Hemophilia B is a rare X-linked genetic disorder manifested by spontaneous and traumatic bleeding, especially in joints (85% of bleeding events) and soft tissue, because of a deficiency in coagulation factor IX (FIX) activity . Treatment consists of FIX replenishment on a routine (prophylaxis) or episodic (on-demand) basis to prevent or treat acute spontaneous or traumatic bleeding events.
Historically, it has been thought that most spontaneous bleeding episodes occur when FIX activity concentrations (FIX:C) are lowest, shortly before the next scheduled dose. However, the relationship between FIX:C and the likelihood of bleeding episodes may be more complex because of the distribution of FIX in perivascular spaces not accounted for when monitoring FIX:C . The pharmacokinetics of FIX:C after replacement therapy is also known to be complex and multicompartmental . The multicompartmental model suggests 1 or 2 larger, extravascular pools of FIX:C being physiologically active and in communication with a smaller circulating pool. First reported in 1983 in a murine model, a large portion of FIX disappears from the circulation soon after intravenous infusion of factor concentrate, with a 2000-molar excess required to inhibit further clearance [4,5]. Briet et al. observed that patients with hemophilia B with no circulating antigen evidenced a more rapid disappearance than did patients with circulating antigen. Clinicians are aware that progressively less FIX is needed during continuous infusions surrounding surgery to maintain desired 100% levels, which also suggests a saturable, extravascular pool .
Nonacog alfa (BeneFIX; Pfizer Inc., Philadelphia, Pennsylvania, USA) is a recombinant coagulation FIX approved for on-demand treatment and for control of bleeding episodes and routine prophylaxis to reduce the frequency of bleeding episodes in patients with hemophilia B. In a multicenter, phase 4, randomized, open-label study, a once-weekly prophylaxis regimen of nonacog alfa (100 IU/kg) demonstrated a reduction in the annualized bleeding rate (ABR) relative to that of on-demand treatment, and the ABR was similar to that of a twice-weekly nonacog alfa 50-IU/kg prophylaxis regimen .
A pivotal study (clinicaltrials.gov identifier: NCT01335061) of nonacog alfa administered prophylactically at a dose of 100 IU/kg once weekly demonstrated a low median ABR of 2.0 for any bleeding episode, and of 1.0 for spontaneous, 1.0 for traumatic, 0.0 for joint, and 0.0 for soft tissue/muscle bleeding events. Additionally, a significant reduction of the ABR with prophylaxis treatment compared with that of on-demand treatment (P < 0.0001) was reported .
The observation that a single weekly dose of nonacog alfa confers efficacy over a full week suggests several hypotheses concerning the larger extravascular pool(s) serving as a depot to replenish circulating FIX:C. Evidence for the extravascular distribution of FIX includes data that demonstrate a displaceable, intimal-bound pool, perivascular Type 4 collagen, intimal basement membrane, and joint fluid. The extravascular distribution of FIX is consistent with the two- compartment and three-compartment model kinetics for FIX described by Björkman et al.[3,10] wherein they reported that, for using the two-compartment model, FIX molecules spent 44% of the mean residence time in the second or extravascular compartment. They concluded that this distribution into the extravascular compartment explained the common clinical finding that postdose FIX activity shortly after dosing is less than that expected. Björkman remarked that the peak FIX activity was not simply the ratio of dose to plasma volume, owing to the existence of the extravascular compartments, which may render strict adherence to the concept of FIX recovery as being somewhat ambiguous.
This post hoc analysis examined the time of onset of bleeding events for patients who experienced spontaneous bleeding events over the study's 52-week prophylaxis treatment period. It also assessed the trough FIX activity levels during the prophylaxis period.
Materials and methods
The pivotal trial during which these data were collected has been described elsewhere . Briefly, this multicenter, open-label, sequential two-period study enrolled 25 male patients aged 12–54 years with moderately severe to severe hemophilia B (FIX:C ≤2%). Patients were required to have had at least 12 bleeding episodes (6 of which must have been joint bleeding events) in the year prior to the screening visit, and at least 100 exposure days to FIX products.
