Factor 4 Research Articles

Soluble expression of recombinant coagulation factor IX protein using Escherichia coli

Hemophilia B is a congenital bleeding disorder caused by factor IX (FIX) deficiency. Generation of recombinant FIX (rFIX) is required for detecting a Hemophilia B indicator, anti-FIX antibody. In this study, we described a method for producing recombinant FIX (rFIX) using Escherichia coli. We constructed a FIX-expressing plasmid without a fusion tag protein-encoding gene and produced rFIX as a soluble form within five days. Dose-dependent curve was obtained from ELISA using anti-FIX antibody, indicating that the rFIX can be used as an antigen to detect anti-FIX antibody with high affinity and sensitivity.

Assessing health plan payer's budget impact of etranacogene dezaparvovec for the treatment of hemophilia B in the United States.

Etranacogene dezaparvovec is a recently approved gene therapy for people with hemophilia B (PwHB). Current standard of care is prophylaxis with factor IX (FIX) to prevent bleeding. Etranacogene dezaparvovec increases blood FIX levels such that FIX prophylaxis could be eliminated. To estimate the budgetary impact of etranacogene dezaparvovec adoption and utilization in a commercial health plan of the United States. A budget impact model was developed to evaluate the introduction of etranacogene dezaparvovec to treat severe or moderately severe hemophilia B. The model considered a hypothetical 1-million-member plan over a 5-year horizon. FIX therapy prophylaxis use was estimated based on a weighted average of relevant brands using US market share data. A scenario of etranacogene dezaparvovec adoption/utilization was compared with one without etranacogene dezaparvovec utilization. Two etranacogene dezaparvovec uptake (market share growth) analyses were performed: one with gradual uptake and alternatively assuming all eligible PwHB received etranacogene dezaparvovec in year 1. The one-time cost of etranacogene dezaparvovec was assumed to be $3.5 million. Other costs (FIX prophylaxis, disease monitoring, bleed management, and adverse events) were estimated from published literature. All costs were in 2022 US dollars. Bleed and adverse event rates were sourced from the HOPE-B trial comparing etranacogene dezaparvovec to previous FIX therapy prophylaxis. The model estimated annual and per-member per-month costs over 5 years. Secondary analyses were performed considering a 10-year horizon. In the 1-million-member health plan, an estimated 1.8 PwHB were eligible for treatment with etranacogene dezaparvovec. Gradual uptake of etranacogene dezaparvovec resulted in cumulative 5-year budget impact of $848,509 compared with a scenario without etranacogene dezaparvovec. In years 1-5, the incremental annual and per-member per-month costs ranged from $79,824 to $271,435 and from $0.007 to $0.023, respectively. In the alternative uptake analysis, etranacogene dezaparvovec became cost saving annually beginning in year 2 and cumulatively beginning in year 5, for a 5-year savings of $754,844. Secondary analyses over 10 years found both uptake analyses cost saving. Other scenarios considered did not affect results substantially. Introducing etranacogene dezaparvovec as treatment for PwHB would have a modest budget increase within 5 years after treatment but may become cost saving if all eligible PwHB were treated in year 1. Initiating PwHB on etranacogene dezaparvovec sooner may produce greater overall savings and earlier annual savings. Etranacogene dezaparvovec is a treatment option that may provide overall cost savings for US commercial health plans, which would increase as the plan size increases.

Haemophilia in the era of novel therapies: Where do inhibitors feature in the new landscape?

The advent of therapeutic recombinant factor VIII (FVIII) and factor IX (FIX) protein infusions revolutionized the care of persons with haemophilia in the 1990s. It kicked off an era with the increasing use of prophylactic factor infusions for patients and transformed conversations around the ideal trough activity levels as well as the ultimate goals in tailored, individualized care. Our knowledge surrounding the immunologic basis of inhibitor development and treatment derives from a time when patients were receiving frequent factor infusions and focused on immune tolerance induction following inhibitor development. More recently, care was revolutionized again in haemophilia A with the approval of emicizumab, a bispecific antibody mimicking activated FVIII function, to prevent bleeding. The use of emicizumab prophylaxis has resulted in a significantly slower accumulation of factor exposure days and continued effective prophylaxis in the case of inhibitor development. While emicizumab is effective at reducing the frequency of bleeding events in patients with haemophilia A, management of breakthrough bleeds, trauma, and surgeries still requires additional treatment. Ensuring that FVIII is a therapeutic option, particularly for life-threatening bleeding events and major surgeries is critical to optimizing the care of persons with haemophilia A. Other novel non-factor concentrate therapies, including rebalancing agents, will dramatically change the landscape for persons with haemophilia B with inhibitors. This review discusses the changing landscape regarding the timing of inhibitor development and management strategies after inhibitor development, stressing the importance of education across the community to continue to vigilantly monitor for inhibitors and be prepared to treat persons with inhibitors.

