Role of Transporters and Enzymes in Metabolism and Distribution of 4-Chlorokynurenine (AV-101).

4-Chlorokynurenine (4-Cl-KYN, AV-101) is a prodrug of a NMDA receptor antagonist and is in clinical development for potential CNS indications. We sought to further understand the distribution and metabolism of 4-Cl-KYN, as this information might provide a strategy to enhance the clinical development of this drug. We used excretion studies in rats, in vitro transporter assays, and pharmacogenetic analysis of clinical trial data to determine how 4-Cl-KYN and metabolites are distributed. Our data indicated that a novel acetylated metabolite (N-acetyl-4-Cl-KYN) did not affect the uptake of 4-Cl-KYN across the blood-brain barrier via LAT1. 4-Cl-KYN and its metabolites were found to be renally excreted in rodents. In addition, we found that N-acetyl-4-Cl-KYN inhibited renal and hepatic transporters involved in excretion. Thus, this metabolite has the potential to limit the excretion of a range of compounds. Our pharmacogenetic analysis found that a SNP in N-acetyltransferase 8 (NAT8, rs13538) was linked to levels of N-acetyl-4-Cl-KYN relative to 4-Cl-KYN found in the plasma and that a SNP in SLC7A5 (rs28582913) was associated with the plasma levels of the active metabolite, 7-Cl-KYNA. Thus, we have a pharmacogenetics-based association for plasma drug level that could aid in the drug development of 4-Cl-KYN and have investigated the interaction of a novel metabolite with drug transporters.

Sequencing of Camelina neglecta, a diploid progenitor of the hexaploid oilseed Camelina sativa.

Camelina neglecta is a diploid species from the genus Camelina, which includes the versatile oilseed Camelina sativa. These species are closely related to Arabidopsis thaliana and the economically important Brassica crop species, making this genus a useful platform to dissect traits of agronomic importance while providing a tool to study the evolution of polyploids. A highly contiguous chromosome-level genome sequence of C. neglecta with an N50 size of 29.1 Mb was generated utilizing Pacific Biosciences (PacBio, Menlo Park, CA) long-read sequencing followed by chromosome conformation phasing. Comparison of the genome with that of C. sativa shows remarkable coincidence with subgenome 1 of the hexaploid, with only one major chromosomal rearrangement separating the two. Synonymous substitution rate analysis of the predicted 34 061 genes suggested subgenome 1 of C. sativa directly descended from C. neglecta around 1.2 mya. Higher functional divergence of genes in the hexaploid as evidenced by the greater number of unique orthogroups, and differential composition of resistant gene analogs, might suggest an immediate adaptation strategy after genome merger. The absence of genome bias in gene fractionation among the subgenomes of C. sativa in comparison with C. neglecta, and the complete lack of fractionation of meiosis-specific genes attests to the neopolyploid status of C. sativa. The assembled genome will provide a tool to further study genome evolution processes in the Camelina genus and potentially allow for the identification and exploitation of novel variation for Camelina crop improvement.

Effect of mGluR2 positive allosteric modulation on frontostriatal working memory activation in schizophrenia.

Negative symptoms and cognitive deficits contribute strongly to disability in schizophrenia, and are resistant to existing medications. Recent drug development has targeted enhanced NMDA function by increasing mGluR2/3 signaling. However, the clinical utility of such agents remains uncertain, and markers of brain circuit function are critical for clarifying mechanisms and understanding individual differences in efficacy. We conducted a double-blind, placebo-controlled, randomized cross-over (14 day washout) pilot study evaluating adjunctive use of the mGluR2 positive allosteric modulator AZD8529 (80 mg daily for 3 days), in chronic stable patients with schizophrenia (n = 26 analyzed). We focused on 3 T fMRI response in frontostriatal regions during an n-back working memory task, testing the hypothesis that AZD8529 produces fMRI changes that correlate with improvement in negative symptoms and cognition. We found that AZD8529 did not produce significant group-average effects on symptoms or cognitive accuracy. However, AZD8529 did increase n-back fMRI activation in striatum (p < 0.0001) and anterior cingulate/paracingulate (p = 0.002). Greater drug-versus-placebo effects on caudate activation significantly correlated with greater reductions in PANSS negative symptom scores (r = -0.42, p = 0.031), and exploratory correlations suggested broader effects across multiple symptom domains and regions of interest. These findings demonstrate that fMRI responses to mGluR2 positive modulation relate to individual differences in symptom reduction, and could be pursued for future biomarker development. Negative clinical results at the group level should not lead to premature termination of investigation of this mechanism, which may benefit an important subset of individuals with schizophrenia. Imaging biomarkers may reveal therapeutic mechanisms, and help guide treatment toward specific populations.