Treatment constituted 6 months of on-demand nonacog alfa therapy (dose and schedule at the investigators’ discretion) followed by 12 months of prophylaxis treatment with nonacog alfa 100 IU/kg once weekly. The primary end point was the ABR on prophylaxis and on demand period (number of bleeding events/days on treatment/365.25). Trough FIX:C measurements (measured as part of recovery assessment in the pivotal trial) that were made between 144 h (6 days) and 196 h (8 days) were collected at week 26 (end of the on-demand period) and week 78 [end of 52-week prophylaxis period (end of study)]. Observed data from this post hoc analysis were summarized using descriptive statistics; no formal statistical analyses were performed.
The study protocol and amendments were approved by the following institutional review boards or ethics committees: Ethics Committee for Multicenter Trials, Sofia, Bulgaria; Ottawa Hospital Research Ethics Boards, Ottawa, ON, Canada; Central Ethics Committee, Agency for Medicinal Products and Medical Devices, Zagreb, Croatia; Institutional Review Board of Eulji University Hospital, Daejeon, Republic of Korea; Medical Research and Ethics Committee, Bangsar, Kuala Lumpur; Comite de Ética, Investigacion y Bioseguridad Privada de Aguascalientes, SC, Aguascalientes, Aguascalientes, Mexico; Comite de Ética e Investigación del Hospital y Clinica OCA, Monterrey, Nuevo Leon, Mexico; Komisja Biotyczna Akademii Medycznej, Wroclaw, Poland; Health Products Regulation Group, Health Sciences Authority, Helios, Singapore; and Ege Universitesi Tip Fakultesi Klinik Arastirmalar Etik Kurulu, Bornova, Izmir, Turkey.
The study was conducted in accordance with the ethical principles of the Declaration of Helsinki and the International Council for Harmonisation Guidelines for Good Clinical Practice, and all patients provided written informed consent prior to study participation.
A total of 25 patients were included in the study, 22 with severe and 3 with moderately severe hemophilia [FIX:C predose: severe (<1%); moderately severe (1 to ≤2%)]. Prior to entry into the study, no patients was treated with prophylaxis therapy. All 25 patients were on an on-demand regimen before crossing over to the 52-week prophylaxis regimen with once-weekly nonacog alfa; 12 (48%) reported no spontaneous bleeding episodes during the prophylaxis period. The remaining 13 patients experienced at least one spontaneous bleeding event during the prophylaxis period (total, 64 spontaneous bleeding events) and form the basis of this analysis. Mean (SD) dose of nonacog alfa during the on-demand period was 39 (10) IU/kg per infusion. Figure 1 depicts the frequency distribution for the elapsed time interval between the weekly prophylactic dose and a spontaneous bleeding event. It demonstrates no obvious pattern to the onset of bleeding episodes over the 7 days between nonacog alfa once-weekly doses.
The ABR reduction for all spontaneous bleeding events and for spontaneous target joint bleeding events between the on-demand and prophylaxis periods is shown in Table 1. A substantial decrease in the number of spontaneous bleeding events when comparing the on-demand and prophylaxis regimens was demonstrated. Of note, however, was the reduction in spontaneous target joint bleeding events achieved with once-weekly nonacog alfa treatment.
Table 1 -
Spontaneous bleeding event profile of patients who experienced a bleeding event during the prophylaxis
period: spontaneous annualized bleeding rate with on-demand treatment versus spontaneous annualized bleeding rate with prophylaxis
||Any spontaneous bleeding events
||Target joint spontaneous bleeding events
||sABR on-demand period (6 months)
||sABR prophylaxis period (1 year)
||sABR on-demand period (6 months)
||sABR prophylaxis period (1 year)
sABR, spontaneous annualized bleeding rate.
A total of 17 FIX:C samples were collected during the time period corresponding to the trough of once-weekly dosing. Eight (47%) of the samples were reported to be greater than 2%. There appeared to be no relationship between trough FIX:C and ABR, as several patients with no bleeding episodes had trough FIX:C less than 1% while others had bleeding episodes despite trough FIX:C greater than 4%.
