Once-weekly prophylactic treatment vs. on-demand treatment with nonacog alfa in patients with moderately severe to severe haemophilia B.

INTRODUCTION: Limited data are available on optimal prophylaxis regimens of factor IX (FIX) replacements for patients with haemophilia B. AIM: This multicentre, open-label study evaluated the efficacy and safety of once-weekly prophylaxis with nonacog alfa compared with on-demand treatment in adolescent and adult patients. METHODS: Males aged 12-65 years with moderately severe to severe haemophilia B (FIX:C ≤ 2%) were eligible for enrolment. Patients received on-demand treatment for 26 weeks, followed by once-weekly prophylaxis of 100 IU kg(-1) for 52 weeks. The primary efficacy end point was the annualized bleeding rate (ABR). Secondary end points included response to on-demand treatment, the number of infusions used to treat bleeding events, and the incidence of less-than-expected therapeutic effect (LETE). FIX:C was measured on day 1 and at weeks 26 and 78. RESULTS: Mean (±SD) ABR was lower during prophylaxis vs. on-demand treatment [3.6 (±4.6) vs. 32.9 (±17.4) events, respectively; P < 0.0001]. The majority (88.4%) of bleeding events had excellent or good responses upon the first infusion; 82.1% of events responded to the first infusion. No incident of LETE occurred. No thrombotic events or FIX inhibitors were reported. Eight of 17 FIX:C approximately 1 week after dosing were >2 IU dL(-1) (min-max of 2.13-10.39 IU dL(-1) ). CONCLUSIONS: Once-weekly prophylaxis of 100 IU kg(-1) was associated with lower ABR compared with on-demand treatment in adolescents and adults with moderately severe to severe haemophilia B. Once-weekly prophylaxis was well tolerated, with a similar safety profile as that reported during the on-demand treatment period. Residual FIX:C may be supportive of effectiveness.

Population pharmacokinetic modelling of factor IX activity after administration of recombinant factor IX in patients with haemophilia B.

PURPOSE: The aim is to develop a pharmacokinetic model for factor IX activity (FIX) after BeneFIX (nonacog alfa, rFIX) administration and assess potential covariates using all available clinical data collected during development. METHODS: The data set for model development combined observations from eight studies. Postdose FIX observations were adjusted by subtracting predose FIX if these were above the lower limit of quantification (BLQ) and all BLQ observations were removed. A population pharmacokinetic model was then developed with 4936 observations from 201 patients. Two additional studies (385 observations from 72 patients) became available and were used to evaluate the model. RESULTS: A two-compartment model, parameterized for clearance (CL), volume of distribution of the central (V1) and peripheral (V2) compartments, and intercompartmental clearance (Q), with an effect of weight on all parameters was the final model. Weight was incorporated as a power function with exponent estimates close to conventional allometric scaling. Including interoccasion variability (IOV) on CL and V1 showed decreases in the objective function. Investigations of a full block omega matrix lead to the retention of a correlation between V2 and Q. Age was not a significant covariate with weight already included in the model. Observations in the studies used for evaluation were found to be higher than simulated values immediately after dosing, as well as a week after dosing. The differences may be due perhaps to differences in the patients enrolled in the evaluation studies (all were adults) as well as the sample collection time after dosing (longer after dosing in the evaluation studies). CONCLUSIONS: FIX is appropriately modelled as a two-compartment model after rFIX administration. When weight is included, no additional effect of age is observed. Longer times of observation after dosing may be helpful in refining the model.

Factor IX oligomerization underlies reduced activity upon disruption of physiological conditions.

Coagulation factor IX (FIX) is a serine protease that plays a pivotal role in the blood coagulation cascade. FIX deficiency leads to a blood clotting disorder known as haemophilia B. FIX, synthesized as a prepro-peptide of 461 amino acids, is processed and secreted into plasma. The protein undergoes numerous modifications, including, but not limited to glycosylation, γ-carboxylation and disulphide bond formation. Upon processing and limited proteolysis, the protein is converted into an active protease. Under physiological conditions, the FIX zymogen is a monomer. The purpose of this work was to analyse the conditions that may affect FIX monomeric state and promote and/or reduce oligomerization. Using native gel electrophoresis and size exclusion chromatography, we found that under decreased pH and ionic strength conditions, the FIX zymogen can oligomerize, resulting in the formation of higher molecular weight species, with a concomitant reduction in specific activity. Similarly, FIX oligomers formed readily with low bovine serum albumin (BSA) concentrations; however, increased BSA concentrations impeded FIX oligomerization. We hypothesize that normal blood physiological conditions are critical for maintaining active FIX monomers. Under conditions of stress associated with acidosis, electrolyte imbalance and low albumin levels, FIX oligomerization is expected to take place thus leading to compromised activity. Furthermore, albumin, which is commonly used as a drug stabilizer, may enhance the efficacy of FIX biological drugs by reducing oligomerization.

Head-to-head comparison of the pharmacokinetic profiles of a high-purity factor IX concentrate (AlphaNine®) and a recombinant factor IX (BeneFIX®) in patients with severe haemophilia B.

