Growth parameters in children with achondroplasia: A 7-year, prospective, multinational, observational study.

PURPOSE: This study was undertaken to collect baseline growth parameters in children with achondroplasia who might enroll in interventional trials of vosoritide, and to establish a historical control. METHODS: In this prospective, observational study, participants (≤17 years) underwent a detailed medical history and physical examination and were followed every 3 months until they finished participating in the study by enrolling in an interventional trial or withdrawing. RESULTS: A total of 363 children were enrolled (28 centers, 8 countries). Mean (SD) follow up was 20.4 (15.0) months. In participants <1 year, mean annualized growth velocity (AGV) was 11.6 cm/year for girls and 14.6 cm/year for boys. By age 1 year, mean AGV decreased to 7.4 cm/year in girls and 7.1 cm/year in boys. By age 10 years, mean AGV decreased to 3.6 cm/year for both sexes. Mean height z-score in participants <1 year was -2.5 for girls and -3.2 for boys and decreased up to the age 5 years (-5.3 for girls; -4.6 for boys). Girls and boys had a disproportionate upper-to-lower body segment ratio. Mean ratio was highest in participants aged <1 year (2.9 for girls; 2.8 for boys) and decreased gradually to approximately 2 in both sexes from 4 years of age onward. CONCLUSION: This study represents one of the largest datasets of prospectively collected medical and longitudinal growth data in children with achondroplasia. It serves as a robust historical control to measure therapeutic interventions against and to further delineate the natural history of this condition.

Global Seroprevalence of Pre-existing Immunity Against AAV5 and Other AAV Serotypes in People with Hemophilia A.

Adeno-associated virus (AAV)-mediated gene therapy may provide durable protection from bleeding events and reduce treatment burden for people with hemophilia A (HA). However, pre-existing immunity against AAV may limit transduction efficiency and hence treatment success. Global data on the prevalence of AAV serotypes are limited. In this global, prospective, noninterventional study, we determined the prevalence of pre-existing immunity against AAV2, AAV5, AAV6, AAV8, and AAVrh10 among people ≥12 years of age with HA and residual FVIII levels ≤2 IU/dL. Antibodies against each serotype were detected using validated, electrochemiluminescent-based enzyme-linked immunosorbent assays. To evaluate changes in antibody titers over time, 20% of participants were retested at 3 and 6 months. In total, 546 participants with HA were enrolled at 19 sites in 9 countries. Mean (standard deviation) age at enrollment was 36.0 (14.87) years, including 12.5% younger than 18 years, and 20.0% 50 years of age and older. On day 1, global seroprevalence was 58.5% for AAV2, 34.8% for AAV5, 48.7% for AAV6, 45.6% for AAV8, and 46.0% for AAVrh10. Considerable geographic variability was observed in the prevalence of pre-existing antibodies against each serotype, but AAV5 consistently had the lowest seroprevalence across the countries studied. AAV5 seropositivity rates were 51.8% in South Africa (n = 56), 46.2% in Russia (n = 91), 40% in Italy (n = 20), 37.2% in France (n = 86), 26.8% in the United States (n = 71), 26.9% in Brazil (n = 26), 28.1% in Germany (n = 89), 29.8% in Japan (n = 84), and 5.9% in the United Kingdom (n = 17). For all serotypes, seropositivity tended to increase with age. Serostatus and antibody titer were generally stable over the 6-month sampling period. As clinical trials of AAV-mediated gene therapies progress, data on the natural prevalence of antibodies against various AAV serotypes may become increasingly important.

The Use of External Controls in FDA Regulatory Decision Making.

