Clinical Immunogenicity Evaluation of Eptinezumab, a Therapeutic Humanized Monoclonal Antibody Targeting Calcitonin Gene-Related Peptide (CGRP) for the Preventive Treatment of Migraine.

Background: Eptinezumab is a humanized monoclonal antibody that selectively binds calcitonin gene-related peptide and is indicated for the preventive treatment of migraine in adults. This analysis characterizes the immunogenic profile of eptinezumab using data from clinical trials of eptinezumab for migraine prevention. Methods: Immunogenicity data were collected from five studies that included 2076 patients with episodic or chronic migraine treated with eptinezumab at dose levels ranging from 10 to 1000 mg, administered intravenously for up to 4 doses at 12-week intervals. Anti-drug antibody (ADA) results were available from 2074 of these patients. Four studies were randomized, double-blind, placebo-controlled trials with ADA monitoring for up to 56 weeks; one was a 2-year, open-label, phase 3 safety study with ADA monitoring for 104 weeks. Patients who had a confirmed ADA-positive result at the end-of-study visit were monitored for up to 6 additional months. Development of ADA and neutralizing antibodies (NAbs) were evaluated to explore three key areas of potential impact: pharmacokinetic exposure profile (eptinezumab trough plasma concentrations), efficacy (change in monthly migraine days), and safety (rates of treatment-emergent adverse events). These studies included methods designed to capture the dynamics of a potential humoral immune response to eptinezumab treatment, and descriptive analyses were applied to interpret the relationship of ADA signals to drug exposure, efficacy, and safety. Results: Pooled across the five clinical trials, treatment-emergent ADAs and NAbs occurred in 15.8 and 6.2% of eptinezumab-treated patients, respectively. Highly consistent profiles were observed across all studies, with initial onset of detectable ADA observed at the week 8 measurement and maximal ADA frequency and titer observed at week 24, regardless of eptinezumab dose level or number of doses. After 24 weeks, the ADA and NAb titers steadily declined despite additional doses of eptinezumab. Interpretation: Collectively, these integrated analyses did not demonstrate any clinically meaningful impact from ADA occurring after treatment with eptinezumab. The ADA profiles were low titer and transient, with the incidence and magnitude of ADA or NAb responses declining after week 24. Development of ADAs and NAbs did not impact the efficacy and safety profiles of eptinezumab.

Tolerability of eptinezumab in overweight, obese or type 1 diabetes patients.

Introduction: In addition to its role in the pathogenesis of migraine, calcitonin gene-related peptide (CGRP) is implicated in the regulation of insulin secretion. However, there are limited data on the use of CGRP inhibitor monoclonal antibodies in individuals who are overweight/obese and those with diabetes. Methods: Two randomized, double-blind, placebo-controlled trials were conducted to assess the safety and metabolic effects of eptinezumab in non-migraine overweight/obese patients (study 1) and patients with type 1 diabetes (T1D; study 2). The primary end-point in overweight/obese patients was safety and changes in basal metabolic rate (BMR), defined as the energy expenditure during the fasting and resting states. In patients with T1D, the primary end-points were safety and insulin sensitivity as assessed by the bodyweight and insulin concentration corrected glucose infusion rate (M/I). Results: A total of 24 patients were enrolled in study 1, and 21 patients were enrolled in study 2. In overweight/obese patients, there was no significant difference in the least squares (LS) mean change in BMR between the eptinezumab- and placebo-treated patients from baseline to day 7 (6.4 vs -25.2 Kcal/day; LS mean difference 31.6 [95% confidence interval -90.6, 153.8]). In patients with T1D, there was no significant difference in insulin sensitivity between the eptinezumab and placebo groups. Eptinezumab was well tolerated in both studies with a similar rate of adverse events between treatment groups, and no new safety signals were identified. Conclusion: Eptinezumab was well tolerated and not associated with adverse metabolic effects in patients who were overweight/obese or had T1D, providing ongoing support for the use of eptinezumab in these subgroups of patients with migraine.

Long-term safety and tolerability of eptinezumab in patients with chronic migraine: a 2-year, open-label, phase 3 trial.