Head-on comparative studies of factor IX (FIX) concentrates performed under standardized conditions are rarely conducted regardless of being a valuable instrument guiding health care providers towards better informed and cost-effective decisions. This study is an extension of a multicentre study that assessed the efficacy, safety and pharmacokinetics (PK) of AlphaNine(®) in 25 previously treated patients with severe haemophilia B (FIX:C ≤ 2%). After a washout period ≥ 7 days following the last PK performed with AlphaNine(®) after a dose of 65-75 IU kg(-1) , an identical PK study was performed with BeneFIX(®) on 22 of the same patients. Venous blood samples for analysis were taken at baseline and at 0.25, 0.5, 1, 3, 6, 9, 24, 48, 72 and 74 h post infusion. The outcomes of the comparison of the PK parameters were as follows: Mean (± SD) in vivo recovery (IVR) was 1.3 ± 0.4 IU dL(-1) per IU kg(-1) for AlphaNine(®) and 1.0 ± 0.3 IU dL(-1) per IU kg(-1) for BeneFIX(®) (P < 0.01). Mean terminal half-life, mean residence time, area under the curve, clearance and volume of distribution of BeneFIX(®) were 36.0 ± 12.8 h, 39.3 ± 13.9 h, 1631 ± 467 IU h dL(-1) , 0.046 ± 0.01 dL kg(-1) min(-1) and 1.75 ± 0.52 mL kg(-1) respectively. These values were not significantly different to those observed in AlphaNine(®), although BeneFIX(®) displayed higher than expected IVR values and lower than expected clearance values. In conclusion, AlphaNine(®) showed a comparable half-life, but an IVR significantly higher than that of BeneFIX(®). This dissimilarity may have implications on dosing requirements for on-demand treatment regimes affecting optimal resource allocation.

Confirmation of longer FIX activity half-life with prolonged sample collection after single doses of nonacog alfa in patients with haemophilia B.

A multicentre, single-dose study enrolled 12 previously treated patients with moderately severe to severe (factor IX [FIX] levels ≤2 IU/dl) haemophilia B to assess FIX pharmacokinetics after nonacog alfa administration and to evaluate the impact of length of sampling time on half-life (t½). After refraining from FIX replacement for four days, patients received 50 IU/kg as an intravenous (IV) infusion over 10 minutes. Blood samples were collected predose and 0.25, 0.5, 1, 3, 6, 9, 24, 50, 72, and 96 h post dose. Tolerability and safety were assessed by monitoring adverse events and were subsequently summary tabulated. FIX activity was measured by a one-stage clotting assay with a lower limit of quantification of 0.010 IU/ml, and inhibitors to FIX were measured using the Bethesda assay. Pharmacokinetic parameters were calculated by noncompartmental analysis and were descriptively summarised. Half-life estimates were calculated first using all available data, then excluding 96-h observations (truncated at 72 h) and, finally, excluding both 72- and 96-h observations (truncated at 50 h). No patient was positive for FIX inhibitors. No treatment-emergent adverse events were reported. Prolonging the duration of the sample collection to 96 h resulted in a terminal t½ estimate of 39.6 ±7.4 h in the eight patients aged 18 years and older, which was longer than the estimates obtained using shorter periods of observation: 29.6 ± 5.5 h (truncated at 72 h) and 27.2 ± 7.0 h (truncated at 50 h). To accurately assess an adult patient's t½, sampling should be continued for at least 96 h.

Pharmacokinetics, Efficacy, and Safety of Nonacog Alfa in Previously Treated Patients with Moderately Severe to Severe Hemophilia B.

PURPOSE: Nonacog alfa, a recombinant factor IX (FIX) product, is used for FIX replacement in the treatment and prevention of bleeding events in patients with hemophilia B. This study aimed to provide supplemental pharmacokinetic (PK), efficacy, and safety data for nonacog alfa when administered as part of usual hemophilia care, including on-demand treatment, routine prophylaxis, and surgical prophylaxis. METHODS: Men with previously treated severe or moderately severe hemophilia B (FIX activity ≤2%) were enrolled in this prospective, open-label, nonrandomized, multicenter study. An initial 72-hour PK assessment was performed wherein patients received a single dose of nonacog alfa (75 IU/kg) as an infusion over 10 minutes. A final 72-hour PK assessment was performed at the patient's last visit, after a minimum washout period of 4 days. Correlations between Cmax after the first dose and body weight and body mass index (BMI) were assessed post hoc using Spearman test after evaluating normality. FINDINGS: In total, 23 patients (age, 12-59 years; weight, 44-173 kg; and BMI, 16.3-45.1) with previous exposure to FIX products (median, 460 days; range, 150-2400 days) were enrolled; 21 were evaluable for efficacy. The median number of exposure days per efficacy-evaluable patient in this study was 48 (range, 31-103). The FIX activity profiles showed multiphasic disposition characteristics, with initial mean (SD) PK profiles as follows: Cmax, 61.4 (12.5) IU/dL; AUC∞, 1055 (227) IU·h/dL; t½, 23.7 (5.6) hours; and recovery, 0.818 (0.167) IU/dL. Mean plasma FIX activity versus time profiles were essentially identical upon initial exposure and after repeated use (n = 17), and bioequivalence was confirmed. No apparent relationship was observed between Cmax and either body weight (P > 0.1732) or BMI (P > 0.1235). IMPLICATIONS: The FIX activity profile after administration of nonacog alfa is predictable and is not altered after repeated exposure during usual hemophilia care. PK parameters are consistent with nonacog alfa use for FIX replacement in on-demand treatment, routine prophylaxis, and surgical prophylaxis in patients with hemophilia B.

Continuous and semi-continuous cell culture for production of blood clotting factors.

Recombinant clotting factors are important biotherapeutics that Pfizer has produced and marketed for over fifteen years. Owing to the complexity of the structure and function of these blood factors, it can be challenging to achieve the required product quality and manufacturing productivity. The article highlights the semi-continuous and continuous cell culture processes employed by Pfizer for the production of BeneFIX and ReFacto AF. The benefits of such processes, the challenges of maintaining an aseptic production culture for extended periods, and batch definition are discussed in this article.