Objective:
PAI-1 is a serine protease inhibitor (SERPIN) whose function in vivo is to downregulate fibrinolysis by inhibiting urokinase- and tissue-type plasminogen activators (uPA and tPA). The goal of this research is to use PAI-1 as a prototypical SERPIN scaffold from which to develop “designer” PAI-1 variants with altered specificity and inhibitory kinetics.
Methods:
PAI-1 variants of interest were identified using a molecular evolution approach in which traditional phage-display technology was coupled with next generation high throughput DNA sequencing. Filamentous phage displaying PAI-1 fused to the p3 coat protein were randomly mutagenized through error prone PCR, reacted with uPA, and selected with an anti-uPA polyclonal antibody in two sets of complimentary experiments to (1) determine the stable half-life of PAI-1 or (2) the rate at which PAI-1 inhibits uPA. At selected time points in each of the described experiments, the PAI-1 encoding portion of the phage genome was analyzed by next- generation sequencing. Evaluation of 7-10 million sequencing reads per time point facilitated massively parallel kinetic analyses to determine the effect of amino acid substitutions at every residue in PAI-1 simultaneously with respect to both half-life and rate of uPA inhibition.
Summary of Results:
This analysis generated data for 74% of all possible single amino acid substitutions, identifying 492 mutations that extended the functional half-life of PAI-1, as well as 1509 destabilizing mutations. These results were validated for representative single amino acid substitutions expressed as individual, purified recombinant proteins.
Conclusions:
These data provide a useful resource for interpreting human mutations identified by future large scale clinical human genome sequencing. In addition, these findings provide new insight into structure-function relationships in PAI-1. Finally, these tools lay the groundwork for future studies aimed at developing novel SERPINs based on the PAI-1 scaffold with altered target protease specific potentially applicable to treatment for a number of disorders of hemostasis and thrombosis, as well as various other SERPIN disorders (eg alpha-1-antitrypsin and C1-inhibitor deficiency).
Objective:
The adolescent/adult pivotal phase 3 pathfinder 2 trial assessed N8-GP (turoctocog alfa pegol, ESPEROCT®) use for routine prophylaxis and treatment of bleeds in previously treated patients (PTPs).
Methods:
pathfinder 2 was a multi-center, multi-national, single-arm study evaluating safety, efficacy and pharmacokinetics. Adolescents/adults (aged ≥12 y) with severe hemophilia A were administered prophylaxis (50 IU/kg Q4D) in the main phase with option for eligible patients (0-2 bleeds in prior 6 months) to randomize (2:1) to 75 IU/kg Q7D or 50 IU/kg Q4D during extension 1 (24 weeks) and continue treatment into extension 2. An on-demand group was included throughout. Current analysis covers January 2012 through December 2018.
Summary:
Of the 186 PTPs (including 46 [25%] from the US) enrolled in the main phase, 150 (81%) started extension 1, 139 (75%) completed extension 1, and 128 (69%) completed the study. Mean age was 31.1 years, weight 75 kg and BMI 24.3.
The complete trial covers 785 patient-years of treatment (66,577 exposure days [ED]) during which there were 2,758 bleeds, including 1,807 (66%) spontaneous bleeds and 1,735 (63%) joint bleeds. Twelve patients treated on-demand for a mean 3.1 years reported nearly half of all bleeds (1,270, 46%), including 971 (54%) spontaneous bleeds and 627 (36%) joint bleeds. Hemostatic efficacy was rated excellent/good in 2,470 (90%) episodes; 2,614 bleeds (95%) were treated with 1-2 injections.
Of 175 patients on prophylaxis, 55 of 110 eligible were randomized in extension 1. For 177 patients treated with 50 IU/kg Q4D prophylaxis for 613 years (57,723 ED), 126 (71%) experienced 1,312 bleeds. For 61 low-bleed patients with 134 years (7,255 ED) on 75 IU/kg Q7D prophylaxis, 53 (87%) experienced 176 bleeds. Median ABRs are shown in the TABLE.
| 50 IU/kg Q4D | 75 IU/kg Q7D | |
| n | 177 | 61 |
| Mean treatment | 3.5 years | 2.2 years |
| Median ABR | 0.8 | 1.7 |
N8-GP mean trough levels were 3.1 IU/dL on 50 IU/kg Q4D and 1.0 IU/dL on 75 IU/kg Q7D.
A total of 1,827 adverse events were reported over 785 exposure years, including 63 serious adverse events. One patient with an intron 22 inversion developed a low-titer inhibitor at 93 ED and was withdrawn when it progressed to >5 BU. Non-neutralizing anti-PEG antibodies were seen at baseline in 12 patients (6.5%) prior to first N8-GP exposure and 11 (5.9%), who had negative anti-PEG at baseline, had positive antibodies after exposure.
Conclusion:
These data support the safety and efficacy of N8-GP in a controlled phase 3 trial setting in adolescents/adults. Prophylaxis with N8-GP with a consistent dose/interval (50 IU/kg Q4D) was effective in preventing bleeds; extended dosing was evaluated as successful for a subgroup of low-bleed patients. No significant safety issues were identified.