In patients with moderately severe to severe hemophilia B, the relationship between FIX:C and bleeding events is complex; lower FIX:C does not always result in bleeding events, and higher FIX levels are not always protective [3,11]. Several additional factors that influence bleeding phenotype, such as gene mutation, co-inherited genetic factors [including factor V Leiden mutation, deficiency of protein C, S, and antithrombin deficiency (AT III)], arthropathy, inflammation, angiogenesis, and physical activity, among others, have been reported. The data in this report further reinforce the uneven relationship between circulating FIX:C and bleeding events by showing that the frequency of spontaneous bleeding episodes did not progressively rise throughout the week; rather, they showed no apparent pattern throughout the week after once-weekly dosing with nonacog alfa.
As previously noted, the complexity of the relationship between FIX:C and probability for bleeding episodes appears to correlate with the distribution of FIX in perivascular compartments. However, the propensity of FIX to bind to tissues and other endogenous substances may also affect biologic activity. Stern et al.  demonstrated endothelial cell binding with a bovine aortic endothelial cell culture, showing that the bound FIX was biologically active, some three times more active than activated FIX in solution. Moreover, in a human umbilical vein endothelial culture, FIX binding was specific, calcium-dependent, and displaceable with excess FIX, and FIX remained biologically active when bound. Autoradiography demonstrated uniform distribution of FIX over the cells. The authors pointed out that bound activated FIX provided a mechanism to localize clot promotion to vessel walls, where it would most logically be needed, when vessel walls were damaged.
The molecular biology of FIX-collagen binding was elucidated by Gui et al., who engineered a number of FIX variants that had higher collagen affinity (FIX-K5R) or lower collagen affinity (FIX-K5A) than wild-type FIX . The authors demonstrated that collagen binding affects FIX recovery, with there being an inverse relationship between collagen affinity and recovery. Thus, higher recovery was observed with the lower collagen affinity FIX-K5A and lower recovery obtained with the high-collagen affinity FIX-K5R compared with wild-type FIX. They also showed that FIX is removed from the circulation, with the majority lost to hepatic binding. This observation, coupled with the earlier work by Stern et al., who showed that this bound pool was easily displaceable with exogenous FIX administration, suggests that, although the FIX rapidly binds, it can be released and reenter the circulation over time to replenish circulating levels. Tight collagen binding may also have played a part in the early FIX gene therapy experiments, wherein intramuscular injection was practiced but little circulating FIX activity was obtained. The production of FIX enveloped in a collagen-rich muscle milieu may have sequestered the FIX as it was produced, rather than having it free for release into the circulation . The location of extravascular FIX was immunohistochemically elucidated, using sections of human popliteal arteries wherein both endothelial surface binding and perivascular, subendothelial, interstitial binding occurred. Such an extravascular pool is consistent with the clinical trial finding described here, wherein protection from spontaneous bleeds (pharmacodynamic observation) departs from the pharmacokinetic observations (low FIX activity levels).
Feng et al. conducted a murine saphenous vein bleeding model experiment 1 week after FIX dosing using wild-type FIX as well as FIX variants designed to have low collagen binding (FIX-K5A) and enhanced collagen binding (FIX-K5R). Their results showed that the enhanced collagen binding variant was associated with less bleeding and low collagen binding was associated with more bleeding than wild type FIX. The authors indicated that tight collagen binding might prolong the physiologic effect of FIX by sustaining for a longer time, a large collagen-bound pool of FIX that would be available to replenish circulating levels. One may speculate that the spontaneous bleeding rate seen in this nonacog alfa trial might be consistent with replenishment, albeit at a low level, of the circulating FIX pool from an extravascular depot.
Joint fluid aspirated from patients with osteoarthritis was found to contain a substantial amount of FIX activity in a concentration that was 9.92% plasma (FII was present at 20.9% of plasma, FXI at 32.0%, and FVIII at 0.58%, the limit of detection). Thrombin generation did occur upon addition of tissue thromboplastin, with 80% of the prothrombin being converted, despite low FVIII levels and modest FIX levels, and was not increased by the addition of FVIII. Low levels of tissue factor pathway inhibitor may account for this. The authors felt that coagulation regulatory mechanisms in extravascular fluid differed from those in the intravascular setting, with slower clearing of activated coagulation factors in the extravascular spaces .
