4-week Recovery Period Research Articles

Obicetrapib exhibits favorable physiochemical and pharmacokinetic properties compared to previous cholesteryl ester transfer protein inhibitors: An integrated summary of results from non-human primate studies and clinical trials.

Anacetrapib, a cholesteryl ester transfer protein (CETP) inhibitor previously under development, exhibited an usually extended terminal half-life and large food effect and accumulated in adipose tissue. Other CETP inhibitors have not shown such effects. Obicetrapib, a potent selective CETP inhibitor, is undergoing Phase III clinical development. Dedicated assessments were conducted in pre-clinical and Phase I and II clinical studies of obicetrapib to examine the pharmacokinetic issues observed with anacetrapib. After 9 months of dosing up to 50 mg/kg/day in cynomolgus monkeys, obicetrapib was completely eliminated from systemic circulation and not detected in adipose tissue after a 13-week recovery period. In healthy humans receiving 1-25 mg of obicetrapib, the mean terminal half-life of obicetrapib was 148, 131, and 121 h at 5, 10, and 25 mg, respectively, and food increased plasma levels by ~1.6-fold with a 10 mg dose. At the end of treatment in Phase II trials, mean plasma levels of obicetrapib ranged from 194.5 ng/mL with 2.5 mg to 506.3 ng/mL with 10 mg. Plasma levels of obicetrapib decreased by 92.2% and 98.5% at four and 15 weeks post-treatment, respectively. Obicetrapib shows no clinically relevant accumulation, is minimally affected by food, and has a mean terminal half-life of 131 h for the 10 mg dose. These data support once daily, chronic dosing of obicetrapib in Phase III trials for dyslipidemia management.

Effects of puerarin on gait disturbance in a 6-hydroxydopamine mouse model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder caused by dopamine (DA) neuronal dysfunction. Although DA agonists and N-methyl-D-aspartate receptor (NMDAR) antagonists are used to treat PD, chronic use causes severe side effects. Puerarin (PUE) is a natural bioactive compound that affects the DA system; however, its effect on PD-associated motor functions is unknown. Therefore, we investigated whether PUE treatment in a 6-hydroxydopamine (6-OHDA) PD mouse model affects motor dysfunction. Adult male ICR mice received unilateral 6-OHDA microinfusion into the right medial forebrain bundle. After a 2-week recovery period, PUE (20 or 50mg/kg) or the vehicle (saline, VEH) was administered intraperitoneally once daily for 21 days. Motor dysfunction was assessed using the locomotion, gait cycle, and rotation tests. Local field potentials (LFPs) were measured in the substantia nigra compacta (SNc), striatum (STR), subthalamic nucleus (STN), and primary motor cortex. 6-OHDA-lesioned PD mice showed increased gait cycle disturbance and unidirectional rotation. PUE treatment ameliorated the gait cycle disturbance, but not unidirectional rotation of PD mice. These effects differed with DA agonist treatment (which improved PD symptoms) and NMDAR antagonist treatment (which aggravated PD symptoms). Moreover, locomotion was increased only in NMDAR antagonist treatment. PUE treatment induced no changes in the attenuated LFP of the beta wave in the STR and STN, and SNc-STN delta-wave coherence was shown in PD animals. This study suggests that PUE is a beneficial co-therapeutic agent for alleviating gait cycle disturbance in PD symptoms.

P16-02 Subchronic intramuscular toxicity and immunogenicity study of a plant-based SARS-CoV-2 vaccine in Sprague-Dawley rats with a 4-week recovery period

P16-02 Subchronic intramuscular toxicity and immunogenicity study of a plant-based SARS-CoV-2 vaccine in Sprague-Dawley rats with a 4-week recovery period

13-Week Repeated Oral Toxicity and Toxicokinetic Studies of Rabeprazole Sodium and Sodium Bicarbonate Combination in Dogs.