Methodologies and Emerging Technologies for the Evaluation of the Hematopoietic System.

Hematology and bone marrow analysis is central to our understanding of the hematopoietic system and how it responds to insults, and this session presented during the 2022 STP symposium provided a review of current and novel approaches for the evaluation of the hematopoietic system in the context of nonclinical investigations. This publication summarizes the information presented on novel approaches for evaluation of the hematopoietic system using automated hematology analyzers, including details around the quantitative assessment of bone marrow cell suspensions as well as introducing several newly available hematology parameters. It was followed by a discussion on intravital microscopy and live cell imaging and how these methods can assist with de-risking hematopoiesis-associated safety concerns, and a review of recent assays using artificial intelligence for the evaluation of bone marrow.

Linearity and Bias of Proton Density Fat Fraction as a Quantitative Imaging Biomarker: A Multicenter, Multiplatform, Multivendor Phantom Study.

Background Proton density fat fraction (PDFF) estimated by using chemical shift-encoded (CSE) MRI is an accepted imaging biomarker of hepatic steatosis. This work aims to promote standardized use of CSE MRI to estimate PDFF. Purpose To assess the accuracy of CSE MRI methods for estimating PDFF by determining the linearity and range of bias observed in a phantom. Materials and Methods In this prospective study, a commercial phantom with 12 vials of known PDFF values were shipped across nine U.S. centers. The phantom underwent 160 independent MRI examinations on 27 1.5-T and 3.0-T systems from three vendors. Two three-dimensional CSE MRI protocols with minimal T1 bias were included: vendor and standardized. Each vendor's confounder-corrected complex or hybrid magnitude-complex based reconstruction algorithm was used to generate PDFF maps in both protocols. The Siemens reconstruction required a configuration change to correct for water-fat swaps in the phantom. The MRI PDFF values were compared with the known PDFF values by using linear regression with mixed-effects modeling. The 95% CIs were calculated for the regression slope (ie, proportional bias) and intercept (ie, constant bias) and compared with the null hypothesis (slope = 1, intercept = 0). Results Pooled regression slope for estimated PDFF values versus phantom-derived reference PDFF values was 0.97 (95% CI: 0.96, 0.98) in the biologically relevant 0%-47.5% PDFF range. The corresponding pooled intercept was -0.27% (95% CI: -0.50%, -0.05%). Across vendors, slope ranges were 0.86-1.02 (vendor protocols) and 0.97-1.0 (standardized protocol) at 1.5 T and 0.91-1.01 (vendor protocols) and 0.87-1.01 (standardized protocol) at 3.0 T. The intercept ranges (absolute PDFF percentage) were -0.65% to 0.18% (vendor protocols) and -0.69% to -0.17% (standardized protocol) at 1.5 T and -0.48% to 0.10% (vendor protocols) and -0.78% to -0.21% (standardized protocol) at 3.0 T. Conclusion Proton density fat fraction estimation derived from three-dimensional chemical shift-encoded MRI in a commercial phantom was accurate across vendors, imaging centers, and field strengths, with use of the vendors' product acquisition and reconstruction software. © RSNA, 2021 See also the editorial by Dyke in this issue.

Ectopic expression of cDNAs from larkspur (Consolida ajacis) for increased synthesis of gondoic acid (cis-11 eicosenoic acid) and its positional redistribution in seed triacylglycerol of Camelina sativa.

