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Keywords:

  • D-dimer;
  • pharmacokinetics;
  • prothrombin fragment 1+2;
  • prothrombin time;
  • recombinant activated FVII (rFVIIa) analog;
  • safety

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Design, subjects, and methods
  5. Results
  6. Discussion and conclusions
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Summary. Background: NN1731 is a recombinant activated factor VII (rFVIIa) analog with enhanced activity. Objectives: This clinical trial aimed to assess the safety and pharmacokinetics of single doses of NN1731 in healthy male subjects. Methods: This was a randomized, placebo-controlled dose-escalation trial with four dose tiers (NN1731 530 μg kg−1). Eight subjects were randomized to either NN1731 (= 6) or placebo (= 2) in each tier. Results: No thromboembolic or serious adverse events were reported and no antibody formation towards NN1731 was detected. NN1731 was demonstrated to be pharmacologically active based on coagulation-related parameters (prothrombin fragment 1+2, activated partial thromboplastin time and prothrombin time). There were five mild/moderate adverse events in three subjects. The FVIIa activity of NN1731 after ascending single-dose administration of NN1731 fits well with a two-compartment model, indicating a bi-exponential decline with a rapid initial distribution of approximately 73% FVIIa activity (half-life = 20 min), followed by a less rapid terminal elimination phase eliminating the remaining 27% (half-life = 3 h). Dose proportionality in healthy male subjects at the dose levels investigated (530 μg kg−1) was supported by the FVIIa activity data. Conclusions: Based on the results of this trial, NN1731 appears safe and well tolerated in healthy subjects at doses up to 30 μg kg−1. No immunogenic or thromboembolic events were reported. The pharmacokinetic profile of NN1731 as measured by FVIIa activity appears to follow two-compartment pharmacokinetics characterized by an initial rapid distribution phase followed by a less rapid elimination phase.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Design, subjects, and methods
  5. Results
  6. Discussion and conclusions
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Recombinant human factor VIIa (rFVIIa; NovoSeven®; Novo Nordisk A/S, Bagsværd, Denmark) is effective in treating bleeding in hemophilia patients with inhibitors [1]. Recombinant FVIIa acts locally at sites of vascular injury where tissue factor (TF) is exposed and activated platelets accumulate. When given in pharmacologic doses, rFVIIa binds to activated platelets and directly activates factor X (FX), leading to the generation of a thrombin burst and formation of a stable hemostatic plug [2]. NN1731 is an analog of rFVIIa with three amino acid substitutions V158D/E296V/M298Q, which stabilize the molecule in its active conformation without TF, resulting in increased activity on the surface of activated platelets [3]. In a cell-based model of coagulation, it was found that while rFVIIa and NN1731 resulted in similar FXa generation on TF-bearing cells, NN1731 showed a significantly greater activity on the surface of activated platelets [4]. In contrast to even high concentrations of rFVIIa, NN1731 could normalize the thrombin generation rate in hemophilia conditions. Importantly, the cell-based model of coagulation showed that NN1731 did not directly activate platelets and also thrombin generation was not detected before platelet activation, suggesting that NN1731 would not lead to thrombin generation in the absence of an injury. The hemostatic properties of NN1731 have also been analyzed in vitro in blood from hemophilia patients using thromboelastography [5]. Both rFVIIa and NN1731 improved the hemophilic pattern; however, NN1731 normalized the propagation phase and reduced the clotting time to a greater extent than rFVIIa. In vivo studies in an antibody-induced hemophilia A model in mice showed that NN1731 significantly shortened the bleeding time and decreased the blood loss without any evidence of adverse effects. The results indicated that NN1731 uses the same mechanism to promote clot formation as rFVIIa, however, with a higher hemostatic potential [6].

The objective of the clinical trial aimed to assess the safety and pharmacokinetics (PK) of single doses of NN1731 in healthy male subjects.

Design, subjects, and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Design, subjects, and methods
  5. Results
  6. Discussion and conclusions
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Study protocol

The protocol, amendments, subject information sheet and informed consent forms were reviewed and/or approved according to local regulations and by a local ethics committee prior to trial initiation. Before entering the trial, the nature of the study was explained to each participant and written informed consent was obtained before any trial-related activities. The trial was conducted at SGS-Aster, a contract research organization in Paris, France, in accordance with GCP and with the Declaration of Helsinki and its amendments in force at the initiation of the trial. The subjects attended the clinical unit in the morning before the day of dosing (Day 1) and remained resident under permanent medical and nursing supervision until 24 h after dosing.