The subchronic toxicity and toxicokinetics of a combination of rabeprazole sodium and sodium bicarbonate were investigated in dogs by daily oral administration for 13 consecutive weeks with a 4-week recovery period. The dose groups consisted of control (vehicles), (5 + 200), (10 + 400), and (20 + 800) mg/kg of rabeprazole sodium + sodium bicarbonate, 20mg/kg of rabeprazole sodium only, and 800mg/kg of sodium bicarbonate only. Esophageal ulceration accompanied by inflammation was observed in only one animal in the male (20 + 800) mg/kg rabeprazole sodium + sodium bicarbonate group. However, the severity of the ulceration was moderate, and the site of occurrence was focally extensive; thus, it was assumed to be a treatment-related effect of rabeprazole sodium + sodium bicarbonate. In the toxicokinetics component of this study, systemic exposure to rabeprazole sodium (AUClast and Cmax at Day 91) was greater in males than females, suggesting sex differences. AUClast and Cmax at Day 91 were increased compared to those on Day 1 in a dose-dependent manner. A delayed Tmax and no drug accumulation were observed after repeated dosage. In conclusion, we suggest under the conditions of this study that the no-observed-adverse-effect level (NOAEL) of the combination of rabeprazole sodium + sodium bicarbonate in male and female dogs is (10 + 400) and (20 + 800) mg/kg, respectively.

Reversible effects on female rat fertility with abrocitinib, a Janus kinase 1 inhibitor.

Abrocitinib is a Janus kinase (JAK) 1 selective inhibitor approved for the treatment of atopic dermatitis. Female reproductive tissues were unaffected in general toxicity studies, but an initial female rat fertility study resulted in adverse effects at all doses evaluated. A second rat fertility study was conducted to evaluate lower doses and potential for recovery. This second study had 4 groups of 20 females each administered abrocitinib (0, 3, 10, or 70 mg/kg/day) 2 weeks prior to cohabitation through gestation day (GD) 7. In addition, 2 groups of 20 rats (0 or 70 mg/kg/day) were dosed for 3 weeks followed by a 4-week recovery period before mating. All mated females were evaluated on GD 14. No effects were observed at ≤10 mg/kg/day. At 70 mg/kg/day (29x human exposure), decreased pregnancy rate, implantation sites, and viable embryos were observed. All these effects reversed 4 weeks after the last dose. Based on these data and literature on the potential role of JAK signaling in implantation, we hypothesize that these effects may be related to JAK1 inhibition and, generally, that peri-implantation effects such as these, in the absence of cycling or microscopic changes in nonpregnant female reproductive tissues, are anticipated to be reversible.

Multiple organs injury and myocardial energy metabolism disorders induced by isoproterenol

The study sought to assess the detrimental effects of isoproterenol (ISO) on major organs and investigate the potential reversibility of these adverse reactions in mice. Male mice were divided into normal control, 0.2 mg/kg.d and 3.0 mg/kg.d ISO groups, and were subcutaneously administered of the respective doses for 14 consecutive days. Subsequently, a recovery period experiment was conducted, replicating the aforementioned procedure, followed by an additional 2-week recovery period for the mice. Following 14 consecutive days of administration, mice treated with ISO exhibited notable cardiac damage manifested by abnormal ECG patterns, dysregulated energy metabolism, elevated cardiac hypertrophy, and increased heart pathological score. Additionally, the administration of ISO resulted in liver and kidney damage, as evidenced by increased pathological score, serum albumin level, and urea level. Lung damage was also observed, indicated by an increase in lung pathological score. Furthermore, the administration of ISO at a dosage of 3.0 mg/kg.d resulted in a decrease in liver mass index, serum iron content, and an increase in lung mass index. After a 2-week recovery period, mice treated with ISO showed abnormalities in ECG patterns and dysregulated myocardial energy metabolism, accompanied by a decrease in serum iron content. Histopathological examinations revealed continued pathological changes in the heart and lung, as well as significant hemosiderin deposition in the spleen. Furthermore, the group treated with ISO at a dosage of 3.0 mg/kg.d showed an increase in serum AST and TP levels. In summary, the study demonstrates that both 0.2 mg/kg.d and 3.0 mg/kg.d doses of ISO can induce damage to the heart, liver, lung, kidney, and spleen, with the higher dose causing more severe injuries. After a 2-week withdrawal period, the liver, kidney, and thymus injuries caused by 0.2 mg/kg ISO shows signs of recovery, while damage to the heart, lung, and spleen persists. The thymus injury mostly recovers, with minimal kidney pathology, but significant damage to the heart, liver, and lung remains even after the withdrawal period for the 3.0 mg/kg ISO dose.

Toxicity of a novel antifungal agent (ATB1651 gel) in Yucatan minipigs (Sus scrofa) following 4weeks of daily dermal administration.