The regulatory standards of the United States Food and Drug Administration (FDA) require substantial evidence of effectiveness from adequate and well-controlled trials that typically use a valid comparison to an internal concurrent control. However, when it is not feasible or ethical to use an internal control, particularly in rare disease populations, relying on external controls may be acceptable. To better understand the use of external controls to support product development and approval, we reviewed FDA regulatory approval decisions between 2000 and 2019 for drug and biologic products to identify pivotal studies that leveraged external controls, with a focus on select therapeutic areas. Forty-five approvals were identified where FDA accepted external control data in their benefit/risk assessment; they did so for many reasons including the rare nature of the disease, ethical concerns regarding use of a placebo or no-treatment arm, the seriousness of the condition, and the high unmet medical need. Retrospective natural history data, including retrospective reviews of patient records, was the most common source of external control (44%). Other types of external control were baseline control (33%); published data (11%); and data from a previous clinical study (11%). To gain further insights, a comprehensive evaluation of selected approvals utilizing different types of external control is provided to highlight the variety of approaches used by sponsors and the challenges encountered in supporting product development and FDA decision making; particularly, the value and use of retrospective natural history in the development of products for rare diseases. Education on the use of external controls based on FDA regulatory precedent will allow for continued use and broader application of innovative approaches to clinical trial design, while avoiding delays in product development for rare diseases. Learnings from this review also highlight the need to update regulatory guidance to acknowledge the utility of external controls, particularly retrospective natural history data.

Efficacy of Antibacterial Peptides Against Peptide-Resistant MRSA Is Restored by Permeabilization of Bacteria Membranes.

Clinical application of antimicrobial peptides (AMPs), as with conventional antibiotics, may be compromised by the development of bacterial resistance. This study investigated AMP resistance in methicillin resistant Staphylococcus aureus, including aspects related to the resilience of the resistant bacteria toward the peptides, the stability of resistance when selection pressures are removed, and whether resistance can be overcome by using the peptides with other membrane-permeabilising agents. Genotypically variant strains of S. aureus became equally resistant to the antibacterial peptides melittin and bac8c when grown in sub-lethal concentrations. Subculture of a melittin-resistant strain without melittin for 8 days lowered the minimal lethal concentration of the peptide from 170 µg ml-1 to 30 µg ml-1. Growth for 24 h in 12 µg ml-1 melittin restored the MLC to 100 µg ml-1. Flow cytometry analysis of cationic fluorophore binding to melittin-naïve and melittin-resistant bacteria revealed that resistance coincided with decreased binding of cationic molecules, suggesting a reduction in nett negative charge on the membrane. Melittin was haemolytic at low concentrations but the truncated analog of melittin, mel12-26, was confirmed to lack haemolytic activity. Although a previous report found that mel12-26 retained full bactericidal activity, we found it to lack significant activity when added to culture medium. However, electroporation in the presence of 50 µg ml-1 of mel12-26, killed 99.3% of the bacteria. Similarly, using a low concentration of the non-ionic detergent Triton X-100 to permeabilize bacteria to mel12-26 markedly increased its bactericidal activity. The observation that bactericidal activity of the non-membranolytic peptide mel12-26 was enhanced when the bacterial membrane was permeablized by detergents or electroporation, suggests that its principal mechanism in reducing bacterial survival may be through interaction with intracellular organelles or processes. Additionally, our results showed that the haemolytic peptide bac8c, had increased antibacterial activity at non-haemolytic concentrations when used with membrane-permeabilizing surfactants.

Investigations into methods to improve the antibacterial activity of Acticoat.

Multiple studies have shown that the antibacterial dressing Acticoat can inhibit growth of bacteria but is unable to completely clear a wound of infection, which could leave patients vulnerable to sepsis. Agar inoculated with four different Staphylococcus aureus strains and overlain with Acticoat showed growth inhibition beneath and within a 1 mm perimeter of the dressing after 24 h. When lifted from inoculated agar and briefly blotted onto fresh agar plates, Acticoat transferred viable bacteria. Scanning electron microscopy of the surface of Acticoat that overlaid meticillin-resistant S. aureus for 24, 48 and 72 h showed dense clusters of apparently undamaged bacteria distributed across the mesh. The number of bacteria growing on inoculated pig skin, underneath and on the surface of Acticoat, was lower than on controls for the first 8 h, but after 24 h the number of bacteria on the skin was 2.3-fold greater than the untreated controls. In contrast, after 24 h the number of bacteria surviving on the surface of the Acticoat was 11.9 % of controls. Acticoat moistened with 10 % glycerol plus antimicrobial peptides (AMPs) mel12-26 or bac8c (50 µg ml- 1) reduced the numbers of bacteria on the dressing and on the skin underneath to below 10 % and 0.01 % of the controls, respectively. When lysozyme (1 mg ml- 1) was added to Acticoat wetted with glycerol and the AMP bac8c, the dressing was able to prevent the survival of bacteria on densely inoculated pig skin and on the surface of Acticoat for up to 24 h. In effect, biocompatible solvents and AMPs significantly enhance the bactericidal efficacy of Acticoat.