BACKGROUND: Eptinezumab, an anti-calcitonin gene-related peptide monoclonal antibody recently approved in the United States for preventive treatment of migraine in adults, was found to be well tolerated in double-blind, placebo-controlled studies in patients with episodic and chronic migraine. The objective of the PREVAIL study was to evaluate the long-term safety, immunogenicity, and impact on patient-reported outcomes of repeat doses of eptinezumab in patients with chronic migraine. METHODS: PREVAIL was an open-label, phase 3 trial comprising a 48-week treatment phase followed by a second 48-week treatment phase. Adults with chronic migraine received eptinezumab 300 mg by 30-min intravenous administration every 12 weeks for up to 8 doses. Patients were followed for 20 weeks after the final infusion (end-of-study visit at week 104). RESULTS: Overall, 128 adults (mean age, 41.5 years) with chronic migraine were included. During the 2 years, the most frequently reported treatment-emergent adverse events were nasopharyngitis (14.1%), upper respiratory tract infection (7.8%), sinusitis (7.8%), influenza (6.3%), bronchitis (5.5%), and migraine (5.5%). The rate of study-drug discontinuation due to adverse events was 6.3%, which included 3 patients with infusion-related hypersensitivity. The incidence of anti-eptinezumab antibodies peaked at week 24 and declined despite continued dosing, to nondetectable levels at week 104. Improvements in patient-reported outcomes were observed at first assessment (week 4) and generally sustained through week 104. CONCLUSION: In adults with chronic migraine, eptinezumab 300 mg demonstrated a favorable safety profile, limited long-term immunogenicity, early and sustained reductions in migraine-related burden, and improvements in health-related quality of life over 2 years. TRIAL REGISTRATION: ClinicalTrials.gov (Identifier: NCT02985398 ).

Eptinezumab for the Prevention of Episodic Migraine: Sustained Effect Through 1 Year of Treatment in the PROMISE-1 Study.

PURPOSE: The Prevention of Migraine via Intravenous ALD403 Safety and Efficacy 1 (PROMISE-1) study was a phase III, randomized, double-blind, placebo-controlled study designed to evaluate the efficacy, tolerability, and pharmacokinetic properties of repeat intravenous (IV) doses of the calcitonin gene-related peptide‒targeted monoclonal antibody eptinezumab (ALD403) for migraine prevention in adults with episodic migraine. Here we present the results of PROMISE-1 through 1 year of treatment (up to 4 doses). METHODS: Patients received up to 4 IV administrations of eptinezumab 30 mg, 100 mg, 300 mg, or placebo every 12 weeks. Patients recorded migraine and headache in an electronic diary daily. Additional assessments, including the patient-reported outcomes, were performed at regularly scheduled clinic visits throughout the 56-week study period. FINDINGS: A total of 888 adults (mean age, 39.8 years; 84.3% female; 83.8% white) received treatment: eptinezumab 30 mg, n = 219; eptinezumab 100 mg, n = 223; eptinezumab 300 mg, n = 224; and placebo, n = 222. During the primary 12-week study evaluation period, single doses of eptinezumab 100 mg and 300 mg led to significant reductions in mean monthly migraine-days versus placebo, beginning as early as the first day after the initial dose. The reduction in mean monthly migraine-days was maintained throughout the study (100 mg, -3.9, -4.5, -4.7, and -4.5 days; 300 mg, -4.3, -4.8, -5.1, and -5.3 days; and placebo, -3.2, -3.8, -4.0, and -4.0 days during weeks 1-12, 13-24, 25-36, and 37-48, respectively). Overall, the number of patients with a ≥50% or ≥75% reduction in migraine for each 12-week interval during the entire study was consistently numerically higher in the eptinezumab groups than in the placebo group. The proportions of patients with ≥50% reduction in migraine were similar across the eptinezumab groups. Eptinezumab was well tolerated throughout the study. Adverse events were similar across dosing periods, and there were no serious tolerability signals identified with continued dosing. IMPLICATIONS: IV eptinezumab administered every 12 weeks for up to 4 doses was associated with early and sustained migraine-preventive effects and a favorable safety profile in adults with episodic migraine. ClinicalTrials.gov identifier: NCT02559895.

Eptinezumab for the prevention of chronic migraine: efficacy and safety through 24 weeks of treatment in the phase 3 PROMISE-2 (Prevention of migraine via intravenous ALD403 safety and efficacy-2) study.

