Disease progression modeling of the North Star Ambulatory Assessment for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a rare genetic disorder caused by decreased or absent dystrophin gene leading to progressive muscle degeneration and weakness in young boys. Disease progression models for the North Star Ambulatory Assessment (NSAA), a functional measurement widely used to assess outcomes in clinical trials, were developed using a longitudinal population modeling approach. The relationship between NSAA total score over time, loss of ambulation, and potential covariates that may influence disease progression were evaluated. Data included individual participant observations from an internal placebo-controlled phase II clinical trial and from the external natural history database for male patients with DMD obtained through the Cooperative International Neuromuscular Research Group (CINRG). A modified indirect response model for NSAA joined to a loss of ambulation (LOA) time-to-event model described the data well. Age was used as the independent variable because ambulatory function is known to vary with age. The NSAA and LOA models were linked using the dissipation rate constant parameter from the NSAA model by including the parameter as a covariate on the hazard equation for LOA. No covariates were identified. The model was then used as a simulation tool to explore various clinical trial design scenarios. This model contributes to the quantitative understanding of disease progression in DMD and may guide model-informed drug development decisions for ongoing and future DMD clinical trials.

Dystrophin and mini-dystrophin quantification by mass spectrometry in skeletal muscle for gene therapy development in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal, degenerative muscle disorder caused by mutations in the DMD gene, leading to severe reduction or absence of the protein dystrophin. Gene therapy strategies that aim to increase expression of a functional dystrophin protein (mini-dystrophin) are under investigation. The ability to accurately quantify dystrophin/mini-dystrophin is essential in assessing the level of gene transduction. We demonstrated the validation and application of a novel peptide immunoaffinity liquid chromatography-tandem mass spectrometry (IA-LC-MS/MS) assay. Data showed that dystrophin expression in Becker muscular dystrophy and DMD tissues, normalized against the mean of non-dystrophic control tissues (n = 20), was 4-84.5% (mean 32%, n = 20) and 0.4-24.1% (mean 5%, n = 20), respectively. In a DMD rat model, biceps femoris tissue from dystrophin-deficient rats treated with AAV9.hCK.Hopti-Dys3978.spA, an adeno-associated virus vector containing a mini-dystrophin transgene, showed a dose-dependent increase in mini-dystrophin expression at 6 months post-dose, exceeding wildtype dystrophin levels at high doses. Validation data showed that inter- and intra-assay precision were ≤20% (≤25% at the lower limit of quantification [LLOQ]) and inter- and intra-run relative error was within ±20% (±25% at LLOQ). IA-LC-MS/MS accurately quantifies dystrophin/mini-dystrophin in human and preclinical species with sufficient sensitivity for immediate application in preclinical/clinical trials.

Antitumor Necrosis Factor-like Ligand 1A Therapy Targets Tissue Inflammation and Fibrosis Pathways and Reduces Gut Pathobionts in Ulcerative Colitis.

BACKGROUND: The first-in-class treatment PF-06480605 targets the tumor necrosis factor-like ligand 1A (TL1A) molecule in humans. Results from the phase 2a TUSCANY trial highlighted the safety and efficacy of PF-06480605 in ulcerative colitis. Preclinical and in vitro models have identified a role for TL1A in both innate and adaptive immune responses, but the mechanisms underlying the efficacy of anti-TL1A treatment in inflammatory bowel disease (IBD) are not known. METHODS: Here, we provide analysis of tissue transcriptomic, peripheral blood proteomic, and fecal metagenomic data from the recently completed phase 2a TUSCANY trial and demonstrate endoscopic improvement post-treatment with PF-06480605 in participants with ulcerative colitis. RESULTS: Our results revealed robust TL1A target engagement in colonic tissue and a distinct colonic transcriptional response reflecting a reduction in inflammatory T helper 17 cell, macrophage, and fibrosis pathways in patients with endoscopic improvement. Proteomic analysis of peripheral blood revealed a corresponding decrease in inflammatory T-cell cytokines. Finally, microbiome analysis showed significant changes in IBD-associated pathobionts, Streptococcus salivarius, S. parasanguinis, and Haemophilus parainfluenzae post-therapy. CONCLUSIONS: The ability of PF-06480605 to engage and inhibit colonic TL1A, targeting inflammatory T cell and fibrosis pathways, provides the first-in-human mechanistic data to guide anti-TL1A therapy for the treatment of IBD.

Anti-TL1A Antibody PF-06480605 Safety and Efficacy for Ulcerative Colitis: A Phase 2a Single-Arm Study.