Changes in Brain Volume with Bapineuzumab in Mild to Moderate Alzheimer's Disease.

BACKGROUND: Bapineuzumab, an anti-amyloid-ß monoclonal antibody, was evaluated in two placebo-controlled trials in APOE*ɛ4 carriers and noncarriers, respectively, with Alzheimer's disease. OBJECTIVES: A volumetric magnetic resonance imaging substudy was performed to determine if bapineuzumab altered brain volume rate of change. METHODS: Bapineuzumab dosages included 0.5 mg/kg in carriers and 0.5 or 1.0 mg/kg in noncarriers, every 13 weeks for 78 weeks. Volumetric outcomes included annualized brain, ventricular, and mean hippocampal boundary shift integrals (BBSI; VBSI; HBSI) up to Week 71. Treatment differences were estimated using mixed models for repeated measures. RESULTS: For BBSI and HBSI, there were no significant treatment-related differences within either study, but, compared to pooled carriers and noncarriers receiving placebo, noncarriers receiving1.0 mg/kg bapineuzumab had greater increases in these measures. Bapineuzumab-treated patients showed significantly greater VBSI rates compared with placebo for 0.5 mg/kg in carriers and 1.0 mg/kg (but not 0.5 mg/kg) in noncarriers. CONCLUSIONS: Bapineuzumab produced an increase in ventricular volume compared with placebo. Etiology for this increase is unclear but may be related to amyloid-ß clearance or its consequences.

Amyloid-ß 11C-PiB-PET imaging results from 2 randomized bapineuzumab phase 3 AD trials.

OBJECTIVE: To evaluate the effects of bapineuzumab on brain ß-amyloid (Aß) burden using (11)C-Pittsburgh compound B ((11)C-PiB)-PET. METHODS: Two phase 3 clinical trials, 1 each in apolipoprotein APOE ε4 carriers and noncarriers, were conducted in patients with mild to moderate Alzheimer disease dementia. Bapineuzumab, an anti-Aß monoclonal antibody, or placebo, was administered by IV infusion every 13 weeks for 78 weeks. PET substudies assessed change in brain fibrillar Aß over 71 weeks using an (11)C-PiB-PET standardized uptake value ratio (SUVr) global cortical average (GCA) comprising the average SUVr from 5 cortical regions of interest with cerebellar gray matter as the reference region. RESULTS: A total of 115 carriers and 39 noncarriers were analyzed. The difference (δ) in mean baseline to 71 week change in (11)C-PiB-PET GCA between bapineuzumab and placebo was significant in carriers (0.5 mg/kg vs placebo δ = -0.101; p = 0.004) and in pooled analyses of both carriers and noncarriers (0.5 mg/kg vs placebo δ = -0.068; p = 0.027; 1.0 mg/kg vs placebo δ = -0.133; p = 0.028) but not in the noncarrier trial separately. Analyses by individual region of interest and in mild disease yielded findings similar to the main trial results. CONCLUSIONS: The (11)C-PiB-PET imaging results demonstrated reduction of fibrillar Aß accumulation in patients with Alzheimer disease treated with bapineuzumab; however, as no clinical benefit was observed, the findings are consistent with the hypotheses that bapineuzumab may not have been initiated early enough in the disease course, the doses were insufficient, or the most critical Aß species were inadequately targeted.

Cognition in MCI and Alzheimer's disease: baseline data from a longitudinal study of the NTB.

Baseline data are summarized from a study examining the psychometric properties of the Neuropsychological Test Battery (NTB) and its subtests, and correlating the NTB with other cognitive and functional assessments. A multicenter, longitudinal, non-interventional study included mild to moderate Alzheimer's disease (AD, n = 196), mild cognitive impairment (MCI, n = 70), or normal cognition participants (NC, n = 75). The NTB, other cognitive assessment tools, functional/behavioral questionnaires, and health outcome assessments were administered. At baseline composite NTB, NTB memory, and NTB executive function z-scores were significantly lower for participants with AD compared with MCI, and for participants with MCI compared with NC. The composite NTB z-score had high test-retest reliability between screening and baseline. The results of this study suggest that NTB exhibits good reliability in patients with mild to moderate AD and MCI.