ATB1651 gel is an antifungal drug candidate that enhances antifungal activity through substitution of several aryl rings, alkyl chains, and methyl groups. To ensure safety of use of ATB1651 gel, assessment of its potentially toxic side effects is necessary. In this study, we examined the repeated-dose toxicity of ATB1651 gel to Yucatan minipigs (Sus scrofa) in accordance with the Good Laboratory Practice guidelines. Five doses of ATB1651 gel (0%, 0.2%, 0.5%, 1.0%, 3.0%) were administered dermally to the left and right flanks of 38 minipigs daily for 4weeks. Mortality, clinical symptoms, dermal scores, body weights, and physiological, biochemical, pathological, and toxicokinetic analyses were performed after the treatment period. No systemic toxicological damage was observed in either male or female minipigs regardless of dose; however, dermal application of ATB1651 gel caused some skin alterations at the application sites. Specifically, erythema and eschar formation, edema, and scabs or raise spots were observed at the application site(s) in males in the 3.0% ATB1651 gel treatment group and in females at ATB1651 gel concentrations ≥ 1.0%, with dermal scores ranging from grade 1 to 2. Additionally, histopathological assay indicated infiltration of different types of inflammatory cells and the presence of pustule/crust at the application site(s) in both males and females at ATB1651 gel concentrations ≥ 0.5%. However, these changes were reversible after a 2-week recovery period and were considered a local irritation effect of ATB1651 gel. The no-observed-adverse-effect level of ATB1651 gel was 3.0% with regard to topical and systemic toxicity in both male and female minipigs. Collectively, our results imply that ATB1651 gel is a safe candidate for clinical development as an antifungal drug with a wide therapeutic window.

Chiral S-flurbiprofen axetil-loaded lipid microspheres for improved analgesic effects: Preparation, characterization, and preclinical evaluation

Flurbiprofen axetil (FPA) is a non-steroidal anti-inflammatory drug commonly used for managing post-surgical and cancer-related pain. However, FPA consists of two enantiomers, namely S-flurbiprofen axetil (S-FPA) and R-flurbiprofen axetil (R-FPA), with distinct inhibitory activities on cyclooxygenase (COX). This study focused on utilizing chiral S-FPA as the active pharmaceutical ingredient for the first time, and developed S-FPA-loaded lipid microspheres (S-FPA@LMs). The average particle size and zeta potential of S-FPA@LMs were 217.33 ± 6.66 nm and −36 ± 3.46 mV, respectively. S-FPA@LMs exhibited excellent stability for at least 6 months when stored at 5 ± 3 °C. COX activity inhibition assay revealed that S-FPA selectively inhibited COX-1, exhibiting a more potent inhibitory effect than the racemic form of FPA. In a rat model of postoperative incision pain, S-FPA@LMs demonstrated dose-dependent analgesic effects with superior efficacy compared to equimolar doses of FPA@LMs. Pharmacokinetic studies revealed comparable profiles for the metabolites, including S-flurbiprofen (S-FP) and R-flurbiprofen (R–FP), when S-FPA@LMs and FPA@LMs were administered in equimolar doses. Additionally, continuous administration for 7 days showed no significant accumulation of S-FP and R–FP. The inversion from S-FP to R–FP after S-FPA@LMs administration was also negligible. In terms of safety, S-FPA@LMs did not induce any signs of hemolysis or coagulation in rabbit erythrocytes. Moreover, no immediate allergic reactions were observed in the active systemic anaphylaxis test. The sub-acute toxicity study indicated dose-dependent adverse reactions primarily related to the gastrointestinal system, with recovery observed after a 2-week recovery period. Notably, S-FPA@LMs displayed a lower mortality rate and less severe pathological changes in the deceased rats compared to FPA@LMs. Overall, our findings highlight the potential of S-FPA@LMs as a promising candidate for acute pain management with satisfactory stability, enhanced analgesic efficacy, reduced dosage, and reduced toxicity compared to the racemic mixture. In summary, the introduction of S-FPA@LMs presents an innovative approach to managing acute pain, warranting further exploration and development in the field of pain therapeutics.

Reversible and monitorable nephrotoxicity in rats by the novel potent transcriptional enhanced associate domain (TEAD) inhibitor, K-975.

