Abstract
AIMS
To investigate the influence of age and gender on the intravenous pharmacokinetics and pharmacodynamics of the plasminogen activator, lanoteplase.
METHODS
Forty healthy subjects (10 each of young males, elderly males, young females and elderly females) received a single bolus 10 kU kg−1 intravenous dose of lanoteplase. Plasma from blood serially collected for 24 h post-dose was analyzed for lanoteplase (antigen), fibrinogen, plasminogen and α2-antiplasmin concentrations, plasma plasminogen activation activity (PPAA) and rapid plasminogen activator inhibitor (PAI-1).
RESULTS
Lanoteplase mean total systemic clearance (CLt) values ranged from 1.9 to 2.8 l h−1 and mean steady-state volume of distribution (Vss) values ranged from 12.3 to 15.6 l. Age-by-gender interactions were observed for lanoteplase CLt (P = 0.04), but no differences were observed for Vss or elimination half-life. Elderly females had a 27% lower mean CLt than young females (95% CI for the difference 0.17, 1.27 l h−1) and 32% lower CLt than elderly males (95% CI for the difference 0.15, 1.65 l h−1). PPAA AUC/dose values did not show an age-by-gender interaction. Haemostasis parameters indicated only a slight degree of systemic plasminogen activation.
CONCLUSIONS
Elderly females had a lower mean lanoteplase CLt than elderly males and young females. However, no difference was observed between young and elderly females for the AUC/dose of PPAA. In addition, there were no age-related or gender-related differences observed in the other pharmacodynamic parameters measured.
Keywords: acute myocardial infarction, age and gender effects on pharmacodynamics, age and gender effects on pharmacokinetics, fibrinolytic therapy, plasminogen activation
WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT
The pharmacokinetics and pharmacodynamics of the plasminogen activator lanoteplase have not been extensively characterized in specific populations and the need for dose adjustment on the basis of age and/or gender has not been established.
WHAT THIS STUDY ADDS
This paper is the first report of lanoteplase administered to healthy subjects in order to address its clinical use in these important specific populations. The results support the use of the same doses of lanoteplase in males and females and young and elderly patients.
Introduction
Thrombolytic agents lyze fibrin-based clots by activating plasminogen to the active enzyme plasmin which acts by breaking down fibrin in the clot to soluble products [1]. In the case of the early stages of acute myocardial infarction, the goal of therapy with thrombolytic agents is the restoration of blood flow through the coronary artery occluded by the thrombus. The rationale for this approach is the reduction of myocardial necrosis and greater preservation of myocardial function with the objective of reducing mortality and associated morbidity [2].
The utility of thrombolytic therapy in acute myocardial infarction was originally demonstrated with streptokinase infusions. Streptokinase clearly improved mortality rates over conventional anticoagulant therapy (aspirin, heparin, etc.) [3]. The benefits of streptokinase in reducing mortality, however, are tempered by its immunogenicity and attendant allergic reactions [4], and its lack of fibrin specificity [5]. The latter characteristic in particular led to the development of more fibrin-specific compounds, the most notable of which is tissue plasminogen activator (t-PA), a serine protease produced by the vascular endothelium [6]. Although t-PA reduces mortality associated with acute myocardial infarction, its application is limited by a cumbersome, three step, 90 min intravenous infusion regime [7]. This has led to the development of a rationally-designed mutant of t-PA, lanoteplase [also known as BMS-200980, ΔFE1X PA, SUN9216 or nPA (novel plasminogen activator)], which can be administered as a convenient weight-adjusted bolus intravenous dose [8]. The modifications to t-PA to produce lanoteplase included deletion of the fibronectin finger-like and EGF-like domains and mutating Asn-117 to Gln-117. These changes conferred a prolonged in vivo half-life without loss of activity when compared with t-PA at therapeutic concentrations [9]. Lanoteplase therefore has potential to afford some advantages over existing pharmacological interventions for thrombolytic therapy in acute myocardial infarction.
As part of the clinical development of lanoteplase, as with any new medicinal product, it is paramount to establish whether different populations can safely and effectively use the drug at the recommended dose or, if differences are observed, what other doses or dosing regimens may be viable as alternatives. Body weight, blood lipid concentrations, insulin concentrations, age and gender have been reported to play a substantial role in modulating individual responses to fibrinolytic therapy [10]. In order to understand better the influence of age and gender on the efficacy of lanoteplase, this study was designed to evaluate its pharmacokinetics, pharmacodynamics and its effects on haemostasis in young and elderly healthy subjects. To avoid confounding the interpretation of the results of this study by simultaneously exploring the impact of multiple variables on lanoteplase pharmacokinetics, pharmacodynamics and haemostasis, the effects of extremes of body weight, blood lipids or insulin were not examined in this study.
