseline activity level, % ke, h–1 t½, h Tmax, h Cmax, % AUClast, % h AUC0–1, % h Cmax/dose, % AUC0–1/dose, % h CL, L/h
Test Formulation, Mean (SD) 93.6 0.008 92.80 0.75 279.24 14,364.1 17,526.38 0.042 2.627 0.40
Reference Formulation, Mean (SD)
(7.4) (0.001) (15.14) (0.49) (35.92) (2325.25) (3150.81) (0.007) (0.526) (0.09)
95.6 0.008 93.46 0.79 249.75 12,962.95 15,957.67 0.037 2.408 0.44
(6.6) (0.004) (29.97) (0.57) (31.96) (1897.52) (3189.21) (0.005) (0.606) (0.12)
Geometric Mean Ratio 0.978 0.964 1.039 0.991 1.118 1.106 1.104 1.118 1.104 0.906
CL ¼ clearance; ke ¼ elimination rate constant. *Corrected from baseline activity.
formulations. Summarized pharmacokinetic parameters of AT-III Ag with both formulations are presented in Table III. 80
Pharmacodynamic Properties of AT-III
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Reference Test
60 AT-III Ag (mg/dL)
For the pharmacokinetic analysis of AT-III, we assessed PT and aPTT as previously reported.1,2 The baseline PT values before treatment were 11.18 (0.50) seconds with the test formulation and 11.02 (0.53) seconds and with the reference formulation (P = 0.162), and the baseline aPTT values before treatment were 26.85 (1.99) seconds with the test formulation and 27.04 (2.52) seconds with the reference formulation (P = 0.210) (Figure 3). The mean (SD) Emax values of PT of the test and reference formulations were 11.59 (0.53) and 11.65 (0.51) seconds, respectively; Tmax was 6 hours with both. The mean (SD) AUEC values of PT were 2363.95 (106.70) and 2372.61 (86.69) sec h with the test and reference formulations. In the case of aPTT, Tmax for both was 1 hour, and the mean (SD) Emax values of aPTT of the test and reference formulations were 29.40 (2.25) and 29.92 (2.58) seconds, respectively. The mean (SD) AUEC values of aPTT were 5608.88 (407.08) and 5666.21 (461.59) sec h with the test and reference formulations. Summarized pharmacodynamic parameters for both formulations are presented in Table IV.
40
20
0 0
24
48
72
96 120 144 168 192 216 Time (h)
Figure 2. Plasma concentration profiles of antithrombin (AT)-III antigen (Ag) after single-dose administration of 2 intravenous formulations of antithrombin III 100 IU/kg in healthy Korean subjects (n ¼ 19).
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Table III. Pharmacokinetic properties* of antithrombin III antigen after single-dose administration 2 intravenous formulations of antithrombin III 100 IU/kg in healthy Korean subjects (n ¼ 19). Parameter
Test Formulation, Mean (SD)
Baseline Ag level, mg/dL ke, h–1 t½, h Tmax, h Cmax, mg/dL AUClast, mg/dL h AUC0–1, mg/dL h Cmax/dose, mg/dL AUC0–1/dose, mg/dL h CL, L/h
24.64 0.009 84.46 0.66 62.58 3051.94 3639.80 0.009 0.542 1.91
Reference Formulation, Mean (SD)
(1.71) (0.003) (23.14) (0.39) (5.66) (401.87) (726.01) (0.001) (0.098) (0.37)
25.05 0.009 84.00 0.64 58.63 2805.08 3340.00 0.009 0.498 2.07
(1.50) (0.002) (14.90) (0.41) (5.27) (272.38) (428.46) (0.001) (0.076) (0.33)
T/R Ratio 0.983 0.988 1.013 1.045 1.088 1.109 1.114 1.088 1.111 0.900
CL ¼ clearance; ke ¼ elimination rate constant. *Corrected from baseline activity.
Pharmacokinetic Equivalence The equivalence statistics of the pharmacokinetic parameters of AT-III, including the Cmax, AUClast, and AUC0–1, for the 2 formulations are summarized in Table V. Considering that AT-III is an endogenous compound and that there are baseline AT-III activities and Ag levels in the body, we assessed the pharmacokinetic equivalence of these 2 formulations. The mean log-transformed ratios of these parameters and their 90% CIs for AT-III activity and Ag levels all fell
within the predefined pharmacokinetic equivalence range of 80% to 125%.
