Analytical comparability demonstrated for an IgG4 molecule, inclacumab, following transfer of manufacturing responsibility from Roche to Global Blood Therapeutics.

BACKGROUND: Inclacumab is a recombinant, fully human, immunoglobulin IgG4 monoclonal antibody that selectively binds to P-selectin. Initially discovered and developed by Roche through phase 2 clinical studies in peripheral arterial disease and coronary artery disease, inclacumab has been in-licensed by Global Blood Therapeutics (GBT) as a potential treatment to reduce the frequency of vaso-occlusive crises in individuals with sickle cell disease. RESEARCH DESIGN AND METHODS: GBT sought to demonstrate the analytical comparability between material produced by Roche and material produced by GBT to ensure that no meaningful differences in identity, safety, purity, potency, or bioavailability exist between the GBT and Roche lots. RESULTS: Inclacumab samples produced by GBT were found to be comparable to the Roche v0.2 inclacumab samples based on (1) comparable primary and higher-order structures; (2) comparable purity profiles; (3) comparable potency, in vitro functional activities, and in vivo plasma exposures and pharmacokinetic profiles; and (4) comparable degradation patterns and kinetics under forced degradation conditions. CONCLUSIONS: Based on the design of this comparability study and the results obtained, the US Food and Drug Administration approved the changes to the manufacturing process and gave clearance for GBT to proceed with phase 3 clinical trials.

Spatiotemporal image quality of virtual reality head mounted displays.

Virtual reality (VR) head mounted displays (HMDs) require both high spatial resolution and fast temporal response. However, methods to quantify the VR image quality in the spatiotemporal domain when motion exists are not yet standardized. In this study, we characterize the spatiotemporal capabilities of three VR devices: the HTC VIVE, VIVE Pro, and VIVE Pro 2 during smooth pursuit. A spatiotemporal model for VR HMDs is presented using measured spatial and temporal characteristics. Among the three evaluated headsets, the VIVE Pro 2 improves the display temporal performance using a fast 120 Hz refresh rate and pulsed emission with a small duty cycle of 5%. In combination with a high pixel resolution beyond 2 k [Formula: see text] 2 k per eye, the VIVE Pro 2 achieves an improved spatiotemporal performance compared to the VIVE and VIVE Pro in the high spatial frequency range above 8 cycles per degree during smooth pursuit. The result demonstrates that reducing the display emission duty cycle to less than 20% is beneficial to mitigate motion blur in VR HMDs. Frame rate reduction (e.g., to below 60 Hz) of the input signal compared to the display refresh rate of 120 Hz yields replicated shadow images that can affect the image quality under motion. This work supports the regulatory science research efforts in development of testing methods to characterize the spatiotemporal performance of VR devices for medical use.

Evaluation Challenges for the Application of Extended Reality Devices in Medicine.

Augmented and virtual reality devices are being actively investigated and implemented for a wide range of medical uses. However, significant gaps in the evaluation of these medical devices and applications hinder their regulatory evaluation. Addressing these gaps is critical to demonstrating the devices' safety and effectiveness. We outline the key technical and clinical evaluation challenges discussed during the US Food and Drug Administration's public workshop, "Medical Extended Reality: Toward Best Evaluation Practices for Virtual and Augmented Reality in Medicine" and future directions for evaluation method development. Evaluation challenges were categorized into several key technical and clinical areas. Finally, we highlight current efforts in the standards communities and illustrate connections between the evaluation challenges and the intended uses of the medical extended reality (MXR) devices. Participants concluded that additional research is needed to assess the safety and effectiveness of MXR devices across the use cases.

Gut-Selective Design of Orally Administered Izencitinib (TD-1473) Limits Systemic Exposure and Effects of Janus Kinase Inhibition in Nonclinical Species.