Methods
Study population
This study was of an open-label parallel group, single dose study design. The subject population consisted of non-smoking young and elderly men and women. Young subjects were required to be 18–40 years of age and elderly subjects to be between 65–80 years of age. The subjects were in good health as judged by physical examination and history, and had the ability to provide informed consent. Young and elderly subjects were required to be within 10% and 20%, respectively, of their ideal body weight for height and frame as determined from the Metropolitan Life Insurance Company tables [11]. Female subjects were required to be at least 1 year post menopausal, surgically sterile, or were to use an adequate barrier method of contraception. All women of childbearing potential were to have a negative serum or urine pregnancy test within 72 h prior to the dosing. Subjects with a history of haemostatic disorder or who had evidence suggestive of a clinically significant haemostatic disturbance were excluded. Subjects were also excluded if they had evidence of renal impairment. Summary demographic characteristics of the subjects enrolled in the study are shown in Table 1.
Table 1.
Demographic characteristics of the study subjects
| Young males (n = 10) | Young females (n = 10) | Elderly males (n = 10) | Elderly females (n = 10) | |
|---|---|---|---|---|
| Age (years) | ||||
| Mean (SD) | 27 (5.8) | 29 (6.7) | 71 (6.2) | 69 (2.7) |
| Range | 20–37 | 18–37 | 65–79 | 65–74 |
| Weight (kg) | ||||
| Mean (SD) | 79.2 (6.1) | 64.9 (6.6) | 82.3 (13.7) | 67.6 (9.9) |
| Range | 70.0–87.0 | 51.0–71.0 | 63.0–101.0 | 49.9–82.0 |
| Height (cm) | ||||
| Mean (SD) | 179.7 (5.6) | 167.8 (4.3) | 174.8 (5.0) | 164.1 (7.0) |
| Range | 173.0–188.0 | 163.0–175.0 | 168.0–182.9 | 155.0–175.0 |
| Race (n) | ||||
| White | 6 | 9 | 10 | 10 |
| Black | 3 | 1 | ||
| Hispanic | 1 |
A total of 40 subjects were enrolled in this study: 10 young males, 10 elderly males, 10 young females, and 10 elderly females. The protocol and informed consent were approved by an Institutional Review Board (Consultants Review Committee, Austin, TX, USA). All subjects gave informed written consent prior to participation in the study.
Dose selection
Prior to this study, lanoteplase had only been administered to patients with acute myocardial infarction at doses of 15 to 120 kU kg−1 with linear pharmacokinetics for lanoteplase antigen systemic exposures over this range [12]. At the 15 kU kg−1 dose there were a small number of cases of bleeding-related adverse events that were considered possibly related to lanoteplase. The linearity of lanoteplase antigen pharmacokinetics indicates that conclusions at a lower dose would be applicable to higher doses so a dose of 10 kU kg−1 was selected to allow for a margin of safety for the healthy subjects in this study.
Formulation
Lanoteplase was produced by cell culture fermentation using a Chinese Hamster Ovary cell line by Suntory Ltd, Osaka, Japan. Lanoteplase was supplied as a sterile, lyophilized powder with each vial containing 14 mg (6 million units). The powder was reconstituted with isotonic saline immediately prior to administration.
Study design
All subjects were admitted to the study site by 18.00 h the day prior to lanoteplase administration in order to have an abbreviated physical examination, serum and urine pregnancy tests for women of childbearing potential, and for drugs of abuse screening. Subjects remained in the study unit for a total of 2 days and 1 night. On the day of dosing, the subjects received a single intravenous bolus dose of 10 kU kg−1 lanoteplase around 08.00 h following an overnight fast (circa 10 h). Blood samples for the pharmacokinetic, pharmacodynamic and haemostatic measurements were collected pre-dose and at 3, 10, 15, 30, 45 min, 1, 1.5, 2, 4, 6, 8, 10, 12 and 24 h post dose. Subjects fasted for an additional 2 h period following drug administration. Vital signs were recorded periodically throughout the study. Adverse events were spontaneously reported or elicited during open-ended questioning, examination or evaluation of a subject. In order to prevent reporting bias, subjects were not questioned regarding the specific occurrence of one or more adverse events. Other than lanoteplase, subjects did not receive any medication for the duration of the study. Before subjects were discharged from the study a full physical examination was carried out (including a 12-lead electrocardiogram) and an additional blood sample for a standard clinical laboratory panel was collected.
Blood collection and sample analysis
Blood samples for lanoteplase concentration determination were collected in evacuated 3 ml collection tubes containing sodium citrate and PPACK (d-phenylalanyl-1-prolyl-1-arginyl chloromethyl ketone, a protease inhibitor). Within 20 min of collection, plasma was collected following centrifugation and the samples were frozen at −70°C until analysis. The plasma concentrations of lanoteplase were quantitated by a validated chemiluminescent assay [13]. This method employs a monoclonal anti-lanoteplase antibody (clone no. H3Wt/1.17.20) adsorbed onto a microtitre plate to capture lanoteplase in plasma samples. The captured lanoteplase was then detected using a biotinylated rabbit anti-lanoteplase polyclonal antibody. Since this assay quantitates the captured lanoteplase, the plasma concentrations of lanoteplase are hereafter referred to as ‘lanoteplase antigen’ concentrations. The between-run, between-plate and within-plate coefficients of variation (CVs) for quality control samples were ≤8.3%. The standard curve for the assay ranged from 3.0 to 200 ng ml−1, defining the lower limit of quantitation (LLQ) and the upper limit of quantitation (ULQ) of the assay, respectively. The coefficients of correlation (r2) for the standard curves for all runs were ≥0.990.