Vascular alterations in PDAPP mice after anti-Aß immunotherapy: Implications for amyloid-related imaging abnormalities.

BACKGROUND: Clinical studies of ß-amyloid (Aß) immunotherapy in Alzheimer's disease (AD) patients have demonstrated reduction of central Aß plaque by positron emission tomography (PET) imaging and the appearance of amyloid-related imaging abnormalities (ARIA). To better understand the relationship between ARIA and the pathophysiology of AD, we undertook a series of studies in PDAPP mice evaluating vascular alterations in the context of central Aß pathology and after anti-Aß immunotherapy. METHODS: We analyzed PDAPP mice treated with either 3 mg/kg/week of 3D6, the murine form of bapineuzumab, or isotype control antibodies for periods ranging from 1 to 36 weeks and evaluated the vascular alterations in the context of Aß pathology and after anti-Aß immunotherapy. The number of mice in each treatment group ranged from 26 to 39 and a total of 345 animals were analyzed. RESULTS: The central vasculature displayed morphological abnormalities associated with vascular Aß deposits. Treatment with 3D6 antibody induced clearance of vascular Aß that was spatially and temporally associated with a transient increase in microhemorrhage and in capillary Aß deposition. Microhemorrhage resolved over a time period that was associated with a recovery of vascular morphology and a decrease in capillary Aß accumulation. CONCLUSIONS: These data suggest that vascular leakage events, such as microhemorrhage, may be related to the removal of vascular Aß. With continued treatment, this initial susceptibility period is followed by restoration of vascular morphology and reduced vulnerability to further vascular leakage events. The data collectively suggested a vascular amyloid clearance model of ARIA, which accounts for the currently known risk factors for the incidence of ARIA in clinical studies.

Predictive mutational bioinformatic analysis of variation in the skin and wool associated corneodesmosin (CDSN) gene in sheep.

Corneodesmosin (CDSN) is an important component of the desmosome in the epidermal cornified stratum and inner root sheath of hair follicles. DNA from a sheep BAC clone previously identified by us to contain CDSN was PCR amplified using cattle-derived primers and the product sequenced. A region of 4579 bp containing CDSN was shown to contain two exons separated by one intron and spanning 3683 bp. The DNA encodes a predicted protein of 546 amino acids. Phylogenetic analysis shows that sheep CDSN falls within a clade containing cattle and other ruminant-like species. Comparison of sequences generated from 12 unrelated merino sheep and the International Sheep Genome Consortium (ISGC) data identified 58 single nucleotide polymorphisms (SNPs) within the 4579 bp region of which 16 are contained within coding sequences (1 in 80 bp). The SNPs identified in this study will add to the Major Histocompatibility Complex (MHC) SNP panel, which will allow extensive haplotyping of the sheep MHC in future studies.

Amyloid-related imaging abnormalities in patients with Alzheimer's disease treated with bapineuzumab: a retrospective analysis.