Cost-Effectiveness Analysis of Etranacogene Dezaparvovec Versus Extended Half-Life Prophylaxis for Moderate-to-Severe Haemophilia B in Germany.

Haemophilia B is a rare genetic disease that is caused by a deficiency of coagulation factor IX (FIX) in the blood and leads to internal and external bleeding. Under the current standard of care, haemophilia is treated either prophylactically or on-demand via intravenous infusions of FIX. These treatment strategies impose a high burden on patients and health care systems as haemophilia B requires lifelong treatment, and FIX is costly. Etranacogene dezaparvovec (ED) is a gene therapy for haemophilia B that has been recently approved by the United States Food and Drug Administration and has received a recommendation for conditional marketing authorization by the European Medicines Agency. We aimed to examine the cost-effectiveness of ED versus extended half-life FIX (EHL-FIX) prophylaxis for moderate-to-severe haemophilia B from a German health care payer perspective. A microsimulation model was implemented in R. The model used data from the ED phase 3 clinical trial publication and further secondary data sources to simulate and compare patients receiving ED or EHL-FIX prophylaxis over a lifetime horizon, with the potential for ED patients to switch treatment to EHL-FIX prophylaxis when the effectiveness of ED waned. Primary outcomes of this analysis included discounted total costs, discounted quality-adjusted life years (QALYs), incremental cost-effectiveness, and the incremental net monetary benefit. The annual discount rate for costs and effects was 3%. Uncertainty was examined via probabilistic analysis and additional univariate sensitivity analyses. Probabilistic analysis indicated that patients treated with ED instead of EHL-FIX prophylaxis gained 0.50 QALYs and experienced cost savings of EUR 1,179,829 at a price of EUR 1,500,000 per ED treatment. ED was the dominant treatment strategy. At a willingness to pay of EUR 50,000/QALY, the incremental net monetary benefit amounted to EUR 1,204,840. Depending on the price, ED can save costs and improve health outcomes of haemophilia patients compared with EHL-FIX prophylaxis, making it a potentially cost-effective alternative. These results are uncertain due to a lack of evidence regarding the long-term effectiveness of ED.

A common protein C inhibitor exosite partially controls the heparin induced activation and inhibition of serine proteases

Protein C inhibitor (PCI) maintains hemostasis by inhibiting both procoagulant and anticoagulant serine proteases, and plays important roles in coagulation, fibrinolysis, reproduction, and anti-angiogenesis. The reactive site loop of PCI traps and irreversibly inhibits the proteases like APC (activating protein C), thrombin (FIIa) and factor Xa (FXa). Previous studies on antithrombin (ATIII) had identified Tyr253 and Glu255 as functional exosites that interact and aid in the inhibition of factor IXa and FXa. Presence of exosite in PCI is not known, however a sequence comparison with the PCI from different vertebrate species and ATIII identified Glu239 to be absolutely conserved. PCI residues analogous to ATIII exosite residues were mutated to R238A and E239A. Purified variant PCI in the presence of heparin (10 μg/ml) showed a 2–4 fold decrease in the rate of inhibition of the proteases. However, the stoichiometry of inhibition of FIIa, APC, and FXa by native PCI, R238A and E239A variants were found to be close to 1.0, which also indicated the formation of stable complexes based on SDS-PAGE and western blot analysis with thrombin and APC. Our findings revealed the possible presence of an exosite in PCI that influences the protease inhibition rates.

Prophylactic Treatment of Children with Hemophilia in Sweden.