Blood samples for plasma plasminogen activation activity (PPAA) determination were collected in evacuated 3 ml collection tubes containing sodium citrate. Within 20 min of collection, plasma was collected following centrifugation and frozen at −70°C until analysis. The activity of lanoteplase in plasma was quantified as PPAA by a validated chromogenic assay [14]. The assay is based on lanoteplase in the sample activating plasminogen to plasmin which subsequently cleaves plasmin to yield a yellow colour. The intensity of the yellow colour was measured at 405 nm to quantitate the activity of lanoteplase. The between-run, between-plate and within-plate coefficients of variation (CVs) for quality control samples were ≤9.9%. The standard curve for the assay ranged from 4.5 to 108 IU ml−1 (1 IU = 2.33 ng), defining the lower limit of quantitation (LLQ) and the upper limit of quantitation (ULQ) of the assay, respectively. The coefficients of correlation (r2) for the standard curves for all runs were ≥0.985.
Blood samples for the determination of plasma concentrations of fibrinogen, plasminogen, α2-antiplasmin and rapid plasminogen activator inhibitor (PAI-1) were collected in evacuated 10 ml collection tubes containing sodium citrate and PPACK. Within 20 min of collection, plasma was collected following centrifugation, aliquoted into separate storage tubes for each assay (0.5–1.0 ml) and frozen at −70°C until analysis. Samples were analyzed by validated assays by Colorado Coagulation Consultants (Aurora, CO, USA).
Pharmacokinetic analysis
The plasma lanoteplase antigen concentration vs. time data and the plasma lanoteplase activity (PPAA) vs. time data were analyzed by non-compartmental pharmacokinetic methods [15, 16] by a validated in-house application (PKMENU) using the Statistical Analysis System (SAS®) software package (v6.12, SAS Institute, Incorporated, Cary, NC, USA). The primary pharmacokinetic parameters for statistical analysis were: area under the plasma concentration vs. time curve extrapolated to infinity beyond the last measured concentration (AUC), total plasma clearance (CLt) for lanoteplase, or plasma AUC/dose (AUC/dose) for PPAA only, steady-state volume of distribution (Vss) and the terminal elimination phase half-life. In addition to the non-compartmental analysis, plasma lanoteplase antigen concentration vs. time data were also fitted to standard intravenous administration compartmental models with various weighting schemes using KineticaΣ (v2.0.1, MicroPharm International, Créteil, France), a commercial pharmacokinetic modelling package. The model selection was performed in the basis of Akaike information criterion [17]. For presentation of mean and SD plasma concentration or activity profiles, values less than the lower limit of quantitation for the assay were treated as zero.
Statistical power and data analysis
The results from a phase I lanoteplase pharmacokinetic study showed a 9–20% between subject CV for lanoteplase antigen AUC following single bolus 0.5–2.0 mg doses of lanoteplase. Assuming a between-subject CV of 20% in AUC for lanoteplase antigen in this study and no significant age-by-gender interaction, the use of 10 subjects per age-gender group would have at least 90% power to declare that the female : male or the young : elderly ratios of the mean AUCs were between 0.8 and 1.25 when the true population mean ratios are 1.00.
An analysis of variance (anova) was performed on each of the non-compartmental pharmacokinetic parameters. The analysis included factors for age (young vs. elderly), gender and age-by-gender interaction. In the analysis of each variable, the absence of an age-by-gender interaction would imply that the effect of age (or gender) was the same in each gender (or age group), so that the estimates of age or gender effects could be pooled across gender or ages. A priori, AUC values were log-transformed. Point and interval estimates of the means and mean differences of AUC were exponentiated to express the results as geometric means and ratios of geometric means on the original scale of measurement. All tests of statistical hypotheses were carried out at the 5% significance level and all interval estimates were two-sided and constructed with 95% confidence. All statistical analyses were carried out using SAS/STAT® (v6.12, SAS Institute, Incorporated).
For each demographic group, the lanoteplase antigen concentrations were plotted against plasma lanoteplase activity concentrations and a correlation analysis was performed (linear least-squares regression).
Results
Plasma lanoteplase antigen concentration and PPAA vs. time data for each demographic group are shown in Figures 1 and 2, respectively.
Figure 1.
Mean (+SD) plasma lanoteplase antigen concentration vs. time profiles in healthy subjects following a single 10 kU kg−1 intravenous bolus dose of lanoteplase. The inset shows the profile over the first hour. n = 10 subjects in each demographic group, except young males, where n = 9. Young males (
); Young females (
); Elderly males (
); Elderly females (
)
Figure 2.