Tolerability No serious or unexpected adverse events were observed in this study. Eight subjects experienced 8 adverse events during the study periods (3 and 5 with the test and reference formulations, respectively). Among them, 2 adverse events were suspected to be unrelated to the formulation (1 case of erythema and 1
12.0
32 Reference Test
Reference Test 30
PT (sec)
aPTT (sec)
11.5
11.0
28
26
10.5 Time (h)
72 96 12 0 14 4 16 8 19 2 21 6
24 48
18
12
6
0
72 96 12 0 14 4 16 8 19 2 21 6
24 48
18
12
6
0
24 Time (h)
Figure 3. Change of (A) PT and (B) aPTT after single-dose administration of 2 intravenous formulations of antithrombin III 100 IU/kg in healthy Korean subjects (n ¼ 19).
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Table IV. Pharmacodynamic properties of 2 intravenous formulations of antithrombin III 100 IU/kg after single-dose administration in healthy Korean subjects (n ¼ 19). Parameter PT Basal, sec Tmax, h* Emax, sec AUEC, sec h aPTT Basal, sec Tmax, h* Emax, sec AUEC, sec h
Test Formulation, Mean (SD)
Reference Formulation, Mean (SD)
Geometric Mean Ratio
11.18 6 11.59 2363.95
(0.50) (0.17–216) (0.53) (106.70)
11.02 6 11.65 2372.61
(0.53) (0.17–120) (0.51) (86.69)
1.014 — 0.995 0.996
26.85 1 29.40 5608.88
(1.99) (0.33–216) (2.25) (407.08)
27.04 1 29.92 5666.21
(2.52) (0.33–8) (2.58) (461.59)
0.995 — 0.984 0.991
AUEC ¼ area under the peak effect curve; Emax ¼ peak effect. *Median (range).
case of sore throat), while 6 adverse events were considered to be related to the formulation tested (3 per formulation). These adverse events included 2 cases of fatigue, 2 cases of headache, and 1 case each of itching sensation, and ecchymosis at the injection site.
DISCUSSION This study revealed the pharmacokinetic characteristics of AT-III in detail in healthy Korean subjects, and that a newly developed AT-III formulation showed similar pharmacokinetic and pharmacodynamic characteristics to those of the currently used AT-III formulation. We demonstrated that the 2 AT-III formulations met the regulatory requirements for pharmacokinetic equivalence. The mean ratios and the 90% CIs for the log-transformed pharmacokinetic parameters of AT-III, including the Cmax and AUC, met the criteria for pharmacokinetic equivalence with respect to AT-III activity and Ag levels. In this study, the average activity values of the Cmax for the test and reference formulations after correction from baseline values were 279.24% and 249.75%, respectively, and those of AUClast for the test and reference formulations were 14,364.10 and 12,962.95 % h. Marzo et al18 reported that the administration of 100 IU/kg of AT-III achieved a Cmax of 262.03% and an AUClast of 10,452 % h in
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12 healthy subjects.18 This finding suggests a 24% to 38% greater AUC of AT-III in our study despite the comparable Cmax after the identical dosing regimen. Interestingly, the study done by Marzo et al showed that the elimination half-life of AT-III activity was 116.9 hours, but our study showed a half-life of 93 hours, which is a 23-hour difference. Based on a literature review, the results of our study and those of
Table V. Comparison of point estimates for logtransformed pharmacokinetic parameters of 2 intravenous formulations of antithrombin III 100 IU/kg after singledose administration in healthy Korean subjects (n ¼ 19). Parameter Activity Cmax AUClast AUC0–1 Antigen Cmax AUClast AUC0–1
Point Estimate
90% CI
1.11494 1.11305 1.11527
1.08994–1.14053 1.05435–1.17503 1.03754–1.19889
1.08802 1.10905 1.11460
1.06258–1.11405 1.05804–1.16242 1.02058–1.21726
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Clinical Therapeutics the Marzo group demonstrated an almost 2-fold longer half-life than other previous findings.16,25,26 Unfortunately, we could not determine which factor determined the half-life of AT-III. However, the presence of heparin decreased the half-life of AT-III from 56.8 to 33.7 hours.16 This means that endogenous compounds, such as heparin, might modulate the levels of AT-III. However, the notion that heparin modulates AT-III levels has a limitation to explain the pharmacokinetic discrepancies with other populations. We thus speculate the plausible mechanisms as