Hemophilia A/B are caused by deficiency or lack of coagulation factors VIII (FVIII) or factor IX (FIX), respectively, in plasma. A person with hemophilia develops bleeding in the joints and muscles at an early age, which, if left untreated, leads to early arthropathy. Preventive treatment can be achieved by regular (prophylactic) administration of FVIII/FIX. In 1958, this was implemented on a small scale in Sweden with FVIII in patients with severe hemophilia A, and in those with hemophilia B in 1972 when FIX became available. However, there were problems with human immunodeficiency virus and hepatitis infection from contaminated blood products. In the 1990s, recombinant FVIII and FIX concentrates were introduced. The major remaining problems then were the development of inhibitors, and the need for a venous route for the injections in very young children. High-titer inhibitors were treated by immune tolerance induction according to a modified model of the original Bonn high-dose protocol. A central venous line, i.e., Port-A-Cath, has enabled early prophylaxis in many children with poor venous access and has enabled the early start of home treatment with adequate injection frequency. Scoring systems for X-rays, magnetic resonance imaging, and function of joints were developed early in Sweden and have been widely disseminated worldwide, partly with modifications. Extended half-life products with half-life increased three to five times have been developed, which can provide superior bleed protection when dosed once-weekly and can maintain therapeutic trough levels when administered less frequently. The ultimate prophylaxis therapy in the future may be gene therapy.

Genetic variant detection in a South African haemophilia B population.

Haemophilia B is characterised by a deficiency of factor IX (FIX) protein due to genetic variants in the FIX gene (F9). Genetic testing may have a vital role in effectively managing haemophilia B. However, in many developing countries, comprehensive genetic variant detection is unavailable. This study aimed to address the lack of genetic data in our country by conducting genetic variant detection on people affected by haemophilia B in our region. Twenty-one participants were screened with a direct Sanger sequencing method to identify variants in the F9 gene. The identified variants were then compared to previously published variants and/or to a reference database. A total of ten F9 genetic changes were detected, with five of them being novel. These identified variants were distributed across different domains of the FIX protein. Only one participant had a history of inhibitor formation against FIX replacement therapy. Notably, this participant had two distinct genetic changes present adjacent to each other. Thus, we hypothesise that the presence of multiple variants within the same functional region of the gene may increase the risk for inhibitor development. The discovery of novel pathogenic variations in the F9 gene highlights the importance of genetic analysis in specific geographical regions. The possible link between a complex variant and inhibitor formation illustrates the potential role that genetic screening has as a pre-treatment tool in predicting treatment reactions and outcomes.

Effect of etranacogene dezaparvovec on quality of life for severe and moderately severe haemophilia B participants: Results from the phase III HOPE-B trial 2 years after gene therapy.

For people with haemophilia B (PwHB), bleeding may occur despite prophylaxis, negatively affecting health-related quality of life (HRQoL). The pivotal phase 3 HOPE-B trial investigating the adeno-associated virus gene transfer product, etranacogene dezaparvovec (EDZ), demonstrated sustained factor IX (FIX) activity and bleed protection in PwHB with baseline FIX levels ≤2%. Assess how EDZ affects HRQoL in HOPE-B trial participants. HRQoL was evaluated using generic and disease-specific patient reported outcomes (PROs) including the EQ-5D-5L and the Hem-A-QoL questionnaires. Mean domain and total scores were compared 6 months pre- and the first 2 years post-EDZ administration using repeated measures linear mixed models. The percentage of participants with minimal clinically important improvements in HRQoL was also evaluated. Two years post-EDZ, there were nominally significant increases in the least squares (LS) mean score for the EQ-5D-5L Index Value (.04; p=.0129), reflecting better HRQoL. Nominally significant decreases in the LS mean scores, reflecting better HRQoL, were also found for the Hem-A-QoL total score (-6.0; p<.0001) and the Treatment (-13.94; p<.0001), Feelings (-9.01; p<.0001), Future (-6.45; p=.0004) and Work/School (-5.21; p=.0098) domains. The percentage of participants with ≥15-point improvement ranged from 45.83% (95% CI: 31.37%, 60.83%) for Treatment to 13.89% (95% CI: 4.67%, 29.50%) for Family Planning. Results were similar for Year 1. In conclusion, gene therapy with EDZ improved HRQoL in the first and second years in several Hem-A-QoL domains, including Treatment, Feelings, Work/School and Future domains, whereas improvement in other aspects of HRQoL were not demonstrated.