Mean (+ SD) plasma plasminogen activation activity (PPAA) concentration vs. time profiles in following a single bolus 10 kU kg−1 intravenous dose of lanoteplase. The inset shows the profile over the first hour. n = 10 subjects in each demographic group, except young males, where n = 9. Young males (
); Young females (
); Elderly males (
); Elderly females (
)
One subject in the young male group had no detectable concentrations of lanoteplase antigen or for PPAA concentrations. All of the samples for this subject were re-analyzed to verify this unexpected finding. The repeat analysis confirmed that lanoteplase antigen and PPAA were all below the LLQ of the assay. As a result, the number of subjects in the young male group was reduced to nine for all further analyses.
The non-compartmental pharmacokinetic parameters for plasma lanoteplase antigen are summarized by age and gender in Table 2, along with the 95% CIs for the differences of their means. The anova analysis for an age-by-gender interaction suggested that the effects of age on AUC (P = 0.029) and CLt (P = 0.036) of lanoteplase antigen depended on gender. Elderly females had a higher mean AUC and lower mean CLt than young females. Mean AUC and CLt values were very similar in elderly and young males. A slightly greater mean half-life value was observed for the elderly compared with young as pooled groups (mean difference of 1.7 h), but the difference did not reach statistical significance. In the elderly group, females had higher mean AUC and lower mean CLt values than males. There were no large difference between the groups in Vss values. In Table 2, the age and gender effects on AUC and CLt are presented by level of the other factor as well as pooled by age and gender because P values for the age-by-gender interactions for these variables were statistically significant. Results for Vss and half-life were pooled over levels of age and gender because P values for the age-by-gender interactions for these variables were not significantly different (0.527 and 0.501, respectively).
Table 2.
Mean (±SD) non-compartmental pharmacokinetic parameters for plasma lanoteplase antigen and summary statistical analysis of the parameters by age and gender following a single bolus 10 kU kg−1 intravenous dose of lanoteplase (n = 10 subjects in each demographic group, except young males, where n = 9)
| Analysis by age | |||
|---|---|---|---|
| Pharmacokinetic parameter | Elderly | Young | Difference between means (95% CI) |
| AUC (µg l−1 h)* | |||
| Males | 751 ± 241 | 797 ± 276 | 46 (−204, 296) |
| Females | 878 ± 163 | 622 ± 194 | −256 (−424, −48)† |
| Both genders | 812 ± 208 | 704 ± 248 | −108 (−256, 40) |
| CLt (l h−1) | |||
| Males | 2.8 ± 1.1 | 2.5 ± 0.7 | −0.3 (−1.2, 0.5) |
| Females | 1.9 ± 0.4 | 2.6 ± 0.7 | 0.7 (0.2, 1.3)† |
| Both genders | 2.4 ± 0.9 | 2.6 ± 0.7 | 0.2 (−0.3, 0.7) |
| Vss (l) | 14.0 ± 3.4 | 13.5 ± 5.7 | 0.5 (−3.5, 2.6) |
| Terminal half-life (h) | 11.2 ± 3.7 | 9.5 ± 4.1 | −1.7 (−4.3, 0.8) |
| Analysis by gender | |||
|---|---|---|---|
| Females | Males | Difference between means (95% CI) | |
| AUC (µg l−1 h)* | |||
| Young | 622 ± 194 | 797 ± 276 | 176 (−53, 404) |
| Elderly | 878 ± 163 | 751 ± 241 | −127 (−320, 60) |
| Both genders | 739 ± 215 | 774 ± 252 | 35 (−117, 187) |
| CLt (l h−1) | |||
| Young | 2.6 ± 0.7 | 2.5 ± 0.7 | −0.2 (−0.8, 0.5) |
| Elderly | 1.9 ± 0.4 | 2.8 ± 1.1 | 0.9 (0.2, 1.7)† |
| Both age groups | 2.3 ± 0.7 | 2.7 ± 0.9 | 0.4 (−0.2, 0.9) |
| Vss (l) | 12.6 ± 4.8 | 14.9 ± 4.1 | 2.3 (−0.6, 5.2) |
| Terminal half-life (h) | 10.2 ± 4.3 | 10.4 ± 3.8 | 0.2 (−2.4, 2.8) |
Data are reported as geometric means.
Statistically significant difference in age-by-gender anova analysis (P < 0.05). Parameters for which a statistically significant age-by-gender interaction was detected by anova are stratified by demographic group. AUC, area under the plasma concentration vs. time curve; CLt, total systemic clearance; Vss, steady-state volume of distribution.