BACKGROUND: PROMISE-2 was a phase 3, randomized, double-blind, placebo-controlled study that evaluated the efficacy and safety of repeat intravenous (IV) doses of the calcitonin gene-related peptide-targeted monoclonal antibody eptinezumab (ALD403) for migraine prevention in adults with chronic migraine. This report describes the results of PROMISE-2 through 24 weeks of treatment. METHODS: Patients received up to two 30-min IV administrations of eptinezumab 100 mg, 300 mg, or placebo separated by 12 weeks. Patients recorded migraine and headache endpoints in a daily eDiary. Additional assessments, including patient-reported outcomes, were performed at regularly scheduled clinic visits throughout the 32-week study period (screening, day 0, and weeks 2, 4, 8, 12, 16, 20, 24, and 32). RESULTS: A total of 1072 adults received treatment: eptinezumab 100 mg, n = 356; eptinezumab 300 mg, n = 350; placebo, n = 366. The reduction in mean monthly migraine days observed during the first dosing interval (100 mg, - 7.7 days; 300 mg, - 8.2 days; placebo, - 5.6 days) was further decreased after an additional dose (100 mg, - 8.2 days; 300 mg, - 8.8 days; placebo, - 6.2 days), with both doses of eptinezumab demonstrating consistently greater reductions from baseline compared to placebo. The ≥50% and ≥ 75% migraine responder rates (MRRs) increased after a second dose, with more eptinezumab-treated patients experiencing migraine response than placebo patients (≥50% MRRs weeks 13-24: 100 mg, 61.0%; 300 mg, 64.0%; placebo, 44.0%; and ≥ 75% MRRs weeks 13-24: 100 mg, 39.3%; 300 mg, 43.1%; placebo, 23.8%). The percentages of patients who improved on patient-reported outcomes, including the Headache Impact Test and Patient Global Impression of Change, increased following the second dose administration at week 12, and were greater with eptinezumab than with placebo at all time points. No new safety concerns were identified with the second dose regarding the incidence, nature, and severity of treatment-emergent adverse events. CONCLUSION: Eptinezumab 100 mg or 300 mg administered IV at day 0 and repeated at week 12 provided sustained migraine preventive benefit over a full 24 weeks and demonstrated an acceptable safety profile in patients with chronic migraine. TRIAL REGISTRATION: ClinicalTrials.gov (Identifier: NCT02974153 ). Registered November 23, 2016.

Efficacy and safety of eptinezumab in patients with chronic migraine: PROMISE-2.

OBJECTIVE: To evaluate the efficacy and safety of eptinezumab, a humanized anti-calcitonin gene-related peptide monoclonal antibody, in the preventive treatment of chronic migraine (CM). METHODS: The Prevention of Migraine via Intravenous ALD403 Safety and Efficacy-2 (PROMISE-2) study was a phase 3, multicenter, randomized, double-blind, placebo-controlled, parallel-group study. Adults with CM were randomly assigned to receive IV eptinezumab 100 mg, eptinezumab 300 mg, or placebo administered on day 0 and week 12. The primary endpoint was change from baseline in mean monthly migraine days (MMDs) over weeks 1 to 12. RESULTS: Among treated participants (n = 1,072), baseline mean number of MMDs was ≈16.1 across groups. Treatment with eptinezumab 100 and 300 mg was associated with significant reductions in MMDs across weeks 1 to 12 compared with placebo (placebo -5.6, 100 mg -7.7, p < 0.0001 vs placebo; 300 mg -8.2, p < 0.0001 vs placebo). Treatment-emergent adverse events (TEAEs) were reported by 43.5% (100 mg), 52.0% (300 mg), and 46.7% (placebo) of patients. Nasopharyngitis was the only TEAE reported for >2% of eptinezumab-treated patients at an incidence of >2% over placebo; it occurred in the 300 mg eptinezumab arm (eptinezumab 9.4%, placebo 6.0%). CONCLUSION: In patients with CM, eptinezumab 100 and 300 mg was associated with a significant reduction in MMDs from the day after IV administration through week 12, was well tolerated, and demonstrated an acceptable safety profile. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that for patients with CM, a single dose of eptinezumab reduces MMDs over 12 weeks of treatment. CLINICALTRIALSGOV IDENTIFIER: NCT02974153.

Population pharmacokinetic and exposure-response analysis of eptinezumab in the treatment of episodic and chronic migraine.