Izencitinib (TD-1473), an oral, gut-selective pan-Janus kinase (JAK) inhibitor under investigation for treatment of inflammatory bowel diseases, was designed for optimal efficacy in the gastrointestinal tract while minimizing systemic exposures and JAK-related safety findings. The nonclinical safety of izencitinib was evaluated in rat and dog repeat-dose and rat and rabbit reproductive and developmental toxicity studies. Systemic exposures were compared with JAK inhibitory potency to determine effects at or above pharmacologic plasma concentrations (≥1× plasma average plasma concentration [Cave]:JAK 50% inhibitory concentration [IC50] ratio). In rats and dogs, 1000 and 30 mg/kg/day izencitinib, respectively, produced minimal systemic findings (ie, red/white cell changes) and low systemic concentrations (approximately 1× plasma Cave:JAK IC50 ratio) with an 8× nonclinical:clinical systemic area under the curve (AUC) margin compared with exposures at the highest clinically tested dose (300 mg, quaque die, once daily, phase 1 study in healthy volunteers). In dogs, it was possible to attain sufficient systemic exposures to result in immunosuppression characteristic of systemic JAK inhibition, but at high AUC margins (43×) compared with systemic exposures observed at the highest tested dose in humans. No adverse findings were observed in the gastrointestinal tract or systemic tissues. Izencitinib did not affect male or female fertility. Izencitinib did not affect embryonic development in rats and rabbits as commonly reported with systemic JAK inhibition, consistent with low maternal systemic concentrations (2-6× plasma Cave:JAK IC50 ratio, 10-33× nonclinical:clinical AUC margin) and negligible fetal exposures. In conclusion, the izencitinib gut-selective approach resulted in minimal systemic findings in nonclinical species at pharmacologic, clinically relevant systemic exposures, highlighting the impact of organ-selectivity in reducing systemic safety findings.

Transverse chromatic aberration in virtual reality head-mounted displays.

We demonstrate a method for measuring the transverse chromatic aberration (TCA) in a virtual reality head-mounted display. The method relies on acquiring images of a digital bar pattern and measuring the displacement of different color bars. This procedure was used to characterize the TCAs in the Oculus Go, Oculus Rift, Samsung Gear, and HTC Vive. The results show noticeable TCAs for the Oculus devices for angles larger than 5° from the center of the field of view. TCA is less noticeable in the Vive in part due to off-axis monochromatic aberrations. Finally, user measurements were conducted, which were in excellent agreement with the laboratory results.

Mapping of Mcs30, a new mammary carcinoma susceptibility quantitative trait locus (QTL30) on rat chromosome 12: identification of fry as a candidate Mcs gene.

Rat strains differ dramatically in their susceptibility to mammary carcinogenesis. On the assumption that susceptibility genes are conserved across mammalian species and hence inform human carcinogenesis, numerous investigators have used genetic linkage studies in rats to identify genes responsible for differential susceptibility to carcinogenesis. Using a genetic backcross between the resistant Copenhagen (Cop) and susceptible Fischer 344 (F344) strains, we mapped a novel mammary carcinoma susceptibility (Mcs30) locus to the centromeric region on chromosome 12 (LOD score of ∼8.6 at the D12Rat59 marker). The Mcs30 locus comprises approximately 12 Mbp on the long arm of rat RNO12 whose synteny is conserved on human chromosome 13q12 to 13q13. After analyzing numerous genes comprising this locus, we identified Fry, the rat ortholog of the furry gene of Drosophila melanogaster, as a candidate Mcs gene. We cloned and determined the complete nucleotide sequence of the 13 kbp Fry mRNA. Sequence analysis indicated that the Fry gene was highly conserved across evolution, with 90% similarity of the predicted amino acid sequence among eutherian mammals. Comparison of the Fry sequence in the Cop and F344 strains identified two non-synonymous single nucleotide polymorphisms (SNPs), one of which creates a putative, de novo phosphorylation site. Further analysis showed that the expression of the Fry gene is reduced in a majority of rat mammary tumors. Our results also suggested that FRY activity was reduced in human breast carcinoma cell lines as a result of reduced levels or mutation. This study is the first to identify the Fry gene as a candidate Mcs gene. Our data suggest that the SNPs within the Fry gene contribute to the genetic susceptibility of the F344 rat strain to mammary carcinogenesis. These results provide the foundation for analyzing the role of the human FRY gene in cancer susceptibility and progression.

Use of genomic data in risk assessment case study: II. Evaluation of the dibutyl phthalate toxicogenomic data set.