Study design

The first human dose trial was a randomized, double-blind, placebo-controlled, parallel-group design with dose escalation. Healthy subjects were chosen for this trial as it is a homogenous group of humans not affected by any underlying diseases or receiving any concomitant medication, thus factors that could affect safety and the pharmacokinetics were minimized. The randomization list was generated by computer and subjects were assigned to the lowest available randomization number. Eight subjects were randomly allocated to a single intravenous (i.v.) injection with either NN1731 (= 6) or placebo (= 2) at each dose tier. The placebo formulation was identical to the active formulation except for the presence of NN1731. NN1731 (0.6 mg mL−1) or placebo was administered as a slow bolus injection over 2 min. The starting dose of NN1731 5 μg kg−1 was based on results obtained from in vitro studies [4,5] and toxicology studies. Although it was planned to administer 5, 10, 20, 30, 40, 50, 60, 80, 120 and 160 μg kg−1 of NN1731 in ascending order, the trial was terminated after completion of the first four dose tiers (5, 10, 20, and 30 μg kg−1), as it was concluded that no further clinically relevant information could be achieved in the investigated healthy subject population by increasing the NN1731 dose.

After completion of each dose level, an evaluation of blinded safety and pharmacokinetic data based on the FVIIa activity was performed to allow progression to the next dose level. After the fourth dose level (NN1731 30 μg kg−1), the following objectives were fulfilled: no safety concerns were identified in man after i.v. administration of up to 30 μg kg−1 NN1731 and the pharmacokinetic assessment of NN1731 indicated dose proportionality in the dose interval 5–30 μg kg−1.

It should be pointed out that the trial was not stopped for safety reasons. No patients experienced any serious adverse events and the observed adverse events are all events that naturally can occur in healthy subjects (headache, nausea). In particular, no thromboembolic serious adverse events or adverse events occurred and there was no indication of myocardial ischemia in the ECG taken or the measured Troponin I samples.

Subjects

Key inclusion criteria  Male Caucasian subjects (between or equal to 18 and 45 years of age) with a body mass index (BMI) between or equal to 18 and 27 kg m−2 and who were considered healthy based on medical history, physical examination (including vital signs and electrocardiogram [ECG]) and clinical laboratory test results.

Key exclusion criteria  History or evidence of potential thromboembolic risk, artherosclerosis or arteriosclerosis; overt bleeding; hepatitis B/C or HIV infection; use of non-steroidal anti-inflammatory drugs (NSAIDs) within 2 weeks prior to trial drug administration; food or drinks which could have an effect on the coagulation system; known or suspected allergy to rFVIIa or related products or components of the formulation; febrile illness within 5 days prior to dosing; or non-febrile illness during the entire trial period.

Safety assessments

The primary safety outcomes were all adverse events, abnormal laboratory safety data (hematology including coagulation factors, coagulation related parameters, clinical chemistry and urinalysis), presence of antibodies against NN1731, abnormalities in physical examination and vital signs (pulse, blood pressure, and body temperature), standard 12-lead ECG and injection site tolerability. Coagulation-related parameters (prothrombin fragment 1+2 [F1+2], prothrombin time [PT], activated partial thromboplastin time [aPTT], D-dimers, platelet count, fibrinogen, and antithrombin III [AT]) were measured at particular time points (pre-dose and 30 min, 1, 5, 12, and 24 h after dosing) and the changes obtained in the placebo group were compared with the changes in each of the NN1731 treatment groups.

Blood sampling and laboratory analysis

Blood was drawn by direct venipuncture with low pressure and centrifuged within 15 min. Plasma was transferred to airtight vials and immediately frozen at −80 °C. PT analysis was performed using STA Stago® with STA-Neoplastin and calcium as reagents. aPTT analysis was performed using STA-R Stago® Plasmatic recalcification time in the presence of cephalin. D-dimer levels were measured by enzyme-linked immunosorbent assay (ELISA) VIDAS® D-Dimer ExclusionTM (bioMérieux, Marcy l’Etoile, France). F1+2 was measured by ELISA: Enzygnost® F1+2 (monoclonal) (Dade Behring, Deerfield, IL, USA). AT was measured by STA-R Stago® using a two-step procedure according to the manufacturer’s instructions. Fibrinogen levels were measured using the STA Stago®– Clauss method.