MAIN CONCLUSION: A ß-ketoacyl-ACP-synthase II (KAS2) like enzyme and a lysophosphatidic acid acyltransferase (LPAT2) from Consolida ajacis catalyze gondoic acid biosynthesis and incorporation into the sn-2 position of seed TAG in engineered Camelina sativa. Gondoic acid (cis-11 eicosenoic acid, 20:1∆11) is the predominant very-long-chain fatty acid (VLCFA) in camelina (Camelina sativa) seed oil accounting for 12-15% of total triacylglycerol fatty acids. To explore the feasibility of engineering increased levels of this fatty acid in camelina seed, oils from a range of plant species were analyzed to identify those producing 20-Carbon (C20) fatty acids as the only VLCFAs in their seed oil. Seeds of Consolida and Delphinium species (Ranunculaceae) were found to contain moderate levels (0.2% to 25.5%) of C20 fatty acids without accompanying longer chain fatty acids. The C20 fatty acids were abundant in both sn-2 and sn-1/3 positions of seed TAG in Consolida, but were largely absent from the sn-2 position in Delphinium seed TAG. Through generation of a developing seed transcriptome, sequences were identified and cDNAs amplified from Consolida ajacis encoding a ß-ketoacyl-ACP-synthase II like protein (CaKAS2B) that lacked a predicted chloroplast transit peptide, and two homologues of Arabidopsis thaliana lysophosphatidic acid acyltransferase 2 (CaLPAT2a and CaLPAT2b). Expression of CaKAS2B in conventional (WT) camelina and a line previously engineered for high seed oleic acid content (HO) resulted in increased seed VLCFA content. Total VLCFA levels were raised from 24 to 35% and from 7 to 23% in T3 seed from representative transformants in the WT and HO backgrounds, respectively. Gondoic acid was the predominant VLCFA in transformed HO lines with low endogenous cytoplasmic fatty acid elongation activity, suggesting limited capacity of CaKAS2B to elongate beyond C20. Expression in camelina of CaLPAT2b resulted in significantly increased C20-VLCFA esterification at the sn-2 position of seed TAG with VLCFA levels of 33.8% in this position in one transformed line compared to 0.3% at sn-2 in the corresponding control line. Only small changes in total seed VLCFA content were observed in transformed lines implying that increased VLCFA esterification capacity in camelina results in positional redistribution of VLCFAs but does not significantly enhance flux through the fatty acid elongation pathway. The full potential of CaKAS2B and CaLPAT2a for the engineering of high gondoic acid levels in camelina remains to be determined. Seed fatty acid composition of Consolida and Delphinium also provides information that may be of value in the systematics of the Ranunculaceae.

Probenecid Increases the Concentration of 7-Chlorokynurenic Acid Derived from the Prodrug 4-Chlorokynurenine within the Prefrontal Cortex.

Recent advances in the understanding of depression have led to increasing interest in ketamine and the role that N-methyl-d-aspartate (NMDA) receptor inhibition plays in depression. l-4-Chlorokynurenine (4-Cl-KYN, AV-101), a prodrug, has shown promise as an antidepressant in preclinical studies, but this promise has not been realized in recent clinical trials. We sought to determine if transporters in the CNS could be playing a role in this clinical response. We used radiolabeled uptake assays and microdialysis studies to determine how 4-Cl-KYN and its active metabolite, 7-chlorokynurenic acid (7-Cl-KYNA), cross the blood-brain barrier (BBB) to access the brain and its extracellular fluid compartment. Our data indicates that 4-Cl-KYN crosses the blood-brain barrier via the amino acid transporter LAT1 (SLC7A5) after which the 7-Cl-KYNA metabolite leaves the brain extracellular fluid via probenecid-sensitive organic anion transporters OAT1/3 (SLC22A6 and SLC22A8) and MRP4 (ABCC4). Microdialysis studies further validated our in vitro data, indicating that probenecid may be used to boost the bioavailability of 7-Cl-KYNA. Indeed, we found that coadministration of 4-Cl-KYN with probenecid caused a dose-dependent increase by as much as an 885-fold increase in 7-Cl-KYNA concentration in the prefrontal cortex. In summary, our data show that 4-Cl-KYN crosses the BBB using LAT1, while its active metabolite, 7-Cl-KYNA, is rapidly transported out of the brain via OAT1/3 and MRP4. We also identify a hitherto unreported mechanism by which the brain extracellular concentration of 7-Cl-KYNA may be increased to produce significant boosting of the drug concentration at its site of action that could potentially lead to an increased therapeutic effect.