Congruent with the observations of Chang et al., Sun et al. injected adeno-associated virus (AAV) gene-transferring vectors expressing FIX into the knee joints of mice with hemophilia B. Fibroblast-like synoviocytes and chondrocytes were shown to produce FIX in situ. Intra-articular AAV-associated FIX production resulted in higher and more sustained FIX activity levels compared with standard intravenous FIX administration. The investigators noted decreases in the progression of hemorrhage-related joint disease (synovitis and blood-induced joint damage), suggesting the anti-inflammatory importance of FIX in joint fluid. The authors speculated that local FIX concentrations might support initial hemostasis, even in the absence of systemic FIX, with extravascular FIX participating in thrombin generation at the intraluminal primary platelet thrombus. Indeed, joint fluid is not the only locus of periarticular FIX, given the demonstrated ability of FIX to bind to specific sites on individual collagen type IV strands .
Recent reports of the terminal half-life of nonacog alfa measured over a longer sampling period revealed a value of 33.8 h . Given that 1 week (168 h) represents roughly five 33.8-h half-lives, simple linear kinetics would indicate that the residual of the peak after 100 IU/kg dosing would be approximately 3% at the end of the week. This level might confer some therapeutic benefit during the latter part of the week and is consistent with the observed, roughly uniform distribution of bleeding events over the course of the week, suggesting that FIX activity is not the only factor associated with optimal prophylaxis treatment.
It is difficult to measure FIX:C in patients while they are experiencing a spontaneous bleeding episode, as these events are unlikely to occur while the patient is at the clinic, when a blood sample could be collected. Korth-Bradley et al. simulated FIX:C profiles after either nonacog alfa 50 IU/kg twice weekly or 100 IU/kg once weekly, as well as the predicted FIX:C, at the time of the reported bleeding episodes for patients who participated in the study reported by Valentino et al.. The profiles and simulated FIX:C at the time of the reported bleeding episodes are shown in Fig. 2. Many of the bleeding episodes occurred at predicted FIX:C that was consistent with mild hemophilia, when they would not be anticipated, offering additional evidence that the relationship between FIX:C and spontaneous bleeding episodes is not simple.
Certain limitations of this study deserve mention, including the post hoc nature of this analysis. Additionally, the study relied upon patients’ self-reporting of bleeding events.
In conclusion, once-weekly prophylaxis treatment with nonacog alfa 100 IU/kg for 52 weeks resulted in 48% of patients experiencing no spontaneous bleeding episodes. The 13 patients who experienced spontaneous bleeding events showed reductions in their ABR and in the number of target joint bleeding episodes during the once-weekly prophylaxis treatment period. Trough FIX:C of more than 2% was noted approximately 1 week after dosing during prophylaxis treatment in 47% of patients. When spontaneous bleeding episodes did occur, no pattern in the day of the spontaneous bleeding episode occurrence was observed.
Editorial support was provided by Teri O’Neill of Peloton Advantage, Parsippany, New Jersey, an OPEN Health company, and was funded by Pfizer Inc.
Author roles: All authors collaborated in the preparation of the manuscript, and critically reviewed and provided revisions to the manuscript. All authors had access to the data and assume responsibility for the completeness and accuracy of the data and data analyses. All authors granted final approval of the manuscript for submission.
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Conflicts of interest
The pivotal trial and this post hoc analysis were sponsored by Pfizer Inc. J.K.-B. and L.M.S. are employees of Pfizer Inc. B.J T., M.E.C., and P.R. were employees of Pfizer Inc. at the time of this study. K.K. has received honoraria and research grants from Pfizer, and has served on an advisory board for Pfizer.
1. Roosendaal G, van den Berg HM, Lafeber FP, Bjilsma JW. Rodriguez-Merchan EC, Goddard NJ, Lee CA. Blood-induced joint damage: an overview of musculoskeletal research in haemophilia. Musculoskeletal Aspects of Haemophilia
. Oxford, UK: Wiley-Blackwell; 2000. 18.