Objective:
Immune tolerance induction (ITI) is the standard of care for inhibitor eradication and restoration of factor VIII (FVIII) responsiveness in subjects with severe hemophilia who develop high-titer inhibitors. Retrospective data support the use of recombinant FVIII Fc fusion protein (rFVIIIFc) in ITI (Carcao et al. Haemophilia. 2018) but this has yet to be confirmed in prospective studies. This study presents preplanned interim results of verITI-8 (NCT03093480).
Methods:
VerITI-8 is a single-arm, nonrandomized, open‐label, ethics-approved study of rFVIIIFc (200 IU/kg/day) for first-time ITI. Eligible subjects had a history of high-titer inhibitors (historical peak ≥5 Bethesda units [BU]/mL) and provided informed consent. The primary endpoint is time to tolerization, defined by negative inhibitor titer (<0.6 BU/mL) at two consecutive visits; incremental recovery ≥66% of expected at two consecutive visits; and rFVIIIFc half‐life ≥7 hours. ITI failure is defined as not meeting the above criteria by Week 48. This interim analysis was planned when ≥10 subjects had received ≥6 months of rFVIIIFc ITI.
Summary:
Fifteen subjects were screened as of the December 5, 2018 cutoff, while 14 subjects enrolled and had received ≥1 dose of rFVIIIFc for ITI. The median (range) age at start of ITI was 2.6 (0.8–16.0) years and historical peak inhibitor titer was 29.6 (6.2–256.0) BU/mL. Six subjects have been successfully tolerized, with a median (range) time to first negative titer, normal incremental recovery, and tolerization of 2.3 (1.7–15.6), 6.0 (4.3–28.1), and 11.7 (8.1–32.0) weeks, respectively. Seven subjects continue to receive rFVIIIFc ITI (median [range] time on ITI: 16.0 [0.1–35.6] weeks) and 1 subject has failed. No adverse events related to rFVIIIFc have been reported.
Conclusions:
Early results from this prospective/ongoing study of first-time ITI indicate that rFVIIIFc may offer rapid time to tolerization in some subjects with severe hemophilia A and high-titer inhibitors. Achieving tolerance faster can improve quality of life and reduce costs.
Objective:
Evaluate clinical characteristics, hemostasis management, and clinical outcomes regarding menstruation, child birth, surgical procedures, dental care, and spontaneous and traumatic bleeds of women and girls with factor VIII (FVIII; hemophilia A) or factor IX (FIX; hemophilia B) deficiency (WGFD).
Methods:
A retrospective chart review is ongoing at three US hemophilia treatment centers (HTC) to collect data on WGFD (obligate or potential carriers of FVIII or FIX deficiency, with or without genetic confirmation). Data are collected on patients who had at least two HTC visits and underwent medical or surgical interventions for hemostasis management between April 2012 and November 2018, with the outcome available in medical charts.
Summary:
Interim results as of April 5, 2019 include charts from two HTCs on 26 (89.7%) patients with FVIII deficiency and 3 (10.3%) patients with FIX deficiency. The median (range) age at factor deficiency diagnosis was 18.5 (0.1–72.0) years. Twenty-four (82.8%) and 8 (27.6%) patients had a family history of hemophilia and other bleeding disorders, respectively. A total of 17 (58.6%) patients initially visited the HTC due to family history/genetic counseling. Other reasons for visiting an HTC were heavy menstrual bleeding (n=12 [41.4%]) or spontaneous or traumatic bleeds (n=12 [41.4%]), including 7 (24.1%) patients reporting both heavy menstrual bleeding and spontaneous or traumatic bleeds. Of the 12 patients with spontaneous or traumatic bleeds, 4 (33.3%) patients had joint bleeds, 6 (50.0%) patients had excessive nose bleeds, and 9 (75.0%) patients had easy bruising. For those with FVIII deficiency, the median (range) FVIII level at diagnosis was 32.5 (2.0–101.1) IU/dL (n=24), median (range) baseline hemoglobin was 12.9 (5.4–14.8) g/dL (n=19), and median (range) baseline von Willebrand factor ristocetin cofactor was 70 (40–150) IU/mL (n=16). The median (range) number of documented bleeds was 1.0 (0.0–24.0) in the first year at the HTC. Final results of this chart review, including data from those with FIX deficiency, HTC interventions, and outcomes for hemostasis management, will be presented.
Conclusions:
This chart review provides further insights into the clinical presentation and hemostasis management of WGFD evaluated at HTCs in the US. Results may contribute to the design of future prospective studies evaluating treatment options for this patient group.
Dr. Meeks is an Associate Professor of Pediatrics in the Department of Pediatrics at the Emory University School of Medicine and the Aflac Cancer and Blood Disorders Center of Children’s Healthcare of Atlanta. She obtained a Bachelor of Science in Mathematics from Duke University where she was elected to Phi Beta Kappa. After earning her medical degree from the University of Mississippi, she completed her clinical training at the University of Virginia and Emory University. Dr. Meeks has a basic, translational, and clinical research interest in the development of inhibitors in hemophilia A. Her work has focused on the early immune response to factor VIII and the diversity of the B-cell response to factor VIII. She is a former NHF clinical fellow who currently has funding to pursue these projects from the Hemostasis and Thrombosis Research Society and the National Institutes of Health.