FVIIa activity was analyzed by the Staclot® VIIa-rTF assay (Diagnostica Stago, Asnières-Sur-Seine, France) applied with minor modifications: a pipes buffer containing 0.1% bovine serum albumin was used to dilute plasma samples, controls and calibrator. rFVIIa (Novo Nordisk A/S) diluted to a final concentration of 300 mIU mL−1 and calibrated against the international FVIIa standard (89/688, International Institute of Biological Standards and Controls, UK) was applied as calibrator. The FVIIa activity assay applies truncated recombinant soluble thromboplastin that does not form complexes with FVII zymogen, thus ensuring assessment of FVIIa exclusively [7].

For the assessment of NN1731 antibodies, plasma was analyzed using a validated radioimmunoassay (Novo Nordisk A/S). In brief, I125 labeled NN1731 was added to the samples, resulting in the formation of antigen–antibody complexes. These immune complexes, together with unspecific immunoglobulins, were then precipitated using protein G sepharose mixed with anti-immunoglobulin antibodies. The radioactivity present in the precipitate is proportional to the amount of NN1731 antibodies in the sample. Inter and intra-assay variation were <10% in human plasma.

The specificity of the assay was confirmed by inhibition of anti-NN1731 antibodies with rFVIIa and NN1731 showing that the assay can identify cross reacting antibodies. Monoclonal antibodies specifically reacting with NN1731 (with no cross reaction against rFVIIa) can also be measured by the anti-NN1731 antibody RIA. This shows that the epitopes specific for NN1731 are accessible for binding and thus also potential NN1731-specific antibodies will be measured.

Any anti-NN1731 antibody positive samples would have been analysed for cross reactivity towards FVII and also for rFVIIa activity neutralizing effect; however, no samples were found positive in the antibody assay.

Pharmacokinetic and statistical analysis

Blood samples for pharmacokinetic assessment based on FVIIa activity were drawn prior to dosing and at 5, 10, 20, and 30 min and 1, 2, 3, 5, 8, 12, and 24 h after dosing. The pharmacokinetic parameters were estimated from individual plasma FVIIa activity profiles by non-compartmental methods and additionally, in a two-compartment pharmacokinetic model. Pharmacokinetic analyses included determination of the area under the FVIIa activity-time profile (AUC) from time 0 to infinity, the maximum FVIIa activity defined as the FVIIa activity in the first sample taken 5 min after dosing (C5min), half-life (t½) and total plasma clearance. Using the two-compartment pharmacokinetic model, estimates of half-lives in the distribution phase (t½,α) and the elimination phase (t½,β) were estimated, as well as the initial phase fraction defined as the AUC in the distribution phase divided by the AUC in the elimination phase.

Dose–response proportionality was investigated for the pharmacokinetic parameters AUC0–24 and C5min using anova on the logarithmically transformed values and with log (dose) as a fixed effect. A slope estimate in this model different from 1 indicates a deviation from dose–response proportionality. The slope estimate was presented with a 95% confidence interval (CI) and a P-value for a test of β = 1. For coagulation-related parameters, the change from baseline to each of the observed time points was analyzed using a linear model with baseline value and dose group as effects. As a result of the low number of subjects per group per time point, the assumption of normal distribution has not formally been evaluated.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Design, subjects, and methods
  5. Results
  6. Discussion and conclusions
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

A total of 32 subjects were randomized and exposed to NN1731 or placebo (Fig. 1). All 32 subjects were included in the safety and pharmacokinetic analyses. One subject did not attend the antibody follow-up visit at 3 months after dosing.

image

Figure 1.  Trial flow chart. The follow-up visit was 3 weeks after dosing; the antibody follow-up visit was 3 months after dosing. One subject was lost for antibody follow up. PK, pharmacokinetic.

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All subjects were male Caucasians with a mean age of 30.5 years (ranged from 20–45 years) and a mean BMI of 22.6 kg m−2 (ranged from 19–26 kg m−2). The subjects were healthy based on a physical examination, hematology and clinical chemistry blood assessments, urinalysis, screening for drug abuse and alcohol, tests for hepatitis B and C and HIV.