2. Iorio A, Fischer K, Blanchette V, Rangarajan S, Young G, Morfini M. Tailoring treatment of haemophilia B: accounting for the distribution and clearance of standard and extended half-life FIX concentrates. Thromb Haemost
3. Bjorkman S. Population pharmacokinetics of recombinant factor IX: implications for dose tailoring. Haemophilia
4. Fuchs HE, Trapp HG, Griffith MJ, Roberts HR, Pizzo SV. Regulation of factor IXa in vitro in human and mouse plasma and in vivo in the mouse. Role of the endothelium and the plasma proteinase inhibitors. J Clin Invest
5. Gui T, Lin HF, Jin DY, Hoffman M, Straight DL, Roberts HR, et al. Circulating and binding characteristics of wild-type factor IX and certain Gla domain mutants in vivo. Blood
6. Briet E, Bertina RM, van Tilburg NH, Veltkamp JJ. Hemophilia B Leyden: a sex-linked hereditary disorder that improves after puberty. N Engl J Med
7. Uprichard J, Adamidou D, Goddard NJ, Mann HA, Yee TT. Factor IX replacement to cover total knee replacement surgery in haemophilia B: a single-centre experience, 2000-2010. Haemophilia
8. Valentino LA, Rusen L, Elezovic I, Smith LM, Korth-Bradley JM, Rendo P. Multicentre, randomized, open-label study of on-demand treatment with two prophylaxis
regimens of recombinant coagulation factor IX in haemophilia B subjects. Haemophilia
9. Kavakli K, Smith L, Kuliczkowski K, Korth-Bradley J, You CW, Fuiman J, et al. Once-weekly prophylactic treatment versus on-demand treatment with nonacog alfa in patients with moderately severe to severe hemophilia B. Haemophilia
10. Bjorkman S, Carlsson M, Berntorp E. Pharmacokinetics of factor IX in patients with haemophilia B. Methodological aspects and physiological interpretation. Eur J Clin Pharmacol
11. Rendo P, Shafer F, Korth-Bradley JM, Sivamurthy K, Korin J. Factors that influence the bleeding phenotype in severe hemophilic patients. Blood Coagul Fibrinolysis
12. Stern DM, Drillings M, Nossel HL, Hurlet-Jensen A, LaGamma KS, Owen J. Binding of factors IX and IXa to cultured vascular endothelial cells. Proc Natl Acad Sci U S A
13. Begbie ME, Mamdani A, Gataiance S, Eltringham-Smith LJ, Bhakta V, Hortelano G, et al. An important role for the activation peptide domain in controlling factor IX levels in the blood of haemophilia B mice. Thromb Haemost
14. Herzog RW, Hagstrom JN, Kung SH, Tai SJ, Wilson JM, Fisher KJ, High KA. Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus. Proc Natl Acad Sci U S A
15. Feng D, Stafford KA, Broze GJ, Stafford DW. Evidence of clinically significant extravascular stores of factor IX. J Thromb Haemost
16. Chang P, Aronson DL, Borenstein DG, Kessler CM. Coagulant proteins and thrombin generation in synovial fluid: a model for extravascular coagulation. Am J Hematol
17. Sun J, Hakobyan N, Valentino LA, Feldman BL, Samulski RJ, Monahan PE. Intraarticular factor IX protein or gene replacement protects against development of hemophilic synovitis in the absence of circulating factor IX. Blood
18. Wolberg AS, Stafford DW, Erie DA. Human factor IX binds to specific sites on the collagenous domain of collagen IV. J Biol Chem
19. Powell JS, Pasi KJ, Ragni MV, Ozelo MC, Valentino LA, Mahlangu JN, et al. B-LONG Investigators. Phase 3 study of recombinant factor IX Fc fusion protein in hemophilia B. N Engl J Med
20. Korth-Bradley JM, Valentino LA, Rendo P, Shafer FE, Smith L, Baumann JA, et al. Pharmacokinetic/pharmacodynamic assessment of reformulated recombinant coagulation factor IX in adults and children with severe hemophilia B. Poster presented at the Annual Meeting of the American Society of Hematology, San Diego, California, USA. 2011.