Plasma lanoteplase antigen vs. time data were best fitted to an open, two-compartment model with bolus input and first-order elimination from the central compartment (plasma concentration = A-αt+ B-βt, where A and B are y-intercepts, and α and β are the absolute values of the slopes describing the distribution and elimination processes, respectively). In order to best fit the data, a weighting factor of (calculated concentration)−2 was used. The mean model-derived pharmacokinetic parameters for lanoteplase antigen are summarized in Table 3. The model-derived pharmacokinetic parameters for AUC, CLt and Vss were similar to those obtained using non-compartmental analysis. The mean α-phase half-life (t1/2,α) values for each demographic group ranged from 27.8 to 32.4 min., and the mean β-phase half-life (t1/2,β) values ranged from 8.5 to 9.8 h.
Table 3.
Mean (±SD) model-derived pharmacokinetic parameters for plasma lanoteplase antigen and lanoteplase activity (plasma plasminogen activation activity; PPAA) by age and gender following a single bolus 10 kU kg−1 intravenous dose of lanoteplase (n = 10 subjects in each demographic group, except young males, where n = 9)
| Two-compartment model-derived pharmacokinetic parameters for plasma lanoteplase antigen | ||||||||
|---|---|---|---|---|---|---|---|---|
| Demographic group | AUC (µg l−1 h) | AUCα (% of total) | AUCβ (% of total) | t1/2,α (min) | t1/2,β (h) | CLt (l h−1) | Vc (l) | Vss (l) |
| Young males | 810 ± 275 | 60 ± 7 | 40 ± 7 | 32 ± 8 | 9.4 ± 4.9 | 2.6 ± 0. 8 | 3.1 ± 0.9 | 14 ± 7 |
| Young females | 616 ± 176 | 63 ± 8 | 37 ± 8 | 30 ± 5 | 8.7 ± 3.9 | 2.8 ± 0.6 | 3.0 ± 0.7 | 13 ± 5 |
| Elderly males | 736 ± 225 | 60 ± 7 | 40 ± 7 | 28 ± 7 | 8.5 ± 3.5 | 3.0 ± 1.0 | 3.0 ± 0.5 | 14 ± 4 |
| Elderly females | 851 ± 162 | 61 ± 6 | 39 ± 6 | 31 ± 5 | 9.8 ± 2.4 | 2.0 ± 0.5 | 2.4 ± 0.6 | 12 ± 2 |
| One-compartment model-derived pharmacokinetic parameters for PPAA | ||||||||
|---|---|---|---|---|---|---|---|---|
| AUC (µg l−1 h) | t1/2 (min) | AUC/dose (h l−1) | Vc (l) | |||||
| Young males | 421 ± 83 | 24 ± 3 | 5.7 ± 0.8 | 2.7 ± 0.5 | ||||
| Young females | 374 ± 62 | 22 ± 5 | 4.4 ± 0.7 | 2.3 ± 0.6 | ||||
| Elderly males | 341 ± 142 | 27 ± 6 | 7.2 ± 4.0 | 4.5 ± 2.3 | ||||
| Elderly females | 369 ± 87 | 28 ± 7 | 4.4 ± 1.1 | 2.9 ± 0.7 | ||||
AUC, area under the plasma concentration vs. time curve; AUC/dose, PPA AUC/dose of lanoteplase; AUCα, distribution-phase AUC; AUCβ, elimination-phase AUC; t1/2,α, distribution-phase half-life; t1/2,β, elimination-phase half-life, t1/2, half-life; CLt, total systemic clearance; Vc, central compartment volume of distribution; Vss, steady-state volume of distribution.
The non-compartmental pharmacokinetic parameters for PPAA are summarized by age and gender in Table 4, along with the 95% CIs for the differences of their means. The anova analysis for an age-by-gender interaction on the non-compartmental pharmacokinetic parameters for PPAA suggested that the effects of age on AUC and AUC/dose may depend on gender (P = 0.10 and 0.064, respectively). In Table 4, the age and gender effects on AUC and AUC/dose for PPAA are presented by level of the other factor as well as pooled by age and gender because P values for the age-by-gender interactions for these variables approached statistical significance. These parameters were also statistically different for lanoteplase as mentioned earlier. Elderly males had a lower mean AUC and higher AUC/dose for PPAA than young males, while these parameters had values that were nearly the same in young and elderly females. The Vss for PPAA had P values of 0.188 for the age-by-gender statistical analysis. The elderly age group had a higher mean Vss than the young age group. Half-life values for PPAA were similar amongst all demographic groups (P = 0.968).
Table 4.