BACKGROUND & AIMS: An immune component of inflammatory bowel disease is up-regulated tumor necrosis factor-like ligand 1A (TL1A). Anti-TL1A antibodies such as PF-06480605, a fully human immunoglobulin G1 monoclonal antibody, may have therapeutic potential. METHODS: This Phase 2a, multicenter, single-arm, open-label study (TUSCANY) evaluated safety, tolerability, efficacy, pharmacokinetics, and immunogenicity in PF-06480605-treated participants with moderate to severe ulcerative colitis (UC). Participants received 500 mg intravenous PF-06480605 every 2 weeks, 7 doses total, with a 3-month follow-up period. Primary safety and efficacy endpoints were the incidence of adverse events (AEs) and week 14 endoscopic improvement (EI) (Mayo endoscopic subscore = 0 or 1), respectively. Secondary endpoints included total soluble TL1A (free/drug-bound) (sTL1A), incidence of anti-drug and neutralizing antibodies, PF-06480605 concentrations, and changes in fecal calprotectin and high-sensitivity C-reactive protein. Histology was assessed at week 14. RESULTS: The study enrolled 50 participants; 42 completed. Of 109 treatment-emergent AEs, 18 were treatment-related. The most common AEs were UC disease exacerbation and arthralgia (6 participants each). Four serious AEs, no deaths, and no malignancies were reported. Week 14 EI was observed in a statistically significant proportion of participants (38.2% [uniformly minimum-variance unbiased estimator, per protocol population]). Minimal histologic disease was observed after treatment (Robarts Histopathology Index ≤5: 33.3%; Geboes Index ≤3.2: 47.6%). sTL1A increase over time from baseline indicated sustained target engagement. Forty-one participants (82%) tested positive for anti-drug antibodies and 5 (10%) for neutralizing antibodies. CONCLUSIONS: PF-06480605 demonstrated an acceptable safety profile and statistically significant EI in participants with moderate to severe UC, warranting further study in a larger participant cohort. Tissue histopathology analyses support this conclusion. TRIAL REGISTRATION NUMBER: https://clinicaltrials.gov/NCT02840721.

Population Pharmacokinetic Analysis of Recombinant Factor VIII Fc Fusion Protein in Subjects With Severe Hemophilia A: Expanded to Include Pediatric Subjects.

Recombinant factor VIII Fc fusion protein (rFVIIIFc) has been indicated for adults and children with hemophilia A. The objective of this article was to build a population pharmacokinetic (PK) model using adult and pediatric data sets and explore relevant dosing scenarios across all ages. The activity-time profiles of rFVIIIFc from 3 clinical studies (all trials registered at https://www.clinicaltrials.gov: NCT01027377, NCT01181128, and NCT01458106) were characterized, and covariates that determine variability of rFVIIIFc PK in children and adults were identified and implemented. Data sets were pooled to estimate population PK parameters. Simulations were conducted to generate activity-time profiles at steady state (SS). The proportion of subjects maintaining SS trough >1 and >3 IU/dL and time >10 IU/dL were estimated. The rFVIIIFc model was a two-compartment model that identified weight and von Willebrand factor as significant covariates. Model-predicted SS peaks and troughs of rFVIIIFc activity-time profiles confirmed the necessity of modifying dosing in pediatric subjects. The model also predicted that the average subject in the adult and adolescent group dosed with 40 IU/kg every 2 days maintained factor VIII activity >10 IU/dL for the entire duration. Children aged <6 years and aged 6 to <12 years receiving this dose maintained factor VIII activity of >10 IU/dL for nearly two-thirds and three-quarters of their time, respectively. In conclusion, these population PK analyses characterize activity-time profiles for rFVIIIFc among pediatric and adult subjects. The model was used for simulation of clinically relevant dosing scenarios, which can provide better protection and better clinical outcomes.

Safety, Tolerability, and Pharmacokinetics of PF-06823859, an Anti-Interferon ß Monoclonal Antibody: A Randomized, Phase I, Single- and Multiple-Ascending-Dose Study.