Six-Year, Real-World Use of Prophylaxis with Recombinant Factor IX-Albumin Fusion Protein (rIX-FP) in Persons with Hemophilia B: A Single-Center Retrospective-Prospective Study.

Background: Extended half-life (EHL) factor IX (FIX) concentrates allow for prophylaxis with prolonged dosing intervals and high bleeding protection in persons with hemophilia B. Long-term real-world studies are lacking. Methods: In a retrospective-prospective study, the six-year use of prophylaxis with the EHL recombinant FIX-albumin fusion protein (rIX-FP) was analyzed, comparing outcomes with previous standard half-life (SHL) FIX in patients already on prophylaxis. Results: Prophylaxis with rIX-FP was prescribed in 15 patients (10 severe, 5 moderate; follow-up: 57 ± 17 months). Based on a pharmacokinetic assessment and clinical needs, the first regimen was 47 ± 7 IU/Kg every 9 ± 2 days. All but one patient remained on rIX-FP prophylaxis, adjusting infusion frequency and/or dose; the last prescribed frequency was ≥10 days in 10/13 patients, being reduced in seven and increased in four vs. the first regimen. The weekly FIX dose was unchanged; FIX trough levels were >5% in all patients. The annual infusion number and FIX IU/Kg significantly decreased (~60%) in eight patients previously on SHL FIX prophylaxis, with similar concentrate costs. Very low bleeding rates (most traumatic bleeds and the last quartile of the infusion interval), improved orthopedic and pain scores, unchanged HEAD-US scores and problem joints, and high treatment adherence (>90%) and satisfaction were registered. Conclusions: Personalized, carefully adjusted rIX-FP regimens contribute to the diffusion and optimization of prophylaxis in persons with severe and moderate hemophilia B, with long-term favorable bleeding, joint, and patient-reported outcomes.

Walking ability in adults with severe hemophilia: A cross-sectional study.

A lack of factor VIII (FVIII) or factor IX (FIX) results in hemophilia, a blood-clotting illness. The mode of inheritance is chromosome X-linked and recessive. The primary symptom of severe hemophilia is spontaneous and recurrent bleeding into joints, muscles, and soft tissues. Unpreventable bleeding may cause arthropathy, chronic discomfort, and muscular atrophy. Therefore, joints' functional loss affects the functional and walking ability. The aim of this study was to determine the walking ability by measuring the 50-m walk test time in severe hemophilic patients, as compared to the normal population. Sixty subjects (males) in the 18-30 year age group were selected and comprised 30 hemophiliacs and 30 in a control group. The 50-m walking ability was measured in seconds. The results showed a normative value of 36.6 sec in the control and 67.2 sec in the hemophilic group. Statistical analysis of the data showed that the walking ability was significantly reduced in the hemophilic group. These normative values illustrate a useful, simple, reproducible, rapid assessment of walking disability in adults with hemophilic arthropathy, and also aid the planning of treatment.

A Post-Authorization Safety Surveillance Study to Report Clinical Experience with Purified Factor IX Concentrate in Pediatric Patients with Hemophilia B.

Purified factor IX (FIX) concentrate (IMMUNINE®, Takeda Manufacturing Austria AG, Vienna, Austria) is indicated for the treatment and prophylaxis of bleeding episodes in patients with congenital hemophilia B. Data on the use of purified FIX concentrate in patients ≤6 years old with congenital hemophilia B are limited. Document real-world clinical experience with purified FIX concentrate in routine practice for pediatric patients with hemophilia B. This prospective post-authorization safety surveillance study enrolled patients ≤6 years old with moderate or severe hemophilia B (baseline FIX ≤5%) who were prescribed purified FIX concentrate, as determined by the treating physician. The planned observation period for each patient was either 12 months or ≥50 exposure days, whichever occurred first. The primary endpoints were the occurrence of treatment-related adverse events (AEs) and serious AEs (SAEs), and inhibitor development. Thirteen male patients (mean ± standard deviation age, 3.80 ± 1.76 years) enrolled and received ≥1 treatment with purified FIX concentrate. Thirty-two AEs were reported in 6 patients; 4 were SAEs. No AEs were considered related to purified FIX concentrate. No patients developed inhibitory antibodies. Inhibitor testing was not conducted in 2 patients. Eighteen bleeding episodes were treated with purified FIX concentrate in 6 patients. Hemostatic efficacy was rated as either "excellent" or "good" in all patients with an available rating. No treatment-related AEs were reported, and purified FIX concentrate was shown to be effective in treating and preventing bleeding episodes in pediatric patients ≤6 years old with hemophilia B.