Eptinezumab is a humanized mAb that targets calcitonin gene-related peptide and is under regulatory review for the prevention of episodic and chronic migraine (EM, CM). It is important to determine whether exposures achieved with intravenous (IV) administration of eptinezumab achieve desired pharmacologic effects. Population pharmacokinetics, including dose- and exposure-response analyses, were performed using patient-level data from the eptinezumab clinical trial program with IV doses ranging from 10 to 1000 mg in pharmacokinetic analyses or 10 to 300 mg in phase 2/3 clinical studies in patients with EM or CM. Exposure-response analysis explored the relationship between eptinezumab exposure metrics and efficacy parameters including monthly migraine days. The pharmacokinetic profile of eptinezumab was characterized by rapid attainment of maximum plasma concentration (ie, end of IV administration) and a terminal half-life of 27 days. Covariate analysis found that patient characteristics had no clinically significant effects on pharmacokinetic parameters and were insufficient to influence dosing. Dose- and exposure-response analyses found exposure with single doses ≥100 mg was associated with greater efficacy compared with doses ≤30 mg and a plateau of effect between 100 and 300 mg. A saturable inhibitory Emax model found the exposure over 12 weeks produced by single-dose eptinezumab 100 and 300 mg exceeded the exposure estimates required to achieve 90% of the maximal efficacy (EC90 ). This pharmacokinetic analysis of eptinezumab supports dosing every 12 weeks with no adjustment for patient characteristics, including exposures associated with 100- or 300-mg doses producing optimal efficacy effects. The similar efficacy profiles support 100 mg as the lowest effective dose of eptinezumab.

Eptinezumab in episodic migraine: A randomized, double-blind, placebo-controlled study (PROMISE-1).

OBJECTIVE: To evaluate the efficacy and safety of eptinezumab, a humanized anti-calcitonin gene-related peptide monoclonal antibody, in the preventive treatment of episodic migraine. METHODS: The PRevention Of Migraine via Intravenous ALD403 Safety and Efficacy-1 (PROMISE-1) study was a phase 3, multicenter, randomized, double-blind, placebo-controlled, parallel-group study. Adults with episodic migraine were randomized to eptinezumab 30 mg, 100 mg, 300 mg, or placebo for up to four intravenous (IV) doses administered every 12 weeks. The primary endpoint was change from baseline in monthly migraine days (MMDs) over weeks 1-12. RESULTS: A total of 888 patients received treatment across 84 study sites. Mean MMDs at baseline was ∼8.6 across treatment groups. Eptinezumab 100 mg and 300 mg met the primary endpoint, significantly reducing MMDs across weeks 1-12 compared with placebo (30 mg, -4.0; 100 mg, -3.9, p = 0.0182; 300 mg, -4.3; placebo, -3.2, p = 0.0001). Treatment-emergent adverse events were reported by 58.4% (30 mg), 63.2% (100 mg), 57.6% (300 mg), and 59.5% (placebo) of patients. Treatment-emergent adverse events reported by ≥2% of eptinezumab-treated patients at an incidence greater than placebo included: upper respiratory tract infection (30 mg, 11.4%; 100 mg, 9.9%; 300 mg, 10.3%; placebo, 7.2%), and fatigue (30 mg, 2.3%; 100 mg, 3.6%; 300 mg, 3.6%; placebo, <1%). CONCLUSION: Eptinezumab (100 mg or 300 mg) significantly reduced migraine frequency, was well tolerated, and had an acceptable safety profile when used for the preventive treatment of migraine in adults with episodic migraine. ClinicalTrials.gov identifier: NCT02559895.

Bioanalytical challenges and unique considerations to support pharmacokinetic characterization of bispecific biotherapeutics.

Compared with conventional (monospecific) therapeutics, bispecific protein therapeutics present unique challenges for pharmacokinetic (PK) characterization - namely, the characterization of multiple functional domains as well as the consideration of biotransformation or interference by the formation of antitherapeutic antibodies against each functional domain. PK characterization is essential to the success of the overall drug development plan and for molecules with multiple binding domains; multiple bioanalytical methods may be needed to answer critical questions for each phase of drug development. The number of bispecific protein therapeutics entering drug development continues to increase, and therefore, a bioanalytical strategy for the PK characterization of bispecific molecules and study of their in vivo structure-function relationship is needed. This review presents case studies and a regulatory perspective.

Pharmacologic Characterization of ALD1910, a Potent Humanized Monoclonal Antibody against the Pituitary Adenylate Cyclase-Activating Peptide.