An evaluation of the toxicogenomic data set for dibutyl phthalate (DBP) and male reproductive developmental effects was performed as part of a larger case study to test an approach for incorporating genomic data in risk assessment. The DBP toxicogenomic data set is composed of nine in vivo studies from the published literature that exposed rats to DBP during gestation and evaluated gene expression changes in testes or Wolffian ducts of male fetuses. The exercise focused on qualitative evaluation, based on a lack of available dose-response data, of the DBP toxicogenomic data set to postulate modes and mechanisms of action for the male reproductive developmental outcomes, which occur in the lower dose range. A weight-of-evidence evaluation was performed on the eight DBP toxicogenomic studies of the rat testis at the gene and pathway levels. The results showed relatively strong evidence of DBP-induced downregulation of genes in the steroidogenesis pathway and lipid/sterol/cholesterol transport pathway as well as effects on immediate early gene/growth/differentiation, transcription, peroxisome proliferator-activated receptor signaling and apoptosis pathways in the testis. Since two established modes of action (MOAs), reduced fetal testicular testosterone production and Insl3 gene expression, explain some but not all of the testis effects observed in rats after in utero DBP exposure, other MOAs are likely to be operative. A reanalysis of one DBP microarray study identified additional pathways within cell signaling, metabolism, hormone, disease, and cell adhesion biological processes. These putative new pathways may be associated with DBP effects on the testes that are currently unexplained. This case study on DBP identified data gaps and research needs for the use of toxicogenomic data in risk assessment. Furthermore, this study demonstrated an approach for evaluating toxicogenomic data in human health risk assessment that could be applied to future chemicals.

Comparative genomics of susceptibility to mammary carcinogenesis among inbred rat strains: role of reduced prolactin signaling in resistance of the Copenhagen strain.

To elucidate the molecular basis for differential susceptibilities to mammary carcinogenesis, we compared the transcriptomes of normal mammary glands from pubescent female rats of the resistant Copenhagen (Cop) strain with those of the susceptible Fischer 344 (F344), August x Copenhagen Irish (ACI), Buffalo/N (Buf/N), Wistar-Furth (WF) strains and F1 (Cop x F344) progeny (F1). Gene expression profiles in mammary tissue within each rat strain were remarkably similar, indicating that gene expression was determined by genetic background. We next identified the subset of genes that were differentially expressed in all susceptible strains relative to the resistant Cop strain. Among these, the messenger RNAs encoding prolactin (Prl) and its cell surface receptor were significantly elevated in all susceptible strains. The expression levels of several Prl-regulated genes were also significantly elevated, indicating the presence of increased Prl signaling in mammary glands of all susceptible strains. Pathway analysis of gene expression profiles further identified the Prl-activated Jak/STAT-signaling pathway among the pathways that most distinguished sensitive rat strains from the resistant Cop rat. To test the hypothesis that reduced levels of the Prl signaling in mammary tissue partially contributed to the genetic resistance to mammary carcinogenesis, we used the neuroleptic drug, perphenazine, to transiently elevate serum Prl levels in the Cop strain. Whereas Cop rats are resistant to N-nitroso-N-methylurea (NMU)-induced mammary carcinogenesis, approximately 5% of pubescent Cop females treated with perphenazine and NMU exposure developed mammary adenocarcinomas with latencies comparable with those of sensitive strains. Together, these finding indicated that in the rat, the molecular mechanisms underlying genetic susceptibility to mammary carcinogenesis include de-regulation of Prl signaling.

Childhood acute lymphocytic leukemia and perspectives on risk assessment of early-life stage exposures.

Recognition that children are a potentially susceptible subpopulation has led to the development of child-specific sensitivity factors. Establishing reliable sensitivity factors in support of risk assessment of early-life stage exposures can be aided by evaluating studies that enhance our understanding both of the biological basis of disease processes and the potential role of environmental exposures in disease etiology. For these reasons, we evaluated childhood acute lymphocytic leukemia (ALL) studies from the point of view of mechanism and etiology. ALL is the most common form of childhood cancer proposed to result from a prenatal primary event and a postnatal second event. This multi-stage model is supported by the observation that chromosomal translocations/fusion genes (e.g., TEL-AML1) involved in producing ALL are detected at birth (prenatal event), and a postnatal event (e.g., TEL deletion) is required for disease manifestation. It appears that a proportion of ALL cases are the result of environmental exposures, in which case preconceptional, prenatal, and postnatal stages are likely to be critical exposure windows. To this end, we recognized postnatal infection-related risk factors as potential candidates associated with the ALL second event. Additionally, we discuss use of ALL-associated fusion genes and genetic polymorphisms, together or separately, as indicators of ALL susceptibility and increased risk. The possibility of using fusion genes alone as biomarkers of response is also discussed because they can serve as predictors of key events in the development of a mode of action (a sequence of key events, starting with interaction of an agent with a cell, ultimately resulting in cancer formation) for particular environmental exposures. Furthermore, we discuss use of an initiated animal model for ALL, namely transgenic mice with TEL-AML1 expression, for exploring mechanisms by which different classes of environmental exposures could be involved in inducing the postnatal step in ALL formation.