Abstract 2008 The Latent State of Protease Domain in Coagulation Factor IXa Discovered by X-Ray Crystallography Provides Exosite-Mediated Allosteric Control of Blood Clotting

Abstract 2008 The Latent State of Protease Domain in Coagulation Factor IXa Discovered by X-Ray Crystallography Provides Exosite-Mediated Allosteric Control of Blood Clotting

Revisiting the effect of cholesteryl sulfate on clotting and fibrinolysis: Inhibition of human thrombin and other human blood proteases

Cholesteryl sulfate (CS) was quantitatively synthesized by microwave-assisted sulfonation of cholesterol followed by sodium exchange chromatography. In vitro effects of CS on human thrombin and other serine proteases of the coagulation and fibrinolysis processes were investigated using a series of biochemical and biophysical techniques. CS was found to inhibit thrombin with an IC50 value of 140.8 ± 21.8 μM at pH 7.4 and 25 ○C. Michaelis-Menten kinetics indicated that thrombin inhibition by CS is non-competitive (allosteric) in nature. Fluorescence-based binding studies indicated that CS binds to thrombin with a KD value of 180.9 ± 18.9 μM. Given the lack of competition with heparins and a hirudin peptide in competitive inhibition assays, it appears that CS does not bind to thrombin's exosites 1 or 2 and it rather recognizes a different allosteric exosite. CS was found to partially inhibit thrombin-mediated fibrinogen activation with an IC50 value of 175.5 ± 17.5 μM and efficacy of ∼26.0 ± 6.6%. Likewise, CS selectively doubled the activated partial thromboplastin time with EC2x of 521 μM. Interestingly, CS was found to also inhibit factors Xa and XIa as well as plasmin with IC50 values of ∼85–250 μM and efficacy of 94–100%. Nevertheless, CS most potently inhibited factor XIIa with an IC50 Value of ∼17 μM and efficacy of 60%. Surprisingly, CS did not inhibit factor IXa. These results encourage further in vitro and in vivo investigation of CS to better understand its (patho-) physiological roles in coagulation and hemostasis.

Клинический случай ингибиторной формы гемофилии В у ребенка

Hemophilia B is a hereditary, X-linked recessive disease caused by a violation of plasma homeostasis, resulting from a deficiency or absence of coagulation factor IX (FIX). The cause is a mutation of the Xq27 gene encoding FIX. A serious complication of this pathology is the inhibitory form, clinically manifested by the presence of IgG allo-antibodies neutralizing exogenous FIX. The article describes a clinical case of an inhibitory form of hemophilia B in a 12-year-old child.

New Triazole-Based Potent Inhibitors of Human Factor XIIa as Anticoagulants.

Factor XIIa (FXIIa) functions as a plasma serine protease within the contact activation pathway. Various animal models have indicated a substantial role for FXIIa in thromboembolic diseases. Interestingly, individuals and animals with FXII deficiency seem to maintain normal hemostasis. Consequently, inhibiting FXIIa could potentially offer a viable therapeutic approach for achieving effective and safer anticoagulation without the bleeding risks associated with the existing anticoagulants. Despite the potential, only a limited number of small molecule inhibitors targeting human FXIIa have been documented. Thus, we combined a small library of 32 triazole and triazole-like molecules to be evaluated for FXIIa inhibition by using a chromogenic substrate hydrolysis assay under physiological conditions. Initial screening at 200 μM involved 18 small molecules, revealing that 4 molecules inhibited FXIIa more than 20%. In addition to being the most potent inhibitor identified in the first round, inhibitor 8 also exhibited a substantial margin of selectivity against related serine proteases, including factors XIa, Xa, and IXa. However, the molecule also inhibited thrombin with a similar potency. It also prolonged the clotting time of human plasma, as was determined in the activated partial thromboplastin time and prothrombin time assays. Subsequent structure-activity relationship studies led to the identification of several inhibitors with submicromolar activity, among which inhibitor 22 appears to demonstrate significant selectivity not only over factors IXa, Xa, and XIa, but also over thrombin. In summary, this study introduces novel triazole-based small molecules, specifically compounds 8 and 22, identified as potent and selective inhibitors of human FXIIa. The aim is to advance these inhibitors for further development as anticoagulants to provide a more effective and safer approach to preventing and/or treating thromboembolic diseases.