Migraine is a debilitating disease that affects almost 15% of the population worldwide and is the first cause of disability in people under 50 years of age, yet its etiology and pathophysiology remain incompletely understood. Recently, small molecules and therapeutic antibodies that block the calcitonin gene-related peptide (CGRP) signaling pathway have reduced migraine occurrence and aborted acute attacks of migraine in clinical trials and provided prevention in patients with episodic and chronic migraine. Heterogeneity is present within each diagnosis and patient's response to treatment, suggesting migraine as a final common pathway potentially activated by multiple mechanisms, e.g., not all migraine attacks respond to or are prevented by anti-CGRP pharmacological interventions. Consequently, other unique mechanisms central to migraine pathogenesis may present new targets for drug development. Pituitary adenylate cyclase-activating peptide (PACAP) is an attractive novel target for treatment of migraines. We generated a specific, high-affinity, neutralizing monoclonal antibody (ALD1910) with reactivity to both PACAP38 and PACAP27. In vitro, ALD1910 effectively antagonizes PACAP38 signaling through the pituitary adenylate cyclase-activating peptide type I receptor, vasoactive intestinal peptide receptor 1, and vasoactive intestinal peptide receptor 2. ALD1910 recognizes a nonlinear epitope within PACAP and blocks its binding to the cell surface. To test ALD1910 antagonistic properties directed against endogenous PACAP, we developed an umbellulone-induced rat model of neurogenic vasodilation and parasympathetic lacrimation. In vivo, this model demonstrates that the antagonistic activity of ALD1910 is dose-dependent, retaining efficacy at doses as low as 0.3 mg/kg. These results indicate that ALD1910 represents a potential therapeutic antibody to address PACAP-mediated migraine.

Bioanalytical strategy used in development of pharmacokinetic (PK) methods that support biosimilar programs.

The development of biosimilar products is expected to grow rapidly over the next five years as a large number of approved biologics reach patent expiry. The pathway to regulatory approval requires that similarity of the biosimilar to the reference product be demonstrated through physiochemical and structural characterization, as well as within in vivo studies that compare the safety and efficacy profiles of the products. To support nonclinical and clinical studies pharmacokinetic (PK) assays are required to measure the biosimilar and reference products with comparable precision and accuracy. The most optimal approach is to develop a single PK assay, using a single analytical standard, for quantitative measurement of the biosimilar and reference products in serum matrix. Use of a single PK assay for quantification of multiple products requires a scientifically sound testing strategy to evaluate bioanalytical comparability of the test products within the method, and provide a solid data package to support the conclusions. To meet these objectives, a comprehensive approach with scientific rigor was applied to the development and characterization of PK assays that are used in support of biosimilar programs. Herein we describe the bioanalytical strategy and testing paradigm that has been used across several programs to determine bioanalytical comparability of the biosimilar and reference products. Data from one program is presented, with statistical results demonstrating the biosimilar and reference products were bioanalytically equivalent within the method. The cumulative work has established a framework for future biosimilar PK assay development.

Characterization of a quantitative method to measure free proprotein convertase subtilisin/kexin type 9 in human serum.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that plays an important role in the regulation of serum low-density lipoprotein (LDL) cholesterol by downregulation of LDL receptor, and as such is considered a novel target in cholesterol lowering therapy. In support of the drug development program for Evolocumab, a fully human IgG2 antibody that targets PCSK9, a quantitative ELISA to measure free PCSK9 in human serum was developed. PCSK9 serves as a biomarker of pharmacological response during treatment, and measuring levels of the free ligand post-dosing was of interest as an aid to establishing the pharmacokinetic and pharmacodynamic properties of the therapeutic. Given the complexities associated with the measurement of free ligand in the presence of high concentrations of circulating drug, it was important to challenge the method with experiments designed to assess ex vivo conditions that have the potential to affect the binding equilibrium of drug and ligand within test samples during routine sampling handling and assay conditions. Herein, we report results of experiments that were conducted to characterize the assay in alignment with regulatory guidance and industry standards, and to establish evidence that the method is measuring the free ligand in circulation at the time serum was collected. A robust supporting data package was generated that demonstrates the method specifically and reproducibly measures the free ligand, and is suitable for its intended use.

Pharmacokinetics and pharmacodynamics of pegylated interferon lambda-1 in cynomolgus monkeys.