Safety

No serious or severe adverse events (including thromboembolic events) were reported and no antibodies to NN1731 were detected up to 3 months post-dosing in any of the dose groups. A total of five non-serious adverse events were reported in three subjects (one subject in each of the groups: placebo, NN1731 10 μg kg−1, and NN1731 20 μg kg−1); all were mild or moderate in severity. One subject dosed with 10 μg kg−1 NN1731 reported three events (headache, nausea and vomiting), whereas two other subjects (one in placebo and one in 20 μg kg−1 dose groups) developed headaches. Overall, there was no dose relationship evident with respect to the incidence or nature of adverse events reported.

No clinically significant abnormal values were reported in any treatment group at any time point for hematological, biochemical and urine laboratory parameters, vital signs, ECG and physical examinations. No clinically relevant changes were observed in plasma levels of platelets, fibrinogen and AT from pre-dose up until 24 h post-dosing and no local injection site reactions were observed.

Pharmacokinetics

The FVIIa activity in plasma obtained after i.v. dosing of NN1731 to healthy male subjects is shown in Fig. 2. The FVIIa activity declines in a bi- or multi-exponential way after i.v. administration of NN1731 and data fit well to a two-compartment pharmacokinetic model. The pharmacokinetic parameters estimated using both non-compartmental methods and a two-compartment model are presented in Table 1. Total and maximum exposure to NN1731, as expressed by AUC0–24 and C5min, increased proportionally with dose in the dose range investigated (5–30 μg kg−1). The terminal half-life (t1/2) and total plasma clearance appeared to be dose independent in the dose range investigated.

image

Figure 2.  Mean pharmacokinetic profiles (FVIIa activity) of NN1731.

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Table 1.   Pharmacokinetic parameters after a single dose of NN1731 to healthy male subjects
NN1731 dose (μg kg−1) Non-compartmental methodTwo-compartment model
AUC (IU*h mL−1)C5min (IU mL−1)t1/2 (h)CL (mL h−1 kg−1)AUC (IU*h mL−1)t1/2,α (h)t1/2,β (h)Initial phase fraction
  1. All data are based on FVIIa activity. AUC, area under the FVIIa activity–time curve from time 0 to infinity; C5min, the FVIIa activity measured 5 min after NN1731 dosing; C5min was used as the maximum FVIIa activity; CL, total plasma clearance; t1/2, terminal half-life; t1/2,α, the half-life of the initial (distribution) phase; t1/2,β, the half-life of the terminal (elimination) phase.

5Mean (SD)8.1 (2.1)11.6 (3.0)3.3 (1.5)112 (25)7.6 (2.0)0.32 (0.10)3.1 (1.6)0.71 (0.06)
Min–Max6–128–172–674–1425.7–11.20.24–0.501.8–6.30.66–0.83
10Mean (SD)15.9 (3.1)26.9 (4.6)3.5 (1.4)112 (18)15.0 (2.9)0.32 (0.07)3.2 (1.3)0.75 (0.09)
Min–Max14–2221–331–579–12912.8–20.70.22–0.421.3–5.00.58–0.84
20Mean (SD)32.7 (7.6)48.8 (8.7)3.6 (1.5)109 (27)31.2 (7.4)0.37 (0.08)3.4 (1.4)0.75 (0.06)
Min–Max25–4241–632–680–13823.3–40.00.25–0.431.9–5.70.68–0.82
30Mean (SD)48.7 (7.3)70.1 (15.0)2.9 (0.8)108 (18)46.1 (6.9)0.31 (0.08)2.7 (0.6)0.72 (0.06)
Min–Max38–5853–882–488–13735.5–53.70.22–0.441.9–3.40.63–0.82

Approximately 73% of NN1731 (based on FVIIa activity) was distributed from plasma in the initial phase with a half-life of approximately 20 min (range: 0.22–0.50 h) and the remaining 27% of NN1731 (based on FVIIa activity) was eliminated in the terminal phase with a half-life of approximately 3.1 h (range: 1.3–6.3 h) (Table 1).

Coagulation-related parameters

A dose-dependent shortening of PT and aPTT was observed immediately after NN1731 administration. Compared with placebo, the change in PT from pre-dose up until 1 h post-dosing was significant for all dose groups. This significant difference persisted up until 5 h post-dosing for the NN1731 30 μg kg−1 dose group (Fig. 3). Values of PT and aPTT returned to baseline levels between the 5–12 h sampling points. The aPTT measurements similarly showed dose-dependent reductions within the first hour post-dosing in the NN1731 10, 20 and 30 μg kg−1 dose groups compared with the placebo group (Fig. 3).

image

Figure 3.  Mean profiles of prothrombin time (PT) (top) and activated partial thromboplastin time (APTT) (bottom). All values are presented relative (%) to pre-dose values. Compared with placebo, the change in PT from pre-dose up until 1 h post-dosing was significantly higher for all dose groups and up until 5 h post-dosing for the 30 NN1731 μg kg−1 dose group. The reductions observed in APTT within the first hour post-dosing in the 10, 20 and 30 μg kg−1 dose groups were significantly higher than the changes observed in the placebo group.