BACKGROUND: Amyloid-related imaging abnormalities (ARIA) have been reported in patients with Alzheimer's disease treated with bapineuzumab, a humanised monoclonal antibody against amyloid ß. ARIA include MRI signal abnormalities suggestive of vasogenic oedema and sulcal effusions (ARIA-E) and microhaemorrhages and haemosiderin deposits (ARIA-H). Our aim was to investigate the incidence of ARIA during treatment with bapineuzumab, and evaluate associated risk factors. METHODS: Two neuroradiologists independently reviewed 2572 fluid-attenuated inversion recovery (FLAIR) MRI scans from 262 participants in two phase 2 studies of bapineuzumab and an open-label extension study. Readers were masked to the patient's treatment, APOE ɛ4 genotype, medical history, and demographics. Patients were included in risk analyses if they had no evidence of ARIA-E in their pre-treatment MRI, had received bapineuzumab, and had at least one MRI scan after treatment. We used Kaplan-Meier survival analysis to examine the distribution of incident ARIA-E from the start of bapineuzumab treatment and proportional hazards regression models to assess risk factors associated with ARIA. FINDINGS: 210 patients were included in the risk analyses. 36 patients (17%) developed ARIA-E during treatment with bapineuzumab; 15 of these ARIA-E cases (42%) had not been detected previously. 28 of these patients (78%) did not report associated symptoms. Adverse events, reported in eight symptomatic patients, included headache, confusion, and neuropsychiatric and gastrointestinal symptoms. Incident ARIA-H occurred in 17 of the patients with ARIA-E (47%), compared with seven of 177 (4%) patients without ARIA-E. 13 of the 15 patients in whom ARIA were detected in our study received additional treatment infusions while ARIA-E were present, without any associated symptoms. Occurrence of ARIA-E increased with bapineuzumab dose (hazard ratio [HR] 2·24 per 1 mg/kg increase in dose, 95% CI 1·40-3·62; p=0·0008) and presence of APOE ɛ4 alleles (HR 2·55 per allele, 95% CI 1·57-4·12; p=0·0001). INTERPRETATION: ARIA consist of a spectrum of imaging findings with variable clinical correlates, and some patients with ARIA-E remain asymptomatic even if treatment is continued. The increased risk of ARIA among APOE ɛ4 carriers, its association with high bapineuzumab dose, and its timecourse in relation to dosing suggest an association between ARIA and alterations in vascular amyloid burden. FUNDING: Elan Corporation, Janssen Alzheimer Immunotherapy, Wyeth Pharmaceuticals, and Pfizer.

11C-PiB PET assessment of change in fibrillar amyloid-beta load in patients with Alzheimer's disease treated with bapineuzumab: a phase 2, double-blind, placebo-controlled, ascending-dose study.

BACKGROUND: Carbon-11-labelled Pittsburgh compound B ((11)C-PiB) PET is a marker of cortical fibrillar amyloid-beta load in vivo. We used (11)C-PiB PET to investigate whether bapineuzumab, a humanised anti-amyloid-beta monoclonal antibody, would reduce cortical fibrillar amyloid-beta load in patients with Alzheimer's disease. METHODS: Patients with mild-to-moderate Alzheimer's disease were randomly assigned to receive intravenous bapineuzumab or placebo in a ratio of seven to three in three ascending dose groups (0.5, 1.0, or 2.0 mg/kg). Each dose group was enrolled after safety review of the previous group. Randomisation was by interactive voice response system; masking was achieved with numbered kit allocation. Patients, investigators, study site personnel, sponsor staff, and carers were masked to treatment. Patients received up to six infusions, 13 weeks apart, and had (11)C-PiB PET scans at baseline and at weeks 20, 45, and 78. The primary outcome was the difference between the pooled bapineuzumab group and the pooled placebo group in mean change from screening to week 78 in (11)C-PiB cortical to cerebellar retention ratio averaged across six cortical regions of interest. Analysis was by modified intention to treat. This study is registered with EudraCT, number 2004-004120-12; ISRCTN17517446. FINDINGS: 28 patients were assigned to bapineuzumab (n=20) or placebo (n=8). 19 patients in the bapineuzumab group and seven in the placebo group were included in the modified intention-to-treat analysis. Estimated mean (11)C-PiB retention ratio change from baseline to week 78 was -0.09 (95% CI -0.16 to -0.02; p=0.014) in the bapineuzumab group and 0.15 (95% CI 0.02 to 0.28; p=0.022) in the placebo group. Estimated mean difference in (11)C-PiB retention ratio change from baseline to week 78 between the bapineuzumab group and the placebo group was -0.24 (95% CI -0.39 to -0.09; p=0.003). Differences between the bapineuzumab group and the placebo group in the individual regions of interest were similar to the overall mean difference. Adverse events were typically mild to moderate in severity and transient. Two patients in the 2.0 mg/kg bapineuzumab group had transient cerebral vasogenic oedema. INTERPRETATION: Treatment with bapineuzumab for 78 weeks reduced cortical (11)C-PiB retention compared with both baseline and placebo. (11)C-PiB PET seems to be useful in assessing the effects of potential Alzheimer's disease treatments on cortical fibrillar amyloid-beta load in vivo. FUNDING: Elan Pharmaceuticals and Wyeth Research.

Isolation and characterization of putative Pseudobutyrivibrio ruminis promoters.