Recombinant factor IX Fc for the treatment of hemophilia B.

Current hemophilia B treatment guidelines recommend routine prophylaxis with factor IX (FIX) replacement products, tailored to maintain plasma activity at levels that will prevent bleeds. However, plasma FIX activity may not be the primary determinant or best indicator of hemostatic efficacy due to its extravascular distribution. FIX replacement therapy has evolved to include extended half-life (EHL) products that provide effective bleed protection when administered at intervals of 7 days or longer. rFIXFc is a recombinant fusion protein with an extended circulation time. rFIXFc has a biodistribution profile consistent with distribution into extravascular space, where it may support hemostasis at sites of vessel injury independent of circulating plasma activity levels. The safety and efficacy of rFIXFc prophylaxis is well established in adults, adolescents and children including previously untreated patients with hemophilia B, with substantial evidence from clinical trials and real-world clinical practice. This review describes the pharmacokinetic characteristics of rFIXFc, summarizes available safety and efficacy data, and evaluates the use of rFIXFc in special populations. Current hemophilia B treatment challenges, including target FIX plasma levels, perioperative use, and management of patients with comorbidities, are discussed together with the potential role of EHL products in the future treatment landscape of hemophilia B.

Effectiveness of emicizumab in preventing bleeding events in severe and moderate hemophilia A: A single-center experience in Bangladesh.

Emicizumab is a monoclonal antibody that bridges activated factor IX (FIX) and factor X (FX) to replace the function of missing activated factor VIII (FVIII) in hemophilia A patients irrespective of FVIII inhibitor status. This study assessed the effectiveness of emicizumab in preventing bleeding episodes in patients with hemophilia A. This observational study included patients with moderate to severe hemophilia A who were undergoing episodic FVIII replacement therapy. The primary endpoint was the difference in annualized bleeding rates (ABR) and the secondary endpoint was the difference in Hemophilia Joint Health Score (HJHS) before and after emicizumab prophylaxis. A total of 30 male hemophilia patients were included, the mean age was 16.7 (SD: ±8.1) years, and most of them had moderate hemophilia A [63.3%]. Before prophylaxis, the median ABR was 48 (interquartile range [IQR]: 35-60), and 93.3% of patients had ABR greater than eight, whereas after prophylaxis the median ABR decreased significantly (median [IQR]: 0 [0.0-0.4], p<0.001), and 56.7% had zero bleeds. ABR was not significantly different in patient with and without FVIII inhibitors. The HJHS scores significantly improved after prophylaxis (10 vs. 2.5, p<0.001). The bleeding events were reduced significantly (23 vs. 0.0, p<0.001), and zero new target joints were reported after prophylaxis. Most of the patients [93.3%] did not face any serious adverse events after prophylaxis. Emicizumab prophylaxis was associated with a significantly lower rate of bleeding events among participants with hemophilia A, regardless of inhibitor status.

Safety and Efficacy of Recombinant Fusion Protein Linking Coagulation Factor IX with Albumin (rIX-FP) in Previously Untreated Patients with Hemophilia B.