Summary statistics for non-compartmental pharmacokinetic parameters for plasma plasminogen activation activity (PPAA) following a single bolus 10 kU kg−1 intravenous dose of lanoteplase (n = 10 subjects in each demographic group, except young males, where n = 9)
| Analysis by age | |||
|---|---|---|---|
| Pharmacokinetic parameter | Elderly | Young | Difference between means (95% CI) |
| AUC (µg l−1 h)* | |||
| Males | 334 ± 133 | 408 ± 82 | 74 (−35, 183) |
| Females | 390 ± 89 | 369 ± 64 | −21 (−105, 63) |
| Both genders | 342 ± 114 | 381 ± 74 | 39 (−24, 102) |
| AUC/dose (h l−1) | |||
| Males | 7.1 ± 3.2 | 4.8 ± 0.8 | −2.3 (−4.6, 0.0) |
| Females | 4.5 ± 1.2 | 4.4 ± 0.7 | −0.1 (−1.0, 0.8) |
| Both genders | 5.8 ± 2.7 | 4.6 ± 0.7 | −1.2 (−2.5, 0.1) |
| Vss (l) | 4.3 ± 3.5 | 2.8 ± 0.6 | −1.5 (−3.2, 0.2) |
| Terminal half-life (h) | 0.58 ± 0.29 | 0.46 ± 0.10 | −0.12 (−0.26, 0.02) |
| Analysis by gender | |||
|---|---|---|---|
| Females | Males | Difference between means (95% CI) | |
| AUC (µg l−1 h)* | |||
| Young | 369 ± 64 | 408 ± 82 | 39 (−32, 110) |
| Elderly | 390 ± 89 | 334 ± 133 | −56 (−162, 50) |
| Both genders | 371 ± 77 | 349 ± 115 | −22 (−85, 41) |
| AUC/dose (h l−1) | |||
| Young | 4.4 ± 0.7 | 4.8 ± 0.8 | 0.4 (−0.3, 1.1) |
| Elderly | 4.5 ± 1.2 | 7.1 ± 3.2 | 2.6 (0.3, 4.9) |
| Both age groups | 4.4 ± 1.0 | 6.0 ± 2.6 | 1.6 (0.3, 2.9) |
| Vss (l) | 2.9 ± 0.6 | 4.3 ± 3.6 | 1.4 (−0.3, 3.1) |
| Terminal half-life (h) | 0.52 ± 0.17 | 0.53 ± 0.28 | −0.01 (−0.14, 0.16) |
data are reported as geometric means. No statistically significant age-by-gender difference was detected by anova for PPAA, but parameters for which a significant difference was found in lanoteplase antigen pharmacokinetic parameters are stratified by demographic group. AUC, area under the plasma concentration vs. time curve; AUC/dose, PPA AUC/dose of lanoteplase; Vss, steady-state volume of distribution.
Pharmacokinetic PPAA vs. time data were best fitted to an open, one-compartment model with bolus input and first-order elimination from the central compartment. In order to best fit the data, a weighting factor of (calculated concentration)−1 was used. The mean model-derived pharmacokinetic parameters for lanoteplase PPAA are summarized in Table 3. The model-derived pharmacokinetic parameters for AUC, AUC/dose and Vss were similar to those obtained using non-compartmental analysis. The mean half-life values for the demographic groups ranged from 21.9 to 27.8 min.
Linear relationships were observed between plasma lanoteplase antigen concentrations and PPAA concentrations for all of the demographic groups. The plots and results of the regression analysis of these data are shown in Figure 3. For each demographic group, the relationships obtained between plasma lanoteplase antigen concentrations and lanoteplase activity concentrations were linear with y-intercepts close to the origin. The mean (SD) of the slopes for the regression lines of best fit was 0.86 ± 0.17 (range: 0.73 to 1.09), suggesting that activity concentrations were slightly lower than their respective drug concentrations.
Figure 3.
Linear regression analysis for lanoteplase antigen and plasminogen activation activity (PPAA) concentrations obtained in healthy subjects following of a single bolus 10 kU kg−1 intravenous dose of lanoteplase. n = 10 subjects in each demographic group, except young males, where n = 9
The percentage change from baseline (pre dose) for the haemostatic parameters measured in this study (plasma concentrations of fibrinogen, plasminogen, α2-antiplasmin and PAI-1) are shown in Figure 4. Administration of a 10 kU kg−1 intravenous bolus of lanoteplase did not appear to cause a reduction of plasma fibrinogen. At 2 h post dose, the plasma concentrations of fibrinogen were 91 ± 16%, 96 ± 22%, 101 ± 29% and 99 ± 24% of baseline in young male, young female, elderly male and elderly female subjects, respectively. Plasma concentrations of plasminogen consistently decreased by 5–10% of baseline in all demographic groups within the first 1–2 h of dosing. At 2 h after dosing, plasminogen concentrations were 94 ± 7%, 90 ± 8%, 93 ± 8% and 90 ± 6% of baseline in young male, young female, elderly male and elderly female subjects, respectively. In all demographic groups, plasma concentrations of plasminogen returned to baseline values within 8 h of dosing.
Figure 4.