Novel plasmids were constructed for the analysis of DNA fragments from the rumen bacterium Pseudobutyrivibrio ruminis. Five previously unidentified promoters were characterized using a novel primer extension method to identify transcription start sites. The genes downstream of these promoters were not identified, and their activity in expression of genomic traits in wild-type P. ruminis remains putative. Comparison with promoters from this and closely related species revealed a consensus sequence resembling the binding motif for the RNA polymerase sigma(70)-like factor complex. Consensus -35 and -10 sequences within these elements were TTGACA and ATAATATA respectively, interspaced by 15-16 bp. The consensus for the -10 element was extended by one nucleotide upstream and downstream of the standard hexamer (indicated in bold). Promoter strengths were measured by reverse transcription quantitative PCR and beta-glucuronidase assays. No correlation was found between the composition and context of elements within P. ruminis promoters, and promoter strength. However, a mutation within the -35 element of one promoter revealed that transcriptional strength and choice of transcription start site were sensitive to this single nucleotide change.

Using the time of maximum effect site concentration to combine pharmacokinetics and pharmacodynamics.

BACKGROUND: To simulate the time course of drug effect, it is sometimes necessary to combine the pharmacodynamic parameters from an integrated pharmacodynamic-pharmacodynamic study (e.g., volumes, clearances, k(e0) [the effect site equilibration rate constant], C(50) [the steady state plasma concentration associated with 50% maximum effect], and the Hill coefficient) with pharmacokinetic parameters from a different study (e.g., a study examining a different age group or sampling over longer periods of time). Pharmacokinetic-pharmacodynamic parameters form an interlocked vector that describes the relationship between input (dose) and output (effect). Unintended consequences may result if individual elements of this vector (e.g., k(e0)) are combined with pharmacokinetic parameters from a different study. The authors propose an alternative methodology to rationally combine the results of separate pharmacokinetic and pharmacodynamic studies, based on t(peak), the time of peak effect after bolus injection. METHODS: The naive approach to combining separate pharmacokinetic and pharmacodynamic studies is to simply take the k(e0) from the pharmacodynamic study and apply it naively to the pharmacokinetic study of interest. In the t(peak) approach, k(e0) is recalculated using the pharmacokinetics of interest to yield the correct time of peak effect. The authors proposed that the t(peak) method would yield better predictions of the time course of drug effect than the naive approach. They tested this hypothesis in three simulations: thiopental, remifentanil, and propofol. RESULTS: In each set of simulations, the t(peak) method better approximated the postulated "true" time course of drug effect than the naive method. CONCLUSIONS: T(peak) is a useful pharmacodynamic parameter and can be used to link separate pharmacokinetic and pharmacodynamic studies. This addresses a common difficulty in clinical pharmacology simulation and control problems, where there is usually a wide choice of pharmacokinetic models but only one or two published pharmacokinetic-pharmacodynamic models. The results will be immediately applicable to target-controlled anesthetic infusion systems, where linkage of separate pharmacokinetic and pharmacodynamic parameters into a single model is inherent in several target-controlled infusion designs.

Family of shuttle vectors for ruminal Bacteroides.

A family of shuttle plasmids was constructed for genetic transformation of Escherichia coli and of ruminal Bacteroides strains AR20 and AR29. Plasmids were based on the replicon from Bacteroides plasmid pBI191 and were designed for studies of chromosomal integration (pBA), for the identification and study of Bacteroides gene promoters (pPPR) and for the expression of heterologous genes in Bacteroides (pBAC). Electroporation efficiency of Bacteroides was up to 10(5) transformants/microg plasmid, depending on the source of the DNA. The largest plasmid, pBA, was maintained at approximately 8 copies per cell in AR20 and did not measurably alter in vitro growth of transformed cells. In the current work, pBA did not integrate into the chromosomes of AR20 or AR29. The ability of plasmid pPPR to select promoter sequences was demonstrated by removal and replacement of promoters that activate the clindamycin resistance gene. The suitability of pBAC for expression of heterologous genes was demonstrated by expression of the Moraxella species fluoroacetate dehalogenase gene H1 to give intracellular activity of 7 nmol fluoride released/min/mg soluble protein in AR20 and 4 nmol/min/mg in AR29. Spontaneous loss of pBAC under non-selective conditions was 0.11-0.165% per generation, significantly less than loss of the native Bacteroides plasmid pBI191, which was lost at 0.53% per generation.