The Hippo pathway plays an important role in the growth, development, and regeneration of cells and organs. Transcriptional enhanced associate domain (TEAD), a transcription activator of the Hippo pathway, forms the complex with a transcriptional coactivator yes-associated protein (YAP) or a transcriptional coactivator PDZ-binding motif (TAZ). Their excessive activations are involved in carcinogenesis such as malignant pleural mesothelioma (MPM), and thus inhibition of the TEAD complex is expected to have potent anticancer activity against MPM. On the other hand, YAP or TAZ conditional knockout mice have been reported to show abnormal findings in various tissues, including the kidney, liver, and lung. In the present study, we evaluated the systemic toxicity of K-975, a novel TEAD inhibitor, in rats. When K-975 was administered orally to rats for 1 week, proteinuria suggestive of nephrotoxicity was observed. Electron microscopy revealed that K-975 at 300 mg/kg induced glomerular podocyte foot process effacement. After a 2-week recovery period, proteinuria with foot process effacement was recovered completely. Urinalysis and urinary biomarker evaluation suggested that the urinary albumin index (urinary albumin/urinary creatinine) was the most sensitive marker for detecting K-975-induced nephrotoxicity. After 3 cycles of 1-week administration followed by 2-week recovery periods, nephrotoxicity was reversible; however, incomplete reversibility was observed in rats with severe proteinuria. In conclusion, this study revealed that in rats, oral K-975 treatment induced severe proteinuria by podocyte foot process effacement, which was reversible and monitorable by the urinary albumin index, suggesting important information for developing K-975 as an anticancer drug.

Exposure of farmed fish to petroleum hydrocarbon pollution and the recovery process: A simulation experiment with tiger puffer Takifugu rubripes

Petroleum hydrocarbon (PH) pollution threatens both wild and farmed marine fish. How this pollution affects the nutrient metabolism in fish and whether this effect can be recovered have not been well-known. The present study aimed to evaluate these effects with a feeding trial on tiger puffer, an important farmed species in Asia. In a 6-week feeding trial conducted in indoor flow-through water, fish were fed a control diet (C) or diets supplemented with diesel oil (0.02 % and 0.2 % of dry matter, named LD and HD, respectively). Following this feeding trial was a 4-week recovery period, during which all fish were fed a same normal commercial feed. At the end of the 6-week feeding trial, dietary PH significantly decreased the fish growth and lipid content. The PH significantly accumulated in fish tissues, in particular the liver, and caused damages in all tissues examined in terms of histology, anti-oxidation status, and serum biochemical changes. Dietary PH also changed the volatile flavor compound profile in the muscle. The hepatic transcriptome assay showed that the HD diet tended to inhibit the DNA replication, cell cycle and lipid synthesis, but to stimulate the transcription of genes related to liver protection/repair and lipid catabolism. The 4-week recovery period to some extent mitigated the damage caused by PH. After the recovery period, the inter-group differences in some parameters disappeared. However, the differences in lipid content, anti-oxidase activity, liver PH concentration, and histological structure still existed. In addition, differences in cellular chemical homeostasis and cytokine-cytokine receptor interaction at the transcriptional level can still be observed, indicated by the hepatic transcriptome assay. In conclusion, 6 weeks of dietary PH exposure significantly impaired the growth performance and health status of farmed tiger puffer, and a short-term recovery period (4 weeks) was not sufficient to completely mitigate this impairment.

Nonclinical safety assessment and immunogenicity of rVSVInd(GML)-mspSGtc vaccine for SARS-CoV-2 in rabbits

The worldwide health, economic, and societal consequences of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic have been devastating. The primary strategy to prevent new infectious diseases is to vaccinate the majority of people worldwide. However, the significant hurdles that are faced include vaccine safety concerns and vaccine reluctance. Among the various types of vaccines, the recombinant vesicular stomatitis virus (rVSV) is a promising candidate owing to its safety and efficacy. Therefore, we investigated the toxicity, immunogenicity, and local tolerance of the rVSVInd(GML)-mspSGtc vaccine against SARS-CoV-2. New Zealand White (NZW) rabbits were administered single or three repeated intramuscular injections of rVSVInd(GML)-mspSGtc every 2 weeks, followed by a 4-week recovery period. Male and female rabbits were randomly assigned into three groups: a control group and two dose-level groups (1 × 109 and 4 × 109 PFU/mL). Treatment-related changes included a temporary increase in body temperature and local inflammation at the injection site. These findings indicated recovery or a trend toward recovery, with no overt systemic toxicity. Immunogenicity analysis results suggested that rVSVInd(GML)-mspSGtc elicited a robust dose-dependent immune response in terms of neutralizing antibodies and IgG antibodies against the SARS-CoV-2 spike protein. In addition, the immune response intensity was increased by repeated vaccine administration. In conclusion, both the approximate lethal dose and the no observed adverse effect level for rVSVInd(GML)-mspSGtc exceeded 4 × 109 PFU/mL in NZW rabbits. Overall, rVSVInd(GML)-mspSGtc induced no adverse effects at the maximum dosage tested; however, its efficacy warrants further clinical evaluation.