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Mean plasma levels of F1+2 were rapidly increased after dose administration for all active dose groups and the increases appeared to be dose dependent, indicating increased thrombin generation with higher doses of NN1731 (Fig. 4). After administration of NN1731 30 μg kg−1, the levels of F1+2 were outside the reference range (80–240 pmol L−1) for five out of six subjects and the individual levels increased up to 5-fold over pre-dose values. Compared with placebo, the change from pre-dose up until 5 h post-dosing was significantly higher for the 10, 20 and 30 μg kg−1 NN1731 dose groups.

image

Figure 4.  Mean plasma levels of prothrombin fragment 1+2 (F1+2) after administration of NN1731. Compared with placebo, the change from pre-dose up until 5 h post-dosing was significantly higher for the 10, 20 and 30 μg kg−1 NN1731 dose groups.

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Mean levels of D-dimers were also elevated after administration of NN1731 (Fig. 5), indicating the pharmacologic effect of increased conversion of fibrinogen to fibrin. The levels of D-dimer increased 2.5-fold compared with predose levels; however, all values were within reference range. Compared with placebo, the changes were not significantly greater for any of the dose groups, except for the 10 μg kg−1 dose group after 5 h (= 0.0389) and there was no clear trend towards dose dependency.

image

Figure 5.  Mean plasma levels of D-dimer after administration of NN1731. The levels of D-dimer increased 2.5-fold compared with pre-dose levels; however, all values were within reference range. There was no clear trend towards dose dependency.

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Discussion and conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Design, subjects, and methods
  5. Results
  6. Discussion and conclusions
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

We report here the first human trial evaluating the safety and pharmacokinetics of ascending single doses of a new rFVIIa analog, NN1731, compared with placebo in healthy male subjects. The main safety concerns associated with NN1731 are the potential risk of thromboembolic complications as a result of the hemostatic effect as well as development of antibodies against NN1731 or FVIIa.

The rationale of the trial was to explore NN1731 in man. As NN1731 has not been given to humans before, the primary and secondary objectives of the trial were to investigate safety and pharmacokinetic properties of ascending single i.v. doses of NN1731 in healthy male subjects. The protocol suggested dose levels were 5, 10, 20, 30, 40, 50, 60, 80, 120 and 160 NN1731 μg kg−1. After completion of each dose level, an evaluation of blinded safety and pharmacokinetic data based on the FVIIa activity assay was performed to allow progression to the next dose level.

The trial was stopped after the fourth dose level (30 μg kg−1) as sufficient safety and PK information was obtained to justify further evaluation of safety and efficacy in a phase 2 trial in hemophilia patients.

In this study, no deaths or serious adverse events including thromboembolic events were observed and overall, there was no dose relationship evident with respect to the incidence or nature of non-serious adverse events. Non-serious adverse events were expected after i.v. administration of protein drugs and the adverse events reported after administration of NN1731 were similar to those reported for rFVIIa and were not any more frequent. An important finding from this study is that no NN1731 antibodies could be detected up to 3 months post-dosing. Furthermore, no local injection site reactions were observed.

Coagulation-related parameters were intensively monitored in the trial. A dose-dependent reduction in PT and aPTT was observed immediately after NN1731 administration. Values of PT and aPTT returned to baseline as the FVIIa activity declined over time.

Also as expected, a dose-dependent increase in thrombin generation was observed and reflected by increased plasma levels of F1+2 and D-dimer. The concentration profile of F1+2 over time (Fig. 4) shows that levels of F1+2 increase rapidly after injection of NN1731, for example highest values are observed 0.5–1 h after administration. A similar increase in F1+2 has been observed after administration of rFVIIa 160 μg kg−1 to healthy subjects [8]; however, the maximal value was not reached until 2–5 h after administration. This tendency to an earlier response after administration of NN1731 compared with rFVIIa was also observed with regard to the formation of D-dimer. These results support that NN1731 has a higher hemostatic potential than rFVIIa and is capable of generating a more rapid thrombin burst as compared with rFVIIa.