follows. First, considering that thrombin is irreversibly bound by AT-III and forms thrombin–AT-III complex,1,2 one may speculate that the extent of thrombin–AT-III complex formation may influence the life of AT-III. Second, study design might be a factor to cause the variability of AT-III half-life. Because AT-III is an endogenous compound, the time interval of assessment of AT-III could make difference in pharmacokinetics. For instance, we collected the blood samples until 216 hours after the start of drug administration, but a previous study revealing the detailed AT-III pharmacokinetics collected samples for 168 hours after treatment.18 Additionally, it has been reported that serum albumin levels anticipate AT-III activity in patients with disseminated intravascular coagulation.26 The test and reference formulations showed similar baseline activity (93.6% vs 95.6%) and Ag (24.64 vs 25.05 mg/dL) values in this study. Because AT-III is an endogenous compound, the difference in baseline activity or Ag can influence the overall exposure of AT-III. However, the data we analyzed here were corrected from baseline values in order to overcome the difference in baseline activity or Ag, so the basal difference would influence the results of the present study. The Cmax and AUC of a test formulation should lie within the 20% deviation of the reference formulation for the acceptance of bioequivalence. The 90% CIs of the log-transformed ratio of Cmax and AUC for AT-III activity of the 2 AT-III formulations (test/ reference) were within the stipulated pharmacokinetic equivalence range of 80% to 125%.22 Furthermore, those of the AT-III Ag levels were also within the stipulated range. The results of this study indicate that the test AT-III formulation exhibits a pharmacokinetic profile, with regard to the extent and rate of absorption, comparable to that of the reference formulation.
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In this study, we evaluated the effects of 2 AT-III formulations on the PT and aPTT prolongation as a pharmacodynamic assessment. AT-III inhibits coagulation serine proteases, factor IX, factor XI, and factor IIa (thrombin).1,2 Therefore, the administration of AT-III might prolong aPTT and PT through the inhibition of the coagulation pathway. In this study, PT was prolonged maximally to 0.41 seconds with the test formulation and 0.63 seconds with the reference formulation at about 6 hours after treatment. In addition, aPTT also showed a similar maximal prolongation at 1 hour after treatment (2.55 and 2.88 seconds with the test and reference formulations, respectively). However, there were no statistical differences in PT and aPTT prolongation between the 2 formulations. This study had some potential limitations. First, it was conducted using an open-label design, and thus there may have been limitations in objectively addressing the effectiveness and safety profiles of the formulations tested. Second, we recruited healthy subjects into this study; because the pharmacokinetics of AT-III may differ in patients, there remains a theoretical uncertainty about generalizing the results to a patient group.
CONCLUSION The results of this study reveal that the test and reference formulations of AT-III tested met the regulatory criteria for bioequivalence in these healthy Korean volunteers.
ACKNOWLEDGMENTS The study was designed, conducted, and analyzed by the Department of Clinical Pharmacology and Toxicology, Anam Hospital, Korea University College of Medicine, Seoul, Korea. Drs. K.-A. Kim and Park contributed to the literature search, figures, study design, writing, data collection, interpretation, and analysis. Ms. Lim and Kim contributed to the writing, interpretation, and analysis.
CONFLICTS OF INTEREST This study was sponsored by SK Chemicals Inc, Gyeonggi-do, Korea. The authors have no other conflicts of interest with regard to the content of this article.
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Address correspondence to: Ji-Young Park, MD, PhD, Department of Clinical Pharmacology and Toxicology, Anam Hospital, Korea University College of Medicine, 126-1, 5-Ga, Anam-dong, Sungbuk-Gu, Seoul 136705, Korea. E-mail: [email protected]
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