Evaluation of the corneal effects of topical ophthalmic fluoroquinolones using in vivo confocal microscopy.

PURPOSE: To compare the effects of several fluoroquinolone antibiotics on the corneal epithelium and stroma using in vivo confocal microscopy. METHODS: Five antibiotic solutions were evaluated: 1) 0.3% ofloxacin (Oflox) solution with 0.005% benzalkonium chloride (BAC); 2) 0.3% gatifloxacin (Gati) solution with 0.005% BAC; 3) 0.3% ciprofloxacin (Cipro) solution with 0.006% BAC; 4) 0.5% levofloxacin (Levo) with 0.005% BAC; and 5) 0.5% moxifloxacin (Moxi) solution with no BAC. Preservative-free artificial tears (Tears) were used as a control. New Zealand white rabbits were used for this study (six per solution group). Ten days prior to exposure to any solution, central corneal epithelial thickness and stromal thickness were measured using in vivo confocal microscopy through focusing. Images of the superficial epithelium were also acquired. Both eyes of each rabbit then received one drop of the assigned solution six times the first day and then four times per day for 6 days. On day 7, in vivo confocal microscopy was repeated. RESULTS: A significant decrease in epithelial thickness was induced by 7 days of exposure to Levo, Gati, Oflox, and Cipro (P < 0.05, two-way repeated-measures ANOVA, Tukey test). Tears and Moxi, which do not contain BAC, did not induce significant changes in epithelial thickness. No significant changes in stromal thickness were detected (P = 0.266), and no keratocyte activation was observed for any of the solutions evaluated. CONCLUSION: We have previously used confocal microscopy to establish a correlation between epithelial thinning (due to superficial cell loss) and slight ocular irritation. The results of this study suggest that Moxi induces less damage to the corneal epithelium than other antibiotic solutions, perhaps because it does not contain BAC.

Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome.

Mutations cause or influence the prevalence of many diseases. In human tissues, somatic point mutations have been observed at fractions at or below 4/10,000 and 5/100,000 in mitochondrial and nuclear DNA, respectively. In human populations, fractions for the multiple alleles that code for recessive deleterious syndromes are not expected to exceed 5 x 10(-4). Both nuclear and mitochondrial point mutations have been measured in human cells and tissues at fractions approaching 10(-6) using constant denaturant capillary electrophoresis (CDCE) coupled with high-fidelity PCR (hifiPCR). However, this approach is only applicable to those target sequences (approximately 100 bp) juxtaposed with a 'clamp', a higher-melting-temperature sequence, in genomic DNA; such naturally clamped targets represent approximately 9% of the human genome. To open up most of the human genome to rare point-mutational analysis, a high-efficiency DNA ligation procedure was recently developed so that a clamp could be attached to any target of interest. We coupled this ligation procedure with prior CDCE/hifiPCR and achieved a sensitivity of 2 x 10(-5) in human cells for the first time using an externally attached clamp. At this sensitivity, somatic mutations, each representing an anatomically distinct cluster of cells (turnover unit) derived from a mutant stem cell, may be detected in a series of tissue samples, each containing as many as 5 x 10(4) turnover units. Additionally, rare inherited mutations may be scanned in pooled DNA samples, each derived from as many as 10(5) persons.

High-efficiency DNA ligation for clamp attachment without polymerase chain reaction.

We coupled ligation with mass action to achieve high-efficiency clamp attachment without polymerase chain reaction (PCR). Using a 10-fold molar excess of a GC-rich clamp of synthesized and hybridized oligonucleotides, we achieved the maximum clamp-ligation efficiency in which the clamp was ligated to >95% of 10(10)-10(12) restriction ends of a PCR-amplified fragment. The maximum efficiency was confirmed by ligating the clamp to 10(11)-10(12) restriction ends of human genomic DNA. Our approach can be added to a constant denaturant capillary electrophoresis (CDCE)-based method of analyzing rare point mutants at fractions as low as 10(-6); such mutants appear as small copy numbers in the initial samples. This CDCE-based method alone is applicable to only those DNA sequences juxtaposed with an internally occurring clamp of a higher melting temperature in genomic DNA. Since such sequences represent 9% of the human genome, the addition of clamp ligation significantly increases the scanning range for the human genome without reducing the initial mutant copy numbers. Furthermore, clamp ligation/attachment without PCR prevents PCR-created mutants from interfering with rare mutational analysis. In addition to those applications seeking high-efficiency DNA ligation, our approach can be generally applied to ligation of restriction ends.