Preclinical Safety Evaluation of the Human-Identical Milk Oligosaccharide Lacto-N-Triose II.

Lacto-N-triose II (LNT II), an essential human milk oligosaccharide and precursor to lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), was evaluated for safety. Genotoxicity was assessed through in vitro tests including Bacterial Reverse Mutation Test and mammalian cell micronucleus test, and a subchronic oral gavage toxicity study was conducted on juvenile Sprague-Dawley rats. In this study, LNT II was administered at dose levels of 0, 1,500, 2,500, or 5,000mg/kg body weight (bw)/day for 90 days, followed by a 4-week treatment-free recovery period. LNT II was non-genotoxic in the in vitro assays. No compound-related effects were observed across all dosage levels based on various measures, including clinical observations, body weight gain, feed consumption, clinical pathology, organ weights, and histopathology. Consequently, the highest dosage of 5,000mg/kg bw/day was established as the no-observed-adverse-effect-level (NOAEL). These results suggest the safe use of LNT II in young children formula and as a food ingredient, within the limits found naturally in human breast milk.

Longitudinal Changes in Cerebral Perfusion, Perivascular Space Volume, and Ventricular Volume in a Healthy Cohort Undergoing a Spaceflight Analog.

A global decrease in brain perfusion has recently been reported during exposure to a ground-based spaceflight analog. Considering that CSF and glymphatic flow are hypothesized to be propelled by arterial pulsations, it is unknown whether a change in perfusion would impact these CSF compartments. The aim of the current study was to evaluate the relationship among changes in cerebral perfusion, ventricular volume, and perivascular space volume before, during, and after a spaceflight analog. Eleven healthy participants underwent 30 days of bed rest at 6° head-down tilt with 0.5% atmospheric CO2 as a spaceflight analog. For each participant, 6 MR imaging brain scans, including perfusion and anatomic-weighted T1 sequences, were obtained before, during, and after the analog period. Global perfusion, ventricular volume, and perivascular space volume time courses were constructed and evaluated with repeated measures ANOVAs. Global perfusion followed a divergent time trajectory from ventricular and perivascular space volume, with perfusion decreasing during the analog, whereas ventricular and perivascular space volume increased (P < .001). These patterns subsequently reversed during the 2-week recovery period. The patterns of change in brain physiology observed in healthy participants suggest a relationship between cerebral perfusion and CSF homeostasis. Further study is warranted to determine whether a causal relationship exists and whether similar neurophysiologic responses occur during spaceflight.

Proton Pump Dysfunction and Upregulation of Caspase-3 Activity via Oxidative-Sensitive Signaling Mediate Rohypnol-Induced Testicular Toxicity

Rohypnol is a benzodiazepine that is used for its anxiolytic, anticonvulsant, muscle relaxant, and hypnotic properties, but commonly abused. It has been reported to induce sexual dysfunction; however, its effect on testicular integrity and sperm quality is yet to be documented. This study evaluated the impact of Rohypnol on testicular integrity, circulatory testosterone, spermatogenesis, and sperm quality. The likely role of oxidative stress-dependent pathway as well as testicular proton pumps and caspase-3 activity in Rohypnol-induced modulation of male reproductive function was explored. In this study, adult male rats were randomized into six groups of six rats each: the control received 1 ml of distilled water as vehicle, while the low- and high-dose Rohypnol-treated groups received 2 mg/kg and 4 mg/kg of Rohypnol for four weeks. The control-, low-, and high-dose recovery groups received 2 mg/kg and 4 mg/kg of Rohypnol for four weeks, respectively, which was followed by a 4-week drug-free recovery period. Rohypnol treatments led to impaired steroidogenesis and spermatogenesis and low sperm quality. This was accompanied by a rise in the markers of testicular injury, oxidative stress, inflammation, and apoptosis. Also, the activities of testicular enzymatic antioxidants and proton pumps were significantly reduced. Although the impacts of low-dose Rohypnol treatment were partially reversible, those of the high-dose Rohypnol treatment were not. Histopathological findings on the testicular tissues were in tandem with the biochemical alterations. Thus, it is safe to infer that Rohypnol induces testicular damage by the suppression of Na+/K+-ATPase and Ca2+-ATPase and the activation of caspase-3 through an oxidative-sensitive signaling pathway.