The decrease in PT and aPTT and the increase in F1+2 and D-dimer indicate an activation of the coagulation system but not to an extent that affects the fibrinogen levels, which were constant in the period from pre-dose to 24 h post-dosing. A similar increase in F1+2 has been observed after administration of rFVIIa to healthy subjects [8]. No clinically relevant changes in plasma levels and thereby no consumption of platelets, fibrinogen and AT were observed in the trial subjects, indicating that the coagulation is fully controlled by physiologic mechanisms without any signs of systemic or uncontrolled coagulation. In normal individuals there is an ongoing repair process in the vasculature for small leaks or damages where TF and/or activated platelets are transiently exposed in the process of normal hemostasis. Circulating levels of rFVIIa/NN1731 would thus contribute to this process by increasing the thrombin burst.

The FVIIa activity in plasma declined very rapidly immediately after i.v. administration of NN1731, indicating two- or multi-compartmental pharmacokinetics with an initial rapid distribution followed by a less rapid elimination phase which might be caused by binding to inhibitors, epithelial cells or distribution to other tissues. About 73% of the administered NN1731 was distributed from plasma in the initial phase with a half-life of about 20 min, whereas the remaining 27% was eliminated in the terminal phase with a half-life of 3.1 h. Dose proportionality for total and maximum exposure to NN1731, as expressed by AUC0–24 and C5min, was supported by the FVIIa activity data in the dose range investigated (5–30 μg kg−1).

The pharmacokinetic profile of NN1731 is different from that of rFVIIa, which can be described by a mono-exponential decline in FVIIa activity having a terminal plasma half-life of about 2.5–3 h [9]. Thus, the terminal elimination half-life is similar for NN1731 and rFVIIa whereas NN1731 is distributed from plasma in the initial phase with a half-life of 20 min. The FVIIa activity after dosing NN1731 30 μg kg−1 is higher than after dosing rFVIIa 90 μg kg−1 in the time interval from dosing to 20 min after dosing. The reason for the initial fast disappearance of FVIIa activity from plasma is currently not known, but could be because of increased binding to plasma inhibitors [10].

The mechanism of action of NN1731 is similar to that of rFVIIa, with a localized thrombin generation at the site of injury. The safety of rFVIIa is probably attributed to its dependence on the presence of activated platelet surface for significant thrombin generation. NN1731 does not activate the platelet directly and is more rapidly inhibited by the natural inhibitors, TFPI and AT/heparin than rFVIIa [10]. Also the plasma clearance of NN1731 is faster than that of rFVIIa. Because the activity of NN1731 is restricted to the activated platelet surface the effects of NN1731 would be localized and limited to the wound site and not expected to result in disseminated activity.

In summary, single i.v. doses of up to 30 μg kg−1 of NN1731 to healthy male subjects did not raise any safety issues and did not cause antibody formation against NN1731 or rFVIIa. The trial demonstrated that NN1731 has a pharmacologic effect in man. NN1731 actively generates thrombin after i.v. administration of 5–30 μg kg−1 as measured by increased plasma levels of prothrombin fragment (F1+2). The generated thrombin is active in formation of cross-linked fibrin, as measured by increased plasma levels of D-dimer. A similar decrease in PT and APTT has been observed. This is all as expected for a pro-haemostatic agent. At the same time, no decrease in platelets, fibrinogen and AT were observed, indicating that coagulation is in full control by normal mechanisms and there are no signs of systemic coagulation or uncontrolled coagulation.

No patients in the trial experienced any serious adverse events and the observed adverse events are all events that can naturally occur in healthy subjects (headache, nausea). More importantly, no thromboembolic SAEs or AEs occurred in the trial and there has been no indication of myocardial ischemia in the ECGs taken or the measured Troponin I samples.

The thrombin burst obtained with NN1731 might help to generate rapid formation of a hemostatic plug during a bleeding situation and thereby offer a fast cessation of bleeding. These specific characteristics of NN1731 may be of great importance to further improve treatment of patients with bleeding disorders.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Design, subjects, and methods
  5. Results
  6. Discussion and conclusions
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

The assessment of NN1731 antibodies performed by M. Loftager is gratefully appreciated.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Design, subjects, and methods
  5. Results
  6. Discussion and conclusions
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References