This double-blind, randomized, placebo-controlled, dose-ascending, first-in-human study (NCT02766621) assessed the safety, tolerability, and pharmacokinetics (PK) of PF-06823859, an anti-interferon ß monoclonal antibody. Healthy subjects were randomized to single ascending doses (SADs) of intravenous PF-06823859 30, 100, 300, 900, or 2000 mg or placebo; to multiple ascending doses (MADs) of subcutaneous PF-06823859 100 or 300 mg or placebo (once every 2 weeks for a total of 3 doses); or to MAD of intravenous PF-06823859 600 mg or placebo (once every 3 weeks or once every 4 weeks for a total of 2 doses). The incidence, severity, and causal relationship of adverse events (AEs) were assessed, along with immunogenicity and PK. In total, 62 subjects were randomized to treatment (SAD, n = 35; MAD, n = 27). There were 76 treatment-emergent all-causality AEs in the SAD (PF-06823859: n = 25; placebo: n = 4) and MAD (PF-06823859: n = 40; placebo: n = 7) cohorts. In the SAD cohorts, all treatment-emergent all-causality AEs were mild in severity; 4 AEs of moderate severity were identified in the MAD cohorts. No dose-limiting AEs, serious AEs, treatment-related discontinuations, dose reductions, or deaths occurred. PF-06823859 exposure increased dose-proportionally, with half-life values ranging between 23 and 35 days. The estimated subcutaneous bioavailability was 43% to 44%. Immunogenicity incidence rates were low (antidrug antibodies, 12.5%; neutralizing antibodies, 2.1%). No immunogenically related clinical responses of concern were observed. In conclusion, PF-06823859 demonstrated an acceptable safety, tolerability, and PK profile that supports clinical development for treating disorders associated with increased interferon ß levels, such as dermatomyositis or systemic lupus erythematosus.

Population pharmacokinetics of recombinant factor VIII Fc fusion protein.

Population pharmacokinetics (PK) of FVIII activity-time profiles following recombinant factor VIII Fc fusion protein (rFVIIIFc) and recombinant factor VIII (rFVIII) dosing were evaluated in previously treated patients with severe hemophilia A (from two clinical trials). Potential covariates that may be determinants of variability in FVIII activity were identified. A 2-compartment model adequately described the PK of both compounds. von Willebrand Factor (VWF) concentration was the major covariate for rFVIIIFc clearance, reflecting its protective role in FVIII activity clearance. The effect of body weight and hematocrit on the central volume of distribution of rFVIIIFc was minor. The results of these analyses confirmed that rFVIIIFc clearance (1.65 dL/h) is much lower than that of rFVIII (2.53 dL/h), while the steady state volumes of distribution were similar. The strong positive correlations between the PK parameters of rFVIIIFc and rFVIII suggest that individuals who have high time-related PK characteristics with rFVIII are likely to have comparable characteristics with rFVIIIFc. Steady-state activity-time profiles for selected rFVIIIFc dosing regimens were simulated accounting for uncertainty in model parameters. These population PK analyses and simulations provide a comprehensive characterization of the PK of rFVIIIFc and rFVIII and may be useful for designing dosing regimens.

Phase 3 study of recombinant factor VIII Fc fusion protein in severe hemophilia A.

This phase 3 pivotal study evaluated the safety, efficacy, and pharmacokinetics of a recombinant FVIII Fc fusion protein (rFVIIIFc) for prophylaxis, treatment of acute bleeding, and perioperative hemostatic control in 165 previously treated males aged ≥12 years with severe hemophilia A. The study had 3 treatment arms: arm 1, individualized prophylaxis (25-65 IU/kg every 3-5 days, n = 118); arm 2, weekly prophylaxis (65 IU/kg, n = 24); and arm 3, episodic treatment (10-50 IU/kg, n = 23). A subgroup compared recombinant FVIII (rFVIII) and rFVIIIFc pharmacokinetics. End points included annualized bleeding rate (ABR), inhibitor development, and adverse events. The terminal half-life of rFVIIIFc (19.0 hours) was extended 1.5-fold vs rFVIII (12.4 hours; P < .001). Median ABRs observed in arms 1, 2, and 3 were 1.6, 3.6, and 33.6, respectively. In arm 1, the median weekly dose was 77.9 IU/kg; approximately 30% of subjects achieved a 5-day dosing interval (last 3 months on study). Across arms, 87.3% of bleeding episodes resolved with 1 injection. Adverse events were consistent with those expected in this population; no subjects developed inhibitors. rFVIIIFc was well-tolerated, had a prolonged half-life compared with rFVIII, and resulted in low ABRs when dosed prophylactically 1 to 2 times per week.

Antiviral activity of the hepatitis C virus polymerase inhibitor filibuvir in genotype 1-infected patients.