Abstract 6342: Preclinical safety &amp; pharmacokinetics of AFVT-2101, a tetravalent FRα x CD16A bispecific innate cell engager for the treatment of solid tumors

Abstract AFVT-2101 is a novel bispecific innate immune cell engager in development to treat solid tumors. It is comprised of bivalent folate receptor alpha (FRα) and CD16A (FcγRIIIA) binding domains with a silenced IgG1 Fc region. AFVT-2101 is engineered to exert potent and selective immune-mediated antitumor activity with minimal off-tumor toxicity by promoting NK cell and macrophage engagement of FRα-positive tumors. A comprehensive package of IND-enabling studies is currently in progress to support the preclinical safety profile of AFVT-2101. In agreement with target sequence homology, binding affinity of AFVT-2101 against human and cynomolgus monkey FRα and CD16A is highly similar, confirming non-human primate (NHP, specifically cynomolgus monkey) as the only pharmacologically relevant preclinical safety model. The toxicology program includes tissue cross-reactivity assessment against a panel of human and cynomolgus cryosections, single- and repeat-dose range finding (DRF) non-GLP studies and a 4-week IND-enabling GLP study with 4-week recovery period in NHPs. In a single-dose study AFVT-2101 demonstrated non-linear pharmacokinetics when administered as a 2-h IV infusion to male NHPs where the mean Cmax (85.1 and 1620 µg/mL) and AUC168hr (3,430 and 97,000 µg.h/mL) at 3 and 30 mg/kg, respectively, increased supra-proportionally with respect to dose, consistent with target-mediated drug disposition. Half-life was 103-155 h across doses. A DRF study was conducted to characterize toxicity, identify target organs, determine toxicokinetics (TK), immunogenicity (anti-drug antibodies, ADA), and to inform the dose selection of the subsequent 4-week study. AFVT-2101 was administered once-weekly to female NHPs via 2-h IV infusion for a total of 4 doses. Mean Cmax (1120, 3320, 7190 µg/mL) and AUC168hr (83,600, 209,000, 592,000 µg.h/mL) at 30, 75, and 150 mg/kg, respectively, increased in a generally dose-proportional manner with a trend for modest accumulation (&amp;lt;2-fold) upon repeated administration with a half-life of 80-230 h. Exposure did not appear to be impacted by any treatment-emergent ADA. AFVT-2101 was well-tolerated with no test article-related adverse effects on clinical observations, bodyweight, clinical chemistry, hematology, coagulation, anatomic pathology, or immunotoxicity parameters. The no observed adverse effect level (NOAEL) was determined to be 150 mg/kg under the conditions of the study. The safety profile is being further evaluated in an ongoing pivotal 4-week GLP study incorporating standard toxicology endpoints, safety pharmacology (CNS, CV, and respiratory), gross and anatomic pathology, as well as cytokine, immunophenotyping, receptor occupancy, TK, and ADA analyses. In conclusion, AFVT-2101 is well-tolerated in NHPs with a pharmacokinetic and safety profile consistent with its intended clinical application. Citation Format: Wendy Luo, Eric J. Gaukel, Janice A. Lansita, Stephen M. Laczko, Peter Sandy, Daniel J. O'Shannessy, Sudhir Penugonda, Bhavik Manocha, Daniel E. Patterson, Izabela Kozłowska, Séverine Sarlang, Stefan Knackmuss, Daniela Penston, Uwe Reusch, Zoë Johnson. Preclinical safety &amp; pharmacokinetics of AFVT-2101, a tetravalent FRα x CD16A bispecific innate cell engager for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6342.