Type III lambda interferons (IFNs) have activity similar to type I IFNs, but a more restricted receptor distribution. A pegylated human IFN lambda-1 (pegIFNλ) is under development for chronic hepatitis C. Induction of receptor signaling (STAT1 phosphorylation) and expression of interferon-stimulated genes (ISGs) by pegIFNλ were assessed in, respectively, cynomolgus monkey leukocyte subsets and hepatocytes stimulated in vitro. ISG induction by pegIFNλ or IFNα was also assessed in peripheral leukocytes and liver biopsies after single and repeat (x3) dosing of pegIFNλ (0.03, 0.3, 3.0 mg/kg) or unpegylated IFNα-2b (10(7) IU/kg). Single-dose pharmacokinetics of pegIFNλ were evaluated. Strong ISG induction occurred in cultured hepatocytes and liver biopsies with both pegIFNλ and IFNα. However, STAT1 phosphorylation, MHC class 1 upregulation, and ISG induction in leukocytes only occurred with IFNα. Serum neopterin was unaffected by pegIFNλ; however, ß-2-microglobulin was elevated at all doses. The terminal half-life of pegIFNλ was 23 h with a 59 mL/kg volume of distribution, consistent with other pegylated IFNs. Serum exposure was dose-proportional across the dosing range. These data demonstrate the suitability of cynomolgus monkeys for the preclinical evaluation of pegIFNλ. Additionally, the absence of pegIFNλ pharmacologic activity in leukocytes is consistent with its low receptor expression in blood.

B-lymphocyte stimulator/a proliferation-inducing ligand heterotrimers are elevated in the sera of patients with autoimmune disease and are neutralized by atacicept and B-cell maturation antigen-immunoglobulin.

INTRODUCTION: B-lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL) are members of the tumor necrosis factor (TNF) family that regulate B-cell maturation, survival, and function. They are overexpressed in a variety of autoimmune diseases and reportedly exist in vivo not only as homotrimers, but also as BLyS/APRIL heterotrimers. METHODS: A proprietary N-terminal trimerization domain was used to produce recombinant BLyS/APRIL heterotrimers. Heterotrimer biologic activity was compared with that of BLyS and APRIL in a 4-hour signaling assay by using transmembrane activator and CAML interactor (TACI)-transfected Jurkat cells and in a 4-day primary human B-cell proliferation assay. A bead-based immunoassay was developed to quantify native heterotrimers in human sera from healthy donors (n = 89) and patients with systemic lupus erythematosus (SLE; n = 89) or rheumatoid arthritis (RA; n = 30). Heterotrimer levels were compared with BLyS and APRIL homotrimer levels in a subset of these samples. RESULTS: The recombinant heterotrimers consisted mostly of one BLyS and two APRIL molecules. Heterotrimer signaling did not show any significant difference compared with APRIL in the TACI-Jurkat assay. Heterotrimers were less-potent inducers of B-cell proliferation than were homotrimeric BLyS or APRIL (EC(50), nMol/L: BLyS, 0.02; APRIL, 0.17; heterotrimers, 4.06). The soluble receptor fusion proteins atacicept and B-cell maturation antigen (BCMA)-immunoglobulin (Ig) neutralized the activity of BLyS, APRIL, and heterotrimers in both cellular assays, whereas B-cell activating factor belonging to the TNF family receptor (BAFF-R)-Ig neutralized only the activity of BLyS. In human sera, significantly more patients with SLE had detectable BLyS (67% versus 18%; P < 0.0001), APRIL (38% versus 3%; P < 0.0002), and heterotrimer (27% versus 8%; P = 0.0013) levels compared with healthy donors. Significantly more patients with RA had detectable APRIL, but not BLyS or heterotrimer, levels compared with healthy donors (83% versus 3%; P < 0.0001). Heterotrimer levels weakly correlated with BLyS, but not APRIL, levels. CONCLUSIONS: Recombinant BLyS/APRIL heterotrimers have biologic activity and are inhibited by atacicept and BCMA-Ig, but not by BAFF-R-Ig. A novel immunoassay demonstrated that native BLyS/APRIL heterotrimers, as well as BLyS and APRIL homotrimers, are elevated in patients with autoimmune diseases.

Preclinical safety of recombinant human thrombin.