UNLABELLED: More effective and better-tolerated therapies are needed for chronic hepatitis C virus (HCV) infection. Among the direct-acting anti-HCV agents in development is the nonstructural 5B protein (NS5B polymerase) non-nucleoside inhibitor filibuvir. We investigated the antiviral activity, pharmacokinetics, safety, and tolerability of multiple doses of filibuvir in treatment-naive and treatment-experienced patients who were chronically infected with HCV genotype 1 in two phase 1b clinical studies (study 1 was a randomized, placebo-controlled dose escalation study and study 2 was a nonrandomized, open-label study). The filibuvir doses evaluated ranged from 200-1400 mg daily, and the duration of dosing ranged from 3-10 days. Genotypic changes in the NS5B nucleotide sequence following short-term filibuvir therapy were also assessed. Filibuvir potently inhibited viral replication in a dose-dependent manner. Mean maximum HCV RNA change from baseline ranged from -0.97 log(10) IU/mL with filibuvir given at 100 mg twice daily to -2.30 log(10) IU/mL with filibuvir given at 700 mg twice daily in treatment-naive patients. In treatment-experienced patients, an HCV RNA reduction of 2.20 log(10) IU/mL was achieved with filibuvir given at 450 mg twice daily. Filibuvir was well tolerated in both studies. Adverse events were mild or moderate in severity. No discontinuations, serious adverse events, or deaths were reported. NS5B sequencing identified residue 423 as the predominant site of mutation after filibuvir dosing. CONCLUSION: Filibuvir administration resulted in significant reductions in HCV RNA concentrations at doses that were well tolerated in patients infected with HCV genotype 1. Filibuvir is currently being evaluated in combination with pegylated interferon alfa 2a plus ribavirin in treatment-naive patients.

Erythropoietin pharmacokinetic/pharmacodynamic analysis suggests higher doses in treating neonatal anemia.

BACKGROUND: The establishment of effective treatment of neonatal anemia using recombinant human erythropoietin (r-HuEPO) requires a thorough understanding of the physiology and mechanism of EPO's pharmacologic effect. The purpose of the present preclinical study in sheep was to elucidate the stimulatory effect of EPO on erythroid progenitors and their differentiation into reticulocytes useful in predicting optimal r-HuEPO dosing. METHODS: Five young adult sheep each underwent two phlebotomies spaced 4-6 weeks apart in which their hemoglobin levels were reduced from 12 g/dL to 3-4 g/dL. Endogenous EPO levels and reticulocyte counts produced in response to anemia were sampled throughout the study and analyzed using a pharmacokinetic/pharmacodynamic (PK/PD) model. RESULTS: The phlebotomy-induced drop in hemoglobin resulted in a increase in EPO levels, which reached a maximum of 764 +/- 55 mU/mL (mean +/- %CV) in 0.5-2.6 days. The reticulocyte counts increased from baseline values of 76.9 x 10(3) +/- 67/microL to 619 x 10(3) +/- 30/microL in 8 days. The PK/PD analysis indicated an increased maturation time for the reticulocytes (4.88 +/- 35 days) and demonstrated that the E(max) model for EPO's activation of the progenitors did not show significant effect saturation at the endogenous EPO levels reached. CONCLUSIONS: In extrapolating from the animal pilot experiment, the present study provides a case for the use of higher r-HuEPO doses in human studies to determine if higher doses are more effective in treatment of neonatal anemia to reduce, and in some less severe cases, eliminate, the need for blood transfusions.

Bronchoscopic assessment of airway retention time of aerosolized xylitol.

BACKGROUND: Human airway surface liquid (ASL) has abundant antimicrobial peptides whose potency increases as the salt concentration decreases. Xylitol is a 5-carbon sugar that has the ability to lower ASL salt concentration, potentially enhancing innate immunity. Xylitol was detected for 8 hours in the ASL after application in airway epithelium in vitro. We tested the airway retention time of aerosolized iso-osmotic xylitol in healthy volunteers. METHODS: After a screening spirometry, volunteers received 10 ml of nebulized 5% xylitol. Bronchoscopy was done at 20 minutes (n = 6), 90 minutes (n = 6), and 3 hours (n = 5) after nebulization and ASL was collected using microsampling probes, followed by bronchoalveolar lavage (BAL). Xylitol concentration was measured by nuclear magnetic resonance spectroscopy and corrected for dilution using urea concentration. RESULTS: All subjects tolerated nebulization and bronchoscopy well. Mean ASL volume recovered from the probes was 49 +/- 23 microl. The mean ASL xylitol concentration at 20, 90, and 180 minutes was 1.6 +/- 1.9 microg/microl, 0.6 +/- 0.6 microg/microl, and 0.1 +/- 0.1 microg/microl, respectively. Corresponding BAL concentration corrected for dilution was consistently lower at all time points. The terminal half-life of aerosolized xylitol obtained by the probes was 45 minutes with a mean residence time of 65 minutes in ASL. Corresponding BAL values were 36 and 50 minutes, respectively. CONCLUSION: After a single dose nebulization, xylitol was detected in ASL for 3 hours, which was shorter than our in vitro measurement. The microsampling probe performed superior to BAL when sampling bronchial ASL.