Preclinical safety assessment of the suction-assisted intradermal injection of the SARS-CoV-2 DNA vaccine candidate pGO-1002 in white rabbit.

pGO-1002, a non-viral DNA vaccine that expresses both spike and ORF3a antigens of SARS-CoV-2, is undergoing phase 1 and phase 2a clinical trials in Korea and the US. A preclinical repeated-dose toxicity study in New Zealand white rabbits in compliance with Good Laboratory Practice (GLP) was conducted to assess the potential toxicity, local tolerance, and immunogenicity of the vaccine and GeneDerm suction device. The dose rate was 1.2mg/head pGO-1002, and this was administered intradermally to a group of animals (eight animals/sex/group) three times at 2-week intervals, followed by a 4-week recovery period. After each administration, suction was applied to the injection site using the GeneDerm device. Mortality, clinical signs, body weight, food consumption, skin irritation, ophthalmology, body temperature, urinalysis, and clinical pathology were also monitored. Gross observations and histopathological evaluation were performed. Overall, pGO-1002 administration-related changes were confined to minor damage or changes at the injection site, increased spleen weight and minimal increased cellularity in white pulp. All changes of injection site were considered local inflammatory changes or pharmacological actions due to the vaccine with the changes in spleen considered consistent with vaccine-induced immune activation. All findings showed reversibility during the 4-week recovery period. Animals vaccinated with pGO-1002, administered by intradermal injection and followed by application of suction with GeneDerm, developed humoral and cellular responses against the SARS-CoV-2 antigens consistent with prior studies in rats. Collectively, it was concluded that the pGO-1002 vaccine was safe and effective under these experimental conditions and these data supported future human study of the vaccine, now known as GLS-5310, for clinical trial use.

Increased Glucagon Immunoreactivity in a Rat Model of Diet-Induced Obesity following Sleeve Gastrectomy

Objective: Bariatric surgery is currently the most effective treatment for obesity, and procedures such as Roux-en-Y gastric bypass and sleeve gastrectomy (SG) also result in rapid improvements in insulin sensitivity and glucose tolerance. In addition, these procedures cause changes in the secretion of various gut-derived hormones. The role these hormones play in the mechanism of the beneficial effects of bariatric surgery is still debated, but nonetheless, their importance provides inspiration for novel obesity-targeted pharmacotherapies. Methods: Male Sprague-Dawley rats were fed either regular chow or a cafeteria diet to induce obesity. A sub-group of the obese animals then underwent either sham surgery or SG. Results: Following a 4-week recovery period, SG rats weighed significantly less than obese or sham-operated rats. Improvements in glucose tolerance and insulin sensitivity also occurred in the SG group, but these were not always statistically significant. We measured the intracellular lipid content of liver samples and found that obese rats showed signs of non-alcoholic fatty liver disease, which were significantly ameliorated by SG. There were significantly higher glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide responses to a standard mixed meal in the SG group, as well as paradoxically higher glucagon secretion. Conclusion: These data highlight the need for more specific anti-glucagon antibodies to characterize the changes in proglucagon-derived peptide concentrations that occur following SG. Further studies are required to determine whether these peptides contribute to the therapeutic effects of SG.

The risk factors and threshold level of subchronic inhalation exposure of reclaimed water

Inhalation of reclaimed water is known to cause lung inflammation, and free endotoxins have been shown to be a major risk factor for acute exposure. Subchronic exposure has also been shown to induce inflammatory responses with visible tissue damage. However, subchronic risk factors have yet to be identified, and a threshold for the protection of occupational populations during urban reuse is necessary. In this study, potential risk factors in reclaimed water were examined by subchronic exposure with fractionated reclaimed water, and the health risk threshold was tested with a series of diluted reclaimed water. Accordingly, following a 12-week exposure, macromolecules and microorganisms were found to be two major risk factors in reclaimed water that could cause pulmonary inflammation, including increased proportion of polymorphonuclear leukocytes in bronchoalveolar fluid, formation of inducible bronchus-associated lymphoid tissue, and elevation of Immunoglobulin A levels. Moreover, inflammation persisted after a 4-week recovery period. The calculated threshold of reclaimed water exposure for mice was 31.8 Endotoxin Unit (EU)/(kg·day) under when exposed to 50% additional relative humidity from reclaimed water at 25°C for 2 hr/day. Meanwhile, the subchronic threshold estimate for humans under the same exposure conditions was found to be 12.2 EU/(kg·day), corresponding to endotoxin levels of 61.7 EU/mL in reclaimed water. The threshold level of endotoxin was lower than that in most non-potable reclaimed water. The findings of this study suggest that occupational exposure of reclaimed water can serve as a potential risk to workers.