Influence of a single bolus 10 kU kg−1 intravenous dose of lanoteplase in healthy subjects on homeostasis parameters (A) fibrinogen, (B) plasminogen, (C) α2-antiplasmin and (D) rapid plasminogen activator inhibitor. Data are mean ± SD n = 10 subjects in each demographic group, except young males, where n = 9. Young males (
); Young females (
); Elderly males (
); Elderly females (
)
Plasma concentrations of α2-antiplasmin decreased by about 10 to 20% of baseline values following dosing with lanoteplase. However, in contrast to the decreases in plasminogen concentrations, the maximum change in α2-antiplasmin concentrations were observed around 6 h. The mean reduction in α2-antiplasmin concentrations at 6 h were 77 ± 18%, 90 ± 10%, 84 ± 16% and 91 ± 12% (excluding one subject whose concentration was 134% of baseline) of baseline in young male, young female, elderly male and elderly female subjects, respectively. In all demographic groups, plasma concentrations of α2-antiplasmin returned to baseline values within 24 h of dosing. No obvious effects of lanoteplase on PAI-1 plasma concentrations were observed, particularly as a high variability was observed in these data.
Safety assessments were based on medical review of adverse events, vital signs, electrocardiograms, physical examinations and clinical laboratory tests. All adverse events were recorded, regardless of the apparent association with lanoteplase. The investigator's clinical judgment, in conjunction with discussions with the sponsor's medical monitor if necessary, was the basis of assigning relatedness to lanoteplase for a particular adverse event. The relationship to five adverse events [arthritis, headache, muscle ache, musculo-skeletal pain, and pallor (one occurrence for each adverse event)] was judged as being ‘unlikely to be related’ to lanoteplase administration. The remaining adverse events [ecchymosis (15 subjects, mainly at the site of blood sampling), rash (one subject), local reaction at administration site (three subjects), haematoma (one subject), swelling (one subject), numbness (one subject), and abnormal urination (two subjects)] were judged as ‘possibly’ related to lanoteplase administration.
Discussion
Lanoteplase is a rationally-designed plasminogen activator and this study represents one of the few clinical studies carried out with this class of compound in healthy subjects. The primary aim of this study was to determine if any dosage adjustment of lanoteplase was necessary based on a patient's age or gender by examining its pharmacokinetics and pharmacodynamics in both sexes and in different age groups. A limitation of this study was that subjects were excluded from this study if they had evidence of renal impairment. Therefore, the range of renal function in this study was not sufficient to establish the influence of renal function on lanoteplase antigen pharmacokinetics. Furthermore, the renal clearance of lanoteplase antigen was not measured, so differentiating between renal and non-renal clearance with the data generated in this study was not possible.
One subject in the young male group had no detectable concentrations of lanoteplase antigen or for PPAA concentrations. The reason for these observations remains unknown but the findings are consistent with the dose of lanoteplase mistakenly not being administered to a vein.
Age-by-gender interactions were observed for lanoteplase antigen in AUC and CLt values, but not for Vss and terminal half-life values. Elderly females had a 28% lower mean CLt than young females and 32% lower mean CLt than elderly males. No statistically significant differences were observed for CLt between young and elderly male subjects. Within young subjects, the mean CLt in females was only 6% higher than males. Mean CLt values for lanoteplase antigen ranged from 1.9 l h−1 in elderly females to 2.8 l h−1 in elderly males. Based on an average hepatic blood flow value of 87 l h−1[18], the relatively lower CLt of lanoteplase antigen would not be expected to be markedly influenced by differences in hepatic blood flow associated with age, or with changes in hepatic blood flow which could occur in patients with acute myocardial infarction. Since lanoteplase was dosed on a body weight-adjusted basis and there were no apparent differences between the demographic groups in Vss values, it is presently unclear why elderly females had a statistically significantly lower CLt and higher AUC values for lanoteplase antigen than the other groups.
No statistically significant age-by-gender differences for PPAA pharmacokinetic values were found. However, the mean AUC/dose value for PPAA was 1.5 to 1.6-fold higher for elderly males than the values observed in other demographic groups. In addition the 95% confidence intervals for the difference of their means suggest that AUC/dose for PPAA may be significantly higher in elderly males (Table 4). However, the between-individual variability for AUC/dose in elderly males was relatively high (CV of 45%), and the difference between elderly males and the other demographic groups was not significant when analyzed by anova. The significant differences observed in lanoteplase antigen CLt and AUC values in elderly females did not appear to have an effect on the pharmacodynamics of the compound, as assessed by similar PPAA pharmacokinetic parameter values observed in this group and in the young males and females.
The compartmental pharmacokinetic parameters for lanoteplase antigen and PPAA were comparable to those calculated non-compartmentally; suggesting that the models selected to describe lanoteplase antigen and activity were appropriate. In addition, the dose-independent pharmacokinetic parameters for lanoteplase antigen and PPAA were comparable with those observed in a study in acute myocardial infarction patients administered 15–120 kU kg−1 doses of lanoteplase [12]. Lanoteplase antigen concentration data were best described by a two-compartment open model with elimination from the central compartment, whereas a one-compartment was adequate to describe PPAA kinetics. This apparent discrepancy may be attributable to in vivo complexation of lanoteplase with plasma protein inhibitors such as PAI-1, α2-antiplasmin and/or α2-macroglobulin. Such a difference in the pharmacokinetics of antigen activity due to in vivo complexation has also been reported for t-PA [19]. Additionally, the PPAA assay measures only the ‘free’ lanoteplase activity, whereas total lanoteplase (free + bound to inhibitors) is measured by the antigen assay. This, along with difference in the LLQ of the assays (3 and 10.5 ng ml−1 for the antigen and activity assays, respectively), may have lead to the β- (terminal or elimination) phase of the activity vs. time profile not being characterized.