Introduction Recombinant fusion protein linking coagulation factor IX (FIX) with albumin (rIX-FP) has been shown to be an effective, well-tolerated treatment for patients with severe hemophilia B who had previously received factor replacement therapy. This study investigated the safety and efficacy of rIX-FP in previously untreated patients (PUPs). Methods Patients with moderately severe/severe hemophilia B (≤2% FIX) previously untreated with FIX replacement products received rIX-FP (25-75 IU/kg) prophylaxis weekly or on-demand treatment over ≥50 exposure days (EDs). Primary outcomes were the number of patients who developed FIX inhibitors and mean incremental recovery (IR) following a 50 IU/kg dose of rIX-FP. Secondary outcomes included incidence of adverse events (AEs) and annualized bleeding rates (ABRs). Results In total, 12 PUPs with a median age of 0 years (range, 0-11 years) were treated with rIX-FP for a median of 50 EDs (6/12 prophylaxis; 6/12 on-demand then prophylaxis). Overall, 11/12 patients did not develop FIX inhibitors; one 11-year-old patient developed an inhibitor against FIX after 8 EDs and was ultimately withdrawn. Mean (standard deviation) IR was 1.2 (0.4, n = 8) (IU/dL)/(IU/kg). Of the 137 treatment-emergent AEs recorded, five were attributed to rIX-FP. On the prophylaxis regimen, median ABR was 1.0 (range, 0-3.9, n = 12). No thromboembolic events or deaths occurred during the study. Conclusion This study provides data to support the safety and efficacy of rIX-FP in PUPs requiring on-demand or prophylactic treatment for moderately severe/severe hemophilia B, consistent with results in previously treated patients. Overall, 1/12 patients developed an inhibitor against FIX.

Docking-based computational analysis of guava (Psidium guajava) leaves derived bioactive compounds as a coagulation factor IXa inhibitor.

Thrombotic disorders pose a global health threat, emphasizing the urgent need for effective management strategies. This study explores the potential of bioactive compounds derived from guava leaves in inhibiting coagulation factor IXa (CFIXa) using in silico methods. Using GC-MS, bioactive compounds extracted from guava leaf through ethanol maceration were identified. Pharmacokinetic properties were elucidated using SwissADME. Molecular docking with AutoDock Vina was used to investigate the interactions with CFIXa. CFIXa was modeled with pysimm/LAMMPS and analyzed with CastP for active site identification. The setup with a higher solvent concentration and lower surface area yielded the highest percent yield (78.541 g, 39.27%). Among the 28 identified bioactive compounds, predominantly terpenoids, only seven exhibited suitable pharmacokinetic properties for oral ingestion and drug development. Docking analysis revealed favorable binding of these compounds to CFIXa (-7.6:-5.3). This study shows inhibition of coagulation factor IXa, thus bridging the ambiguity surrounding the effect of guava leaves on hemostasis. These findings also reveal that guava leaf extract harbors bioactive compounds with potential as coagulation pathway inhibitors, promising novel avenues for thrombotic disorder management.

Inhibition of Ehrlich ascites carcinoma growth by melatonin: Studies with micro-CT.

Melatonin is a versatile indolamine synthesized and secreted by the pineal gland in response to the photoperiodic information received by the retinohypothalamic signaling pathway. Melatonin has many benefits, such as organizing circadian rhythms and acting as a powerful hormone. We aimed to show the antitumor effects of melatonin in both in vivo and in vitro models through the mammalian target of rapamycin (mTOR) signaling pathway and the Argyrophilic Nucleolar Regulatory Region (AgNOR), using the Microcomputed Tomography (Micro CT). Ehrlich ascites carcinoma (EAC) cells were administered into the mice by subcutaneous injection. Animals with solid tumors were injected intraperitoneally with 50 and 100 mg/kg melatonin for 14 days. Volumetric measurements for the taken tumors were made with micro-CT imaging, immunohistochemistry (IHC), real-time polymerase chain reaction (PCR) and AgNOR. Statistically, the tumor tissue volume in the Tumor+100 mg/kg melatonin group was significantly lower than that in the other groups in the data obtained from micro-CT images. In the IHC analysis, the groups treated with Tumor+100 mg/kg melatonin were compared when the mTOR signaling pathway and factor 8 (F8) expression were compared with the control group. It was determined that there was a significant decrease (p < 0.05). Significant differences were found in the total AgNOR area/nuclear area (TAA/NA) ratio in the treatment groups (p < 0.05). Furthermore, there were significant differences between the amount of mTOR mRNA for the phosphatidylinositol 3-kinase (PI3K), AKT Serine/Threonine Kinase (PKB/AKT) genes (p < 0.05). Cell apoptosis was evaluated with Annexin V in an in vitro study with different doses of melatonin; It was observed that 100 µg/mL melatonin dose caused an increase in the apoptotic cell death. In this study, we have reported anti-tumor effects of melatonin in cell culture studies as well as in mice models. Comprehensive characterization of the melatonin-mediated cancer inhibitory effects will be valuable in advancing our fundamental molecular understanding and translatability of pre-clinical findings to earlier phases of clinical trials.