Screening For Bone Marrow Cellularity Changes in Cynomolgus Macaques in Toxicology Safety Studies Using Artificial Intelligence Models.

Many compounds affect the cellularity of hematolymphoid organs including bone marrow. Toxicologic pathologists are tasked with their evaluation as part of safety studies. An artificial intelligence (AI) tool could provide diagnostic support for the pathologist. We looked at the ability of a deep-learning AI model to evaluate whole slide images of macaque sternebrae to identify and enumerate bone marrow hematopoietic cells. The AI model was trained and able to differentiate the hematopoietic cells from the other sternebrae tissues. We compared the model to severity scores in a study with decreased hematopoietic cellularity. The mean cells/mm2 from the model was lower for each increase in severity score. The AI model was trained by 1 pathologist, providing proof of concept that AI model generation can be fast and agile, without the need of a cross disciplinary team and significant effort. We see great potential for the role of AI-based bone marrow screening.

Potential Induced Radioactivity in Materials Processed with X-ray Energy Above 5 MeV.

Section 5.1.2 of ANSI/AAMI/ISO 11137-1 states that "the potential for induced radioactivity in product shall be assessed." This article describes how compliance with this requirement may be achieved using qualified test methods. Materials of consideration are conceptually discussed, and results of testing conducted on products processed with a 7.5-MeV X-ray irradiation process are provided. As X-ray becomes more widely used in healthcare sterilization, having standard assessment protocols for activation coupled with a shared database of material test results will benefit manufacturers seeking to utilize this innovative technology.

A Randomized Trial of the N-Methyl-d-Aspartate Receptor Glycine Site Antagonist Prodrug 4-Chlorokynurenine in Treatment-Resistant Depression.

BACKGROUND: Ketamine has rapid-acting antidepressant effects but is associated with psychotomimetic and other adverse effects. A 7-chlorokynurenic acid is a potent and specific glycine site N-methyl-d-aspartate receptor antagonist but crosses the blood-brain barrier inefficiently. Its prodrug, L-4-chlorokynurenine (4-Cl-KYN), exerts acute and sustained antidepressant-like effects in rodents and has no reported psychotomimetic effects in either rodents or healthy volunteers. This study examined whether 4-Cl-KYN has rapid antidepressant effects in individuals with treatment-resistant depression. METHODS: After a 2-week drug-free period, 19 participants with treatment-resistant depression were randomized to receive daily oral doses of 4-Cl-KYN monotherapy (1080 mg/d for 7 days, then 1440 mg/d for 7 days) or placebo for 14 days in a randomized, placebo-controlled, double-blind, crossover manner. The primary outcome measure was the Hamilton Depression Rating Scale score, assessed at several time points over a 2-week period; secondary outcome measures included additional rating scale scores. Pharmacokinetic measures of 7-chlorokynurenic acid and 4-Cl-KYN and pharmacodynamic assessments were obtained longitudinally and included 1H-magnetic resonance spectroscopy brain glutamate levels, resting-state functional magnetic resonance imaging, and plasma and cerebrospinal fluid measures of kynurenine metabolites and neurotrophic factors. RESULTS: Linear mixed models detected no treatment effects, as assessed by primary and secondary outcome measures. No difference was observed for any of the peripheral or central biological indices or for adverse effects at any time between groups. A 4-Cl-KYN was safe and well-tolerated, with generally minimal associated adverse events. CONCLUSIONS: In this small crossover trial, 4-Cl-KYN monotherapy exerted no antidepressant effects at the doses and treatment duration studied.ClinicalTrials.gov identifier: NCT02484456.