Recombinant human thrombin (rhThrombin) is being developed as an alternative to thrombin products purified from pooled human or bovine plasma, which are currently marketed for topical hemostasis. Preclinical studies of rhThrombin were conducted prior to its evaluation as a topical adjunct to surgical hemostasis in clinical trials. No overt clinical pathology or signs were observed in cynomolgus monkeys following implantation of a gelatin sponge containing either rhThrombin or bovine thrombin to a surgical liver wound, and similar gross and microscopic wound healing characteristics were observed over an eight-week recovery period with either compound. Repeated subcutaneous injections of rhThrombin or bovine thrombin to cynomolgus monkeys produced no treatment-related effects. Whereas no monkeys demonstrated anti-rhThrombin antibody seroconversion, specific anti-bovine antibodies were detected in all tested monkeys exposed to bovine thrombin. Addition of rhThrombin or bovine thrombin to mouse fibroblast cells resulted in expected detachment and shape change. Topical application of rhThrombin to rabbits did not cause irritation to the eye, normal skin, or abraded skin. These studies showed that topical, subcutaneous, or implanted rhThrombin was minimally immunogenic, safe, and well tolerated in nonclinical models, and supported the clinical evaluation of rhThrombin in a variety of surgical settings.

Safety and pharmacokinetics of recombinant factor XIII-A2 administration in patients with congenital factor XIII deficiency.

Congenital factor XIII (FXIII) deficiency is associated with a tendency for severe bleeding, a risk for spontaneous abortion, and a high rate of spontaneous intracranial hemorrhage. This phase 1 escalating-dose study was developed to evaluate the safety and pharmacokinetics of a single administration of human recombinant FXIII-A2 (rFXIII-A2) homodimer in adults with congenital FXIII deficiency. Pharmacokinetics and activity of rXIII and changes in endogenous B subunit levels were assessed. Recombinant FXIII-A2 homodimer were complexed with endogenous FXIII-B subunits to form an FXIII-A2B2 heterotetramer with a half-life of 8.5 days, similar to that of endogenous FXIII. The median dose response was a 2.4% increase in FXIII activity based on unit per kilogram rFXIII administered. After the administration of rFXIII-A2, clot solubility normalized as measured by clot lysis in urea. Clot strength and resistance to fibrinolysis, as assessed by thromboelastography, also improved. Safety reviews were conducted before each dose escalation; no serious adverse events, including bleeding or thrombosis, were noted during the study. In addition, there was no evidence of the generation of specific antibodies to rFXIII or yeast proteins. Recombinant FXIII appears to be a safe and potentially effective alternative for FXIII replacement in patients with FXIII deficiency.

Preclinical safety and pharmacokinetics of recombinant human factor XIII.

Factor XIII (FXIII) is a thrombin-activated protransglutaminase responsible for fibrin clot stabilization and longevity. Deficiency in FXIII is associated with diffuse bleeding and wound-healing disorders in humans. This report summarizes results from several studies conducted in adult cynomolgus monkeys (M. fascicularis) to evaluate the safety and pharmacokinetics of recombinant human factor XIII A(2) dimer (rFXIII). Intravenous slow bolus injection of rFXIII resulted in the expected formation of the heterotetramer rA(2)cnB(2), prolonged circulating half-life (5-7 days), and increased plasma transglutaminase activity. Recombinant FXIII was well tolerated as a single dose up to 20 mg/kg rFXIII (2840 U/kg), as repeated daily doses up to 6 mg/kg (852 U/kg) for 14 days, and as 3 repeated doses of 8 mg/kg (1136 U/kg) separated by 14 days. Overt toxicity occurred after a single intravenous injection of = 22.5 mg/kg rFXIII (3150 U/kg), or with 2 doses of = 12.5 mg/kg (1775 U/kg) administered within 72 hours. The rFXIII-mediated toxicity was expressed as an acute systemic occlusive coagulopathy. Evaluation of plasma samples from dosed animals demonstrated formation of cross-linked fibrin/fibrinogen oligomers and higher-order protein aggregates, which are hypothesized to be responsible for the observed vessel occlusion and associated embolic sequelae. These results demonstrate that rFXIII-mediated toxicity results from exaggerated pharmacological activity of the molecule at supraphysiological concentrations. The absence of observed toxicological effect with repeated intravenous doses up to 8 mg/kg (1136 U/kg) was used to support an initial clinical dose range of 0.014 to 0.35 mg/kg (2-50 U/kg).