The estimated initial volume of distribution (Vc, see Table 3) for lanoteplase antigen approximated plasma volume (circa 3 l), while Vss was about 15 l. These values suggest that the distribution of lanoteplase, a protein with a molecular mass of circa 58 000, is probably confined to blood and well-perfused tissues. The mean Vss estimated from the PPAA data ranged from 2.6 l in young females to 5.6 l in elderly male subjects, consistent with PPAA being a central compartment measurement.
The activity half-life of lanoteplase (PPAA) was about 30 min., and was comparable among all four demographic groups. Similarly, the half-lives of the α- (distribution) and β- (elimination) phases of lanoteplase antigen were generally comparable between gender and age groups. The α-phase half-life of lanoteplase antigen was about 6-fold longer compared with t-PA [19, 20], and about 1.5-fold longer than that of TNK-tPA [21], while the β-phase half life was about 10-fold longer than t-PA and TNK-tPA. The plasma AUC for lanoteplase antigen in the β-phase represented about 50% of the total area (AUC(0,∞)), suggesting a substantial contribution to activity from the β-phase after the initial rapid decline in plasma concentrations. Prolonged concentrations of plasminogen activator (in the terminal phase) may be of clinical benefit through potential for prevention of re-occlusion [22] and this potential effect of lanoteplase clearly warrants further study.
The effects of lanoteplase on systemic haemostasis were analyzed after prevention of in vitro fibrinogenolysis in plasma by addition of PPACK to blood collection tubes. The haemostasis parameters indicated only a very slight systemic plasminogen activation by lanoteplase in these groups of healthy subjects. No gender- or age-specific trends in haemostasis parameters were observed. Circulating fibrinogen concentrations were indistinguishable from baseline (pre dose) values after administration of lanoteplase at this dose (10 kU kg−1). Based on the relatively large variability in all of the groups of the fibrinogen change from baseline data for the lanoteplase treatment and the point estimates being near unity, it appears that the effect of lanoteplase on this parameter in either direction is small and undetectable with the number of subjects in the study. However, unspecific activation of plasminogen to plasmin by lanoteplase did occur as evidenced by a slight (5–10%) but consistent fall in plasminogen following dosing in all subjects and a 10–20% decrease in α2-antiplasmin. This larger reduction in α2-antiplasmin concentrations was likely to be due to the two-fold lower molar concentration in human plasma of α2-antiplasmin than that of plasminogen (1 µm[23]vs. 2 µm[24], respectively). Similarly, a decrease in plasma concentrations of plasminogen and α2-antiplasmin has been reported for other plasminogen activators, such as t-PA and BM 06.022 [25]. No age- or gender-specific effects of lanoteplase on PAI-1 concentrations could be discerned.
The safety results of this study show that lanoteplase was generally well-tolerated by the healthy subjects after a single intravenous 10 kU kg−1 dose, regardless of age or gender. The types of adverse events reported in this study were mainly associated with the intravenous route of administration and the collection of blood samples. The most frequent treatment-emergent adverse event, ecchymosis, was mainly observed at the blood sampling sites. All of the adverse events were considered to be of mild intensity and were resolved without treatment. Overall, single 10 kU kg−1 doses of lanoteplase were well tolerated by healthy young and elderly subjects in this study and there was no visible sign of bleeding, haematuria or allergic reaction.
Considering the contrasting effects of age observed within female subjects for lanoteplase antigen and activity results, and no age- or gender-specific adverse events at the relatively low dose given in this study, the observed statistically significant age-by-gender interaction for lanoteplase antigen AUC and CLt is not likely to be clinically important, but studies in larger numbers of patients will be needed to confirm this. Additionally, the applicability of the pharmacokinetic, pharmacodynamic and haemostasis findings in this study at lanoteplase doses higher than 10 kU kg−1 will also need to be confirmed in larger phase 3 studies.
In summary, while safety and efficacy studies in larger numbers of patients are needed, on the basis of this study 10 kU kg−1 intravenous lanoteplase was well-tolerated overall by healthy young and elderly subjects. Elderly females had a 28 and 32% lower mean CLt than young females, and elderly males, respectively, but no statistically significant difference was observed within females for CLt of lanoteplase activity. No significant age- or gender-related differences were observed in the haemostatic pharmacodynamics of lanoteplase in healthy subjects.
Competing Interests
At the time that this research was conducted, all of the authors were stockholders and/or employees of Bristol-Myers Squibb Company, the sponsor of this study.
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