Comparative pulmonary and genotoxic responses to inhaled nickel subsulfide and nickel sulfate in F344 rats.

Inhalation studies with nickel (Ni) subsulfide (Ni3 S2 ) and Ni sulfate hexahydrate (NiSO4 ·6H2 O) investigated differences in mode of action that could explain why the former induced lung tumors in rats and the latter did not. Male rats were exposed to ≤0.22 mg Ni/m3 NiSO4 ·6H2 O or 0.44 mg Ni/m3 Ni3 S2 , 6 h/day, 5 days/week for 3 and 13 weeks; subsets of the rats exposed for 13 weeks were held for an additional 13 weeks. Analyses of bronchoalveolar lavage fluid, isolated cells, and whole lung tissue were conducted to compare the extent and persistence of any induced lung effects. Histological findings were qualitatively identical for both compounds and consistent with lesions reported in earlier studies. After 13 weeks of exposure, the incidence and severity of pulmonary inflammation and epithelial cell hyperplasia were greater among Ni3 S2 -exposed rats, whereas the reverse response was seen for apoptosis. Only Ni3 S2 exposure significantly increased epithelial and non-epithelial cell proliferation after 13 weeks of exposure. Both compounds induced DNA damage in isolated lung cells and DNA hypermethylation of whole lung tissue after 13 weeks of exposure at the highest exposure concentrations. Increases in cell proliferation, DNA damage, and tissue DNA hypermethylation did not persist during the 13-week recovery period. In summary, the highest concentrations of each compound produced marked pulmonary toxicity, but the lowest concentrations produced minimal or no effects. Differences in the proliferative and apoptotic responses between the two compounds may help explain differences in carcinogenicity, whereas the identification of no observed adverse effect concentrations (NOAECs) contributes to the risk characterization for inhalation exposure to nickel compounds.

Models of microglia depletion and replenishment elicit protective effects to alleviate vascular and neuronal damage in the diabetic murine retina

Microglia, the resident phagocytes of the retina, are believed to influence the development of retinopathy, but their exact contributions to vascular integrity and neuronal loss are unknown. Therefore, utilizing two models of microglia depletion, we aimed to deplete and repopulate microglia to clarify the contribution of microglia to neuronal loss and vascular damage in the diabetic retina in an STZ-induced model of hyperglycemia. Here, we report that 2 weeks exposure to diphtheria toxin (DTx) in diabetic CX3CR1CreER:R26iDTR transgenic mice induced a 62% increase in Iba1+ microglia associated with an increase in TUJ1+ axonal density and prevention of NeuN+RBPMS+ neuronal loss. Conversely, diabetic PBS controls exhibited robust TUJ1+ axonal and NeuN+RBPMS+ neuronal loss compared to non-diabetic controls. A 2-week recovery period from DTx was associated with a 40% reduction in angiogenesis and an 85% reduction in fibrinogen deposition into the diabetic retina in comparison to diabetic PBS-treated controls. Analysis of microglia morphology and marker expression revealed that following a 2-week recovery period microglia displayed a P2RY12+Ly6C– phenotype and high transformation index (TI) values complimented by a ramified-surveillant morphology closely resembling non-diabetic controls. In contrast, diabetic PBS-treated control mice displayed P2RY12+Ly6C+ microglia, with a 50% reduction in TI values with an amoeboid morphology. To validate these observations were due to microglia depletion, we used PLX-5622 to assess vascular and neuronal damage in the retinas of diabetic mice. Confocal microscopy revealed that PLX-5622 also induced an increase in TUJ1+ axonal density and prevented fibrinogen extravasation into the diabetic retina. mRNAseq gene expression analysis in retinal isolates revealed that PLX-5622-induced microglia depletion and repopulation induced a downregulation in genes associated with microglial activation and phagocytosis, B2m, Cx3cr1, and Trem2, and complement-associated synaptic pruning, C1qa, C1qb, and C1qc. Although the levels of microglia depletion induced with DTx in the CX3CR1CreER:R26iDTR model and those induced with the CSF-1R antagonists are distinct, our results suggest that microglia depletion and replenishment is neuroprotective by inducing the proliferation of a homeostatic microglia pool that supports neuronal and vascular integrity.