Repeated-dose Study Research Articles

Serum Pharmacokinetics and Bone Penetration of Novel Broad-Spectrum Anti-MRSA Agent Levonadifloxacin in Wistar Rats.

Objectives: Levonadifloxacin (IV) and alalevonadifloxacin (oral) are novel broad-spectrum anti-methicillin-resistant Staphylococcus aureus (S. aureus) agents based on novel benzoquinolizine core. Both are recently approved in India for the treatment of acute bacterial skin and skin structure infections, including diabetic foot infections and concurrent bacteremia. The present investigation reports the findings from preclinical pharmacokinetic (PK) studies that support the development of levonadifloxacin as a treatment option for bone and joint infections (BJIs). Methods: PK profiles of levonadifloxacin were obtained in serum, and/or various anatomical segments of femoral bone such as whole bone (WB), hard bone (HB), and bone marrow (BM) following subcutaneous administration of levonadifloxacin single doses at 50, 100, 200, and 400 mg/kg, as well as multiple doses at 200 mg/kg (BID (two times a day), 6 hours apart) for 5 days in Wistar rats. Results: The distribution of levonadifloxacin in bone was rapid, and the extent of distribution (B/S ratio; bone-to-serum area under the concentration-time curve ratio) was nearly comparable across bone segments. In single-dosage PK studies, the mean B/S ratio in WB, HB, and BM was 0.40, 0.33, and 0.34, respectively; however, in 5 days' repeated dose studies, it increased to 1.01, 1.14, and 0.61, respectively. Conclusions: On the basis of bone PK data in Wistar rat and ever-growing clinical experience in terms of safety and efficacy, levonadifloxacin has the potential to offer a well-differentiated therapy for the treatment of BJIs.

Evaluations of the in vitro and in vivo antidiabetic activity of 70 % ethanolic fruit extracts of Rosa abyssinica

BackgroundDiabetes mellitus is becoming major health challenge with continually increasing burden. High costs of conventional medicines and numerous side effects associated with them, on the other hand, easy availability and accessibility of traditional herbal medicines calls upon experimental investigations to validate their effect on lowering blood glucose level. MethodsThe dried fruit of Rosa abyssinica was macerated with 70 % ethanol and the extract's in vitro antidiabetic activity was investigated using dinitrosalisylic acid method for alpha amylase inhibitory activity. Furthermore, the in vivo hypoglycemic and Antihyperglycemic effects of various doses of the extract (100, 200 and 400 mg/kg) was determined on normoglycemic, glucose loaded (1500 mg/kg) and Streptozotocine (180 mg/kg)-induced diabetic mice models. ResultsThe acute oral toxicity study revealed the plant showed no toxic effect on swiss albino mice at 2000 mg/kg. The in vitro alpha amylase inhibitory activity study showed that the extract has comparable IC50 value of 21.37 ± 4.252 μg/ml with the standard drug acarbose (IC50 value of 26.72 ± 3.59 μg/ml). On the other hand, in normal mice, none of the dose levels except at 400 mg/kg significantly reduces blood glucose level. This is in contrast to the oral glucose tolerance test, which the extract produced significant reduction at 60, 90 and 120 min following glucose challenge. The 70 % ethanolic fruit extracts of Rosa abyssinica also experienced profound antidiabetic activity in streptozotocin-induced diabetic model. In the single-dose study, both RAFE200 and RAFE400 demonstrated a significant (P˂0.05) reduction in blood glucose levels at 1, 2, 3, and 4 h. Similarly, in the repeated-dose study, RAFE200 and RAFE400 not only significantly reduced blood glucose levels but also produced a notable improvement in animal body weight. ConclusionThe 70 % ethanolic fruit extracts of Rosa abyssinica have shown significant in vitro alpha amylase inhibition effect and an in vivo blood glucose level lowering effects in diabetic mice.Therefore, this study supports the traditional use of Rosa abyssinica in the management of diabetes mellitus.

Honey bee larval toxicity study designs: Applicability of the current study protocols and endpoints asa predictor of pesticide hazard for pollinators.

The assessment of pesticide risks to bees in North America currently relies in part on Tier 1 honey bee laboratory toxicity studies to support the registration and registration review processes for crop protection chemicals. For immature stages, the studies follow two standardized test designs recommended by the Organization for Economic Cooperation (OECD), evaluating acute (seven-day single-dose, TG OECD 237) and chronic (22-day repeated-dose, GD OECD 239) toxicity in bee larvae. In this article, we aim to evaluate the current approach for generating and interpreting honey bee larval toxicity data, enhancing pesticide risk assessment for pollinators. First, by considering that the repeated-dose larval study covers all stages of honey bee brood development up to adult emergence, we compared endpoints (larval LD/ED50 and LC/EC50 values) from seven-day acute exposure studies with the 22-day chronic exposure studies. Our goal was to identify the study design offering greater sensitivity in assessing pesticide toxicity to immature bees. Our second objective involved analyzing available weight data from emerged adults and comparing it to survival endpoints (e.g., NOEL and LD50) to determine if the weight after adult emergence would accurately represent a sensitive indicator of pesticide effects on developing honey bees. Our analysis determined that the use of a single 22-day chronic exposure study adequately covers all immature stages and that the toxicity values based on cumulative dose are more accurate and representative measures of exposure for immature bees than using endpoints based on estimated daily doses. Furthermore, our analysis suggests that measuring the weight of emerged adults was a more sensitive indicator than mortality of treatment-related effects in 22% of the compounds included in our analysis. Here we also discuss the importance of standardized protocols for proper collection of weight after emergence and the need for further discussion on the relevance of this parameter at risk assessment scheme. Integr Environ Assess Manag 2024;20:2283-2293. © 2024 Pollinator Research Task Force. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Tissue distribution of renadirsen sodium, a dystrophin exon-skipping antisense oligonucleotide, in heart and diaphragm after subcutaneous administration to cynomolgus monkeys

The pharmacokinetics and tissue distribution of renadirsen sodium, a dystrophin exon-skipping phosphorothioate-modified antisense oligonucleotide with 2’-O,4’-C-ethylene-bridged nucleic acid (ENA), after subcutaneous or intravenous administration to cynomolgus monkeys were investigated. The plasma concentration of renadirsen after subcutaneous administration at 1, 3, and 10 mg/kg increased with the dose. The absolute bioavailability at 3 mg/kg after subcutaneous administration was calculated as 88.6%, and the time to reach maximum plasma concentration of renadirsen was within 4 h, indicating the efficient and rapid absorption following subcutaneous administration. The exposure of muscle tissues to renadirsen was found to increase with repeated dosing at 6 mg/kg, and higher exposure was observed in the diaphragm and heart than in the quadriceps femoris and anterior tibialis muscles. Renadirsen achieved more exon 45-skipped dystrophin mRNA in the diaphragm and heart than in the quadriceps femoris and anterior tibialis muscles. Renadirsen also showed a cumulative skipping effect in a repeated-dose study. The findings on exon 45-skipped dystrophin mRNA in these muscle tissues were consistent with the concentration of renadirsen in these tissues. Because it is not feasible to directly evaluate drug concentration and exon skipping in the heart and diaphragm in humans, the pharmacokinetics and pharmacodynamics of renadirsen in these tissues in monkeys are crucial for the design and interpretation of clinical settings.

Nonclinical safety and immunogenicity assessment of a combined DTacP vaccine in animal models.

The (diphtheria, tetanus, and pertussis [acellular, component] [DTacP]) vaccine is a combined vaccine designed to prevent three potentially fatal diseases including pertussis, tetanus, and diphtheria in both children and adults. We utilized advanced technology to develop a novel DTacP vaccine that was previously unavailable in China. The nonclinical studies were performed to evaