Discovery of sulfone containing metallo-ß-lactamase inhibitors with reduced bacterial cell efflux and histamine release issues.

The design, syntheses and antibacterial evaluation of sulfone analogues of previously disclosed metallo-ß-lactamase inhibitors (MBLis) are described. The novel derivatives were overall more effective in gram-negative bacterial cell-based assays when combined with imipenem and relebactam. The major contributors to the improved anti-bacterial activity are enhanced enzyme-inhibitor interactions and reduced bacterial cell efflux monitored via an efflux assay involving isogenic Pseudomonas aeruginosa efflux + and efflux - tool strains.

Structure Guided Discovery of Novel Pan Metallo-ß-Lactamase Inhibitors with Improved Gram-Negative Bacterial Cell Penetration.

The use of ß-lactam (BL) and ß-lactamase inhibitor combination to overcome BL antibiotic resistance has been validated through clinically approved drug products. However, unmet medical needs still exist for the treatment of infections caused by Gram-negative (GN) bacteria expressing metallo-ß-lactamases. Previously, we reported our effort to discover pan inhibitors of three main families in this class: IMP, VIM, and NDM. Herein, we describe our work to improve the GN coverage spectrum in combination with imipenem and relebactam. This was achieved through structure- and property-based optimization to tackle the GN cell penetration and efflux challenges. A significant discovery was made that inhibition of both VIM alleles, VIM-1 and VIM-2, is essential for broad GN coverage, especially against VIM-producing P. aeruginosa. In addition, pharmacokinetics and nonclinical safety profiles were investigated for select compounds. Key findings from this drug discovery campaign laid the foundation for further lead optimization toward identification of preclinical candidates.

The TH 22-mediated IL-22 deficiency associated with premature ovarian insufficiency.

RESEARCH QUESTION: Is deficiency of IL-22 associated with premature ovarian insufficiency (POI)? DESIGN: Levels of IL-22 and IL-22BP, IL-22-producing T cells, and IL22RA1/IL10R2 expression were measured and compared among 29 patients with POI, 42 with precursor stage of POI (pre-POI) and 46 control women. Correlation of serum IL-22 and IL-22+ CD4+ T subsets with ovarian reserve markers were further analyzed. RESULTS: IL-22 levels in serum significantly differed among control women and patients with pre-POI and POI, with the lowest concentrations in POI group (p = .019). Significant reduction of peripheral CD4+ IL-22+ T cells was observed in patients with POI (p = .010), which mainly contributed by decrease of CD4+ IL-22+ IL-17- TH 22 cells (p = .012) but not TH 17 cells (p = .125). Levels of serum IL-22 and IL-22-producing CD4+ T subsets were significantly correlated with ovarian reserve markers, including AMH, bilateral AFC, follicle-stimulating hormone (FSH), and E2 (p < .05). The specific receptor IL22RA1 expression was marginally reduced in granulosa cells from patients with pre-POI (p = .051). No difference of IL-22BP was observed either in serum (p = .216) or follicular fluid (p = .856) among groups. CONCLUSIONS: Our study first demonstrated the significant association between TH 22-mediated IL-22 deficiency and ovarian insufficiency, which provide new insights into the autoimmune disturbance and opens new avenues for exogenous IL-22 administration as potential intervention of POI.

Analysis of Prognostic Factors in Osteoporotic Patients Who Were Treated with Percutaneous Vertebroplasty for Refracture After Vertebral Augmentation.

OBJECTIVE: To retrospectively analyze prognostic factors in osteoporotic patients who treated with percutaneous vertebroplasty for refracture after vertebral augmentation. METHODS: A retrospective analysis was performed of 61 patients with refractures after vertebral augmentation who received percutaneous vertebroplasty treatment again from January 2019 to December 2021. Based on the presence of back pain at the last follow-up, 17 patients were placed in the pain group, and 44 patients were placed in the pain-free group. The following covariates were reviewed: age; bone mineral density; bone cement dosage; bone cement leakage; body mass index; and rate of anterior vertebral height (AVH) loss in the target before surgery, 1 week after surgery, and at last follow-up. Patients were assessed using visual analogue scale score and Oswestry Disability Index. RESULTS: Binary logistic regression analysis revealed that the rate of AVH loss after surgery was associated with postoperative back pain. According to the receiver operating characteristic curve analysis, the area under the curve of AVH loss rate at 1 week after surgery was 0.6845, and the cutoff value was 0.18; the area under the curve of AVH loss rate at the last follow-up was 0.7306, and the cutoff value was 0.2815. Kaplan-Meier survival analysis showed that patients with lower AVH loss rates had lower incidence of postoperative back pain and better prognosis. CONCLUSIONS: Occurrence of postoperative back pain was strongly associated with AVH loss after surgery. Patients with a lower rate of AVH loss had a lower incidence of postoperative back pain and a better prognosis.

The Impact of Moderately High Preconception Thyrotropin Levels on Ovarian Reserve Among Euthyroid Infertile Women Undergoing Assisted Reproductive Technology.

Background: Thyroid dysfunction is prevalent in reproductive-age women and has been identified as a risk factor for female infertility. However, it remains largely unclear whether subtle thyroid dysfunction, as estimated by moderately high thyrotropin (TSH) levels within the normal range, is associated with ovarian reserve in infertile women before assisted reproductive technology (ART) treatment. Methods: This cross-sectional study involved 3501 euthyroid infertile women, including 2189 women with TSH levels ≤2.5 µIU/mL and 1312 women with high-normal TSH levels (2.51-4.20 µIU/mL). Ovarian reserve markers were compared between women with low- and high-normal TSH levels. Correlation analysis and regression models were used to estimate the association of TSH levels with ovarian reserve. In addition, the association of subtle thyroid dysfunction with ovarian reserve was further evaluated after stratification for different infertility diagnoses and statuses of thyroid autoimmunity (TAI). Results: In the total population, women with high-normal TSH levels had significantly decreased anti-Müllerian hormone (AMH) concentrations (p < 0.001), a lower bilateral antral follicle count (AFC) (p < 0.001), and a higher prevalence of diminished ovarian reserve (DOR) (p = 0.018) than women with low-normal TSH levels. The TSH levels showed a negative association with both AMH levels (r = -0.050, p = 0.003) and bilateral AFC (r = -0.071, p < 0.001). Furthermore, the association of high-normal TSH levels with decreased AMH and AFC was more prominent in infertile women with ovulation dysfunction (p = 0.002, p = 0.002), unexplained infertility (p = 0.020, p = 0.028), or negative TAI (both p < 0.001). Conclusions: These data suggested that subtle thyroid dysfunction was associated with DOR in infertile women before ART treatment, which will add evidence that strengthens the systematic screening of TSH levels/TAI in infertile women and will contribute to the discussion of specific TSH cutoff values in predicting ovarian reserve.

Guiding Chemically Synthesized Peptide Drug Lead Optimization by Derisking Mast Cell Degranulation-Related Toxicities of a NaV1.7 Peptide Inhibitor.

Studies have shown that some peptides and small molecules can induce non IgE-mediated anaphylactoid reactions through mast cell activation. Upon activation, mast cells degranulate and release vasoactive and proinflammatory mediators, from cytoplasmic granules into the extracellular environment which can induce a cascade of severe adverse reactions. This study describes a lead optimization strategy to select NaV1.7 inhibitor peptides that minimize acute mast cell degranulation (MCD) toxicities. Various in vitro, in vivo, and PKPD models were used to screen candidates and guide peptide chemical modifications to mitigate this risk. Anesthetized rats dosed with peptides demonstrated treatment-related decreases in blood pressure and increases in plasma histamine concentrations which were reversible with a mast cell stabilizer, supporting the MCD mechanism. In vitro testing in rat mast cells with NaV1.7 peptides demonstrated a concentration-dependent increase in histamine. Pharmacodynamic modeling facilitated establishing an in vitro to in vivo correlation for histamine as a biomarker for blood pressure decline via the MCD mechanism. These models enabled assessment of structure-activity relationship (SAR) to identify substructures that contribute to peptide-mediated MCD. Peptides with hydrophobic and cationic characteristics were determined to have an elevated risk for MCD, which could be reduced or avoided by incorporating anionic residues into the protoxin II scaffold. Our analyses support that in vitro MCD assessment in combination with PKPD modeling can guide SAR to improve peptide lead optimization and ensure an acceptable early in vivo tolerability profile with reduced resources, cycle time, and animal use.

Two distinct resident macrophage populations coexist in the ovary.

Introduction: Tissue-resident macrophages (TRMs) are highly heterogeneous and have a complex and important role in tissue support, homeostasis, and function. The heterogeneity, maintenance, and function of TRMs, as one of the major immune cells in the ovary, are not well understood. Methods: Application of flow cytometry, Parabiosis, Fate mapping, Macrophage depletion, etc. Results: Here, we described two distinct macrophage subsets, F4/80hiCD11bint and F4/80intCD11bhi, with different phenotypic characteristics in the ovary of mice. The F4/80hiCD11bint population contained a distinct CD206+ subgroup and highly expressed CD81, while the F4/80intCD11bhi subset showed higher expression of CCR2 and TLR2. Notably, Ly6c+ macrophages were present almost exclusively in the F4/80intCD11bhi subpopulation. Combining in vivo fate mapping and parabiotic mouse models, we characterized the longevity and replenishment of the two macrophage populations. We found that both the F4/80hiCD11bint and F4/80intCD11bhi subsets were ovary-resident. Importantly, the F4/80hiCD11bint macrophages acted as a self-maintaining and long-lived population with a modest monocyte contribution at a steady state, and the F4/80intCD11bhi subpopulation had a relatively short lifespan with a greater contribution from monocytes. After macrophage ablation, disturbance of estradiol secretion and ovarian hemorrhage due to damaged vascular integrity was observed in mice. Discussion: Our data provide critical insights into ovarian macrophage heterogeneity and highlight the strategic role of TRMs in ovarian homeostasis and physiology.

Development of ProTx-II Analogues as Highly Selective Peptide Blockers of Nav1.7 for the Treatment of Pain.

Inhibitor cystine knot peptides, derived from venom, have evolved to block ion channel function but are often toxic when dosed at pharmacologically relevant levels in vivo. The article describes the design of analogues of ProTx-II that safely display systemic in vivo blocking of Nav1.7, resulting in a latency of response to thermal stimuli in rodents. The new designs achieve a better in vivo profile by improving ion channel selectivity and limiting the ability of the peptides to cause mast cell degranulation. The design rationale, structural modeling, in vitro profiles, and rat tail flick outcomes are disclosed and discussed.

A Series of Novel, Highly Potent, and Orally Bioavailable Next-Generation Tricyclic Peptide PCSK9 Inhibitors.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. Starting from second-generation lead structures such as 2, we were able to refine these structures to obtain extremely potent bi- and tricyclic PCSK9 inhibitor peptides. Optimized molecules such as 44 demonstrated sufficient oral bioavailability to maintain therapeutic levels in rats and cynomolgus monkeys after dosing with an enabled formulation. We demonstrated target engagement and LDL lowering in cynomolgus monkeys essentially identical to those observed with the clinically approved, parenterally dosed antibodies. These molecules represent the first report of highly potent and orally bioavailable macrocyclic peptide PCSK9 inhibitors with overall profiles favorable for potential development as once-daily oral lipid-lowering agents. In this manuscript, we detail the design criteria and multiparameter optimization of this novel series of PCSK9 inhibitors.

Treg deficiency-mediated TH 1 response causes human premature ovarian insufficiency through apoptosis and steroidogenesis dysfunction of granulosa cells.

Immune dysregulation has long been proposed as a component of premature ovarian insufficiency (POI), but the underlying mediators and mechanisms remain largely unknown. Here we showed that patients with POI had augmented T helper 1 (TH 1) responses and regulatory T (Treg ) cell deficiency in both the periphery and the ovary compared to the control women. The increased ratio of TH 1:Treg cells was strongly correlated with the severity of POI. In mouse models of POI, the increased infiltration of TH 1 cells in the ovary resulted in follicle atresia and ovarian insufficiency, which could be prevented and reversed by Treg cells. Importantly, interferon (IFN) -γ and tumor necrosis factor (TNF) -α cooperatively promoted the apoptosis of granulosa cells and suppressed their steroidogenesis by modulating CTGF and CYP19A1. We have thus revealed a previously unrecognized Treg cell deficiency-mediated TH 1 response in the pathogenesis of POI, which should have implications for therapeutic interventions in patients with POI.

Copper Nanoparticles Induce Oxidative Stress via the Heme Oxygenase 1 Signaling Pathway in vitro Studies.

PURPOSE: The toxicity of copper nanoparticle (CuNP) exposure in the ovaries has attracted attention recently, but the precise molecular mechanism involved requires further investigation. We investigated the cytotoxicity of CuNPs in ovarian granulosa cells and the protective effect of heme oxygenase 1 (HO-1) against CuNP-induced damage. METHODS: Human ovarian granulosa cells (COV434) were treated with CuNPs, and cytotoxicity was evaluated using Cell Counting Kit-8 and flow cytometry assays. Oxidative stress was identified using biochemical markers of oxidation and anti-oxidation. The protein levels of mitogen-activated protein kinase 14 (MAPK14), phospho-MAPK14, nuclear factor erythroid 2-related factor 2 (Nrf2), and HO-1 were measured by immunoblotting. Subsequently, for oxidative stress parameter detection, the cells were pre-treated with hemin to induce HO-1 expression prior to CuNP treatment. RESULTS: Exposure to CuNPs decreased cell viability and the mitochondrial membrane potential, increased the apoptosis rate, and induced oxidative stress. Furthermore, hemin pretreatment induced HO-1 expression in cells, which partially reduced the accumulation of reactive oxygen species induced by CuNPs and increased the levels of antioxidant enzymes. CONCLUSION: CuNPs exert cytotoxic effects on human ovarian granulosa cells by inducing oxidative stress, and may induce HO-1 expression via the MAPK14-Nrf2 signaling pathway. Moreover, HO-1 protects against oxidative stress induced by CuNPs.

Humanin Alleviates Insulin Resistance in Polycystic Ovary Syndrome: A Human and Rat Model-Based Study.

Polycystic ovary syndrome (PCOS), the most common endocrine disorder in women of reproductive age, is characterized by hyperandrogenism and insulin resistance (IR); however, the pathogenesis of local ovarian IR in PCOS remains largely unclear. Humanin, a mitochondria-derived peptide, has been reported to be associated with IR. Our previous study confirmed that humanin is expressed in multiple cell types in the ovary and is present in follicular fluid. However, it remains unknown whether humanin participates in the pathogenesis of local ovarian IR or whether humanin supplementation can improve IR in PCOS patients. In this study, we compared humanin concentrations in follicular fluid from PCOS patients with and without IR. We further investigated the effect of humanin analogue (HNG) supplementation on IR in a rat model of dehydroepiandrosterone-induced PCOS. Humanin concentrations in the follicular fluid were found to be significantly lower in PCOS patients with IR than in those without IR. HNG supplementation attenuated both the increases in the levels of fasting plasma glucose and fasting insulin in rats with PCOS and the decreases in phosphorylation of IRS1, PI3K, AKT, and GLUT4 proteins in the granulosa cells of these rats. Combined supplementation with HNG and insulin significantly improved glucose consumption in normal and humanin-siRNA-transfected COV434 cells. In conclusion, downregulated humanin in the ovaries may be involved in the pathogenesis of IR in PCOS, and exogenous supplementation with HNG improved local ovarian IR through modulation of the IRS1/PI3K/Akt signaling pathway in a rat model. This finding supports the potential future use of HNG as a therapeutic drug for PCOS.

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling.

Macrocyclic peptides have the potential to address intracellular protein-protein interactions (PPIs) of high value therapeutic targets that have proven largely intractable to small molecules. Here, we report broadly applicable lessons for applying this modality to intracellular targets and specifically for advancing chemical matter to address KRAS, a protein that represents the most common oncogene in human lung, colorectal and pancreatic cancers yet is one of the most challenging targets in human disease. Specifically, we focused on KRpep-2d, an arginine-rich KRAS-binding peptide with a disulfide-mediated macrocyclic linkage and a protease-sensitive backbone. These latter redox and proteolytic labilities obviated cellular activity. Extensive structure-activity relationship studies involving macrocyclic linker replacement, stereochemical inversion, and backbone α-methylation, gave a peptide with on-target cellular activity. However, we uncovered an important generic insight - the arginine-dependent cell entry mechanism limited its therapeutic potential. In particular, we observed a strong correlation between net positive charge and histamine release in an ex vivo assay, thus making this series unsuitable for advancement due to the potentially fatal consequences of mast cell degranulation. This observation should signal to researchers that cationic-mediated cell entry - an approach that has yet to succeed in the clinic despite a long history of attempts - carries significant therapy-limiting safety liabilities. Nonetheless, the cell-active molecules identified here validate a unique inhibitory epitope on KRAS and thus provide valuable molecular templates for the development of therapeutics that are desperately needed to address KRAS-driven cancers - some of the most treatment-resistant human malignancies.

The role of miR-128-3p through MAPK14 activation in the apoptosis of GC2 spermatocyte cell line following heat stress.

BACKGROUND: MicroRNAs play a crucial role in the regulation of spermatogenesis. For example, miR-128-3p expression is known to decrease significantly after testicular hyperthermia, but the regulatory effect of this change on the spermatogenesis damage caused by heat stress remains unclear. OBJECTIVES: This study aimed to verify whether the target gene of miR-128-3p is MAPK14, which affects spermatogenic cell proliferation and apoptosis under testicular hyperthermia. MATERIALS AND METHODS: Mouse testis and GC2 spermatocyte cell line heat stress models were established. miR-128-3p expression before and after heat stress was analyzed by reverse transcription polymerase chain reaction. MAPK14 and p-MAPK14 expression was detected by Western blot, and cell apoptosis was analyzed by Annexin V-FITC/PI. Subsequently, miR-128-3p inhibitors and mimics were used to interfere with spermatocytes before and after heat stress, respectively, for correlation detection. RESULTS: Compared with the control group, the heat stress group showed decreased miR-128-3p expression, increased p-MAPK14 expression, and decreased cell proliferation activity. In the GC2-spd cell line in vitro, miR-128-3p inhibitors were found to upregulate p-MAPK14 expression, reduce cell proliferation activity, and increase apoptosis, consistent with the results obtained in the heat treatment alone. Furthermore, miR-128-3p mimics transfected in the GC2 cells after heat stress reduced p-MAPK14 expression, alleviated the decrease in cell proliferation, and decreased the apoptosis level. CONCLUSIONS: The downregulation of miR-128-3p expression plays an important role in spermatogenesis damages after testicular hyperthermia, which is probably attributable to the activation of the MAPK signaling pathway. Downregulated miR-128-3p expression induces the apoptosis and inhibits the proliferation of spermatogenic cells by promoting MAPK14 phosphorylation.

Humanin regulates oxidative stress in the ovaries of polycystic ovary syndrome patients via the Keap1/Nrf2 pathway.

Polycystic ovary syndrome (PCOS) is the most common endocrinological pathology among women of reproductive age, whereas the pathogenesis is still not fully understood. Systemic and ovarian oxidative stress (OS) imbalance is a pivotal feature of PCOS. Humanin, a mitochondria-derived peptide, has been reported to function as an antioxidant in cardiomyocytes, pancreatic beta cells and other cells, but how this function is regulated remains unclear. In this study, we investigated whether humanin expression differs in the granulosa cells (GCs) of PCOS patients versus controls, and whether humanin alleviates OS in PCOS ovaries. Sixteen PCOS patients and 28 age- and BMI-matched controls undergoing IVF were recruited, and their serum, follicular fluid and GCs were collected for humanin analysis. Dehydroepiandrosterone-induced rat PCOS models, and vitamin K3-induced OS COV434 cell lines were applied to investigate the mechanism. Humanin expression was significantly down-regulated in the ovaries of PCOS patients relative to those of non-PCOS patients. Exogenous humanin supplementation significantly attenuated body weight gain, ovarian morphological abnormalities, endocrinological disorders and ovarian and systemic OS in PCOS rat models. Our study further demonstrated that this attenuation effect was involved in the modulation of the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor-erythroid 2-related factor 2 (Nrf2) signalling pathway. In summary, this study reported for the first time that decreased expression of humanin in the GCs was associated with oxidative imbalance in PCOS. Humanin alleviates OS in ovarian GCs of PCOS patients via modulation of the Keap1/Nrf2 signalling pathway.

Cold-induced RNA-binding protein (CIRBP) regulates the expression of Src-associated during mitosis of 68 kDa (Sam68) and extracellular signal-regulated kinases (ERK) during heat stress-induced testicular injury.

In this study, the ability of cold-induced RNA-binding protein (CIRBP) to regulate the expression of Src-associated during mitosis of 68 kDa (Sam68) and extracellular signal-regulated kinases (ERK) in the mouse testis and mouse primary spermatocytes (GC-2spd cell line) before and after heat stress was examined to explore the molecular mechanism by which CIRBP decreases testicular injury. A mouse testicular hyperthermia model, a mouse primary spermatocyte hyperthermia model and a low CIRBP gene-expression cell model were constructed and their relevant parameters were analysed. The mRNA and protein levels of CIRBP and Sam68 were significantly decreased in the 3-h and 12-h testicular heat-stress groups, extracellular signal-regulated kinase 1/2 (ERK1/2) protein expression was not significantly affected but phospho-ERK1/2 protein levels were significantly decreased. GC-2spd cellular heat-stress results showed that the mRNA and protein concentrations of CIRBP and Sam68 were reduced 48h after heat stress. In the low CIRBP gene-expression cell model, CIRBP protein expression was significantly decreased. Sam68 mRNA expression was significantly decreased only at the maximum transfection concentration of 50nM and Sam68 protein expression was not significantly affected. These findings suggest that CIRBP may regulate the expression of Sam68 at the transcriptional level and the expression of phospho-ERK1/2 protein, both of which protect against heat-stress-induced testicular injury in mice.

Series of Novel and Highly Potent Cyclic Peptide PCSK9 Inhibitors Derived from an mRNA Display Screen and Optimized via Structure-Based Design.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. In this paper, we describe a series of novel cyclic peptides derived from an mRNA display screen which inhibit the protein-protein interaction between PCSK9 and LDLR. Using a structure-based drug design approach, we were able to modify our original screening lead 2 to optimize the potency and metabolic stability and minimize the molecular weight to provide novel bicyclic next-generation PCSK9 inhibitor peptides such as 78. These next-generation peptides serve as a critical foundation for continued exploration of potential oral, once-a-day PCSK9 therapeutics for the treatment of cardiovascular disease.

A T-cell-dependent antibody response study using a murine surrogate anti-PD-1 monoclonal antibody as an alternative to a non-human primate model.

The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint which may be engaged by cells in a tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda®) is a potent and highly selective humanized monoclonal antibody (mAb) of the IgG4/κ isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. The current work was focused on developing a mouse T-Dependent Antibody Response (TDAR) model using a murinized rat anti-mouse PD-1 antibody (muDX400; a rodent surrogate for pembrolizumab) to evaluate the potential impact of treatment with a PD-1 inhibitor on immune responses to an antigen challenge (e.g. HBsAg in Hepatitis B vaccine). Despite the lower binding affinity and T1/2 compared to pembrolizumab, ligand blocking data indicated muDX400 had appropriate pharmacological activity and demonstrated efficacy in mouse tumor models, thus was suitable for pharmacodynamic and vaccination studies in mice. In a vaccination study in which mice were concomitantly administered muDX400 and the Hepatitis B vaccine, muDX400 was well-tolerated and did not result in any immune-mediated adverse effects. The treatment with muDX400 was associated with a shift in the ratio between naive and memory cells in both CD4+ and CD8+ T-lymphocytes in the spleen but did not affect anti-HBsAg antibody response profile. The mouse TDAR model using the Hepatitis B vaccine and the surrogate anti-PD1 monoclonal antibody was a useful tool in the evaluation of the potential immune-mediated effects of pembrolizumab following vaccination and appears to be a suitable alternative for the nonhuman primate TDAR models utilized for other checkpoint inhibitors.

Characterization of indoleamine-2,3-dioxygenase 1, tryptophan-2,3-dioxygenase, and Ido1/Tdo2 knockout mice.

Indoleamine-2,3-dioxygenase 1 (IDO1) and tryptophan-2,3-dioxygenase 2 (TDO2) degrade tryptophan (Trp) to kynurenine (Kyn), and these enzymes have promise as therapeutic targets. A comprehensive characterization of potential safety liabilities of IDO1 and TDO2 inhibitors using knockout (KO) mice has not been assessed, nor has the dual Ido1/Tdo2 KO been reported. Here we characterized male and female mice with KOs for Ido1, Tdo2, and Ido1/Tdo2 and compared findings to the wild type (WT) mouse strain, evaluated for 14 days, using metabolomics, transcriptional profiling, behavioral analysis, spleen immunophenotyping, comprehensive histopathological analysis, and serum clinical chemistry. Multiple metabolomic changes were seen in KO mice. For catabolism of Trp to Kyn and anthranilic acid, both substrates were decreased in liver of Tdo2 and dual KO mice. Metabolism of Trp to serotonin and its metabolites resulted in an increase in 5-Hydroxyindole-3-acetic acid in the Tdo2 and dual KO mice. Ido1 and dual KO mice displayed a Kyn reduction in plasma but not in liver. Nicotinamide synthesis and conversion of glucose to lactic acid were not impacted. A slight decrease in serum alkaline phosphatase was seen in all KOs, and small changes in liver gene expression of genes unrelated to tryptophan metabolism were observed. Regarding other parameters, no genotype-specific changes were observed. In summary, this work shows metabolomic pathway changes for metabolites downstream of tryptophan in these KO mice, and suggests that inhibition of the IDO1 and TDO2 enzymes would be well tolerated whether inhibited individually or in combination since no safety liabilities were uncovered.

Cross-company evaluation of the human lymphocyte activation assay.

Nonclinical immunotoxicity evaluation is an important component of safety assessment for pharmaceuticals. One in vitro assay that can be applied in a weight of evidence assessment is the human lymphocyte activation (HuLA) assay, an antigen recall assay, similar in many respects to the in vivo T-cell-dependent antibody response (TDAR) in that cooperation of multiple immune cell types are needed to produce responses. This assay uses human cells and is more amenable than the TDAR to compound ranking and mechanistic studies. The HuLA assay requires less time and drug than TDAR assays, uses a relevant antigen (influenza), reflects a human immune response, and applies principles of the 3Rs to non-clinical safety assessment. Peripheral blood mononuclear cells (PBMC) from flu-immunized donors are re-stimulated with flu-vaccine in the presence of test articles, and proliferation is measured. Published data demonstrate the applicability of the HuLA assay, but it has not been evaluated for reproducibility across testing sites. To evaluate assay reproducibility, scientists from a consortium of institutions conducted the assay in parallel, using a common pool of donor PBMC, influenza vaccine, and known immunosuppressant compounds (cyclosporine A and mycophenolic acid). The HuLA assay was highly reproducible in identification of inhibition of antigen-specific responses, and there was significant agreement across testing sites in the half maximal inhibitory concentration (IC50) values. Intra-site variability was the largest contributor to the variability observed within the assay. The HuLA assay was demonstrated to be ideally suited to comparing multiple compounds (i.e. compound ranking or benchmarking) within the same assay. Overall, the data reported herein support the HuLA assay as a useful tool in mechanistic evaluations of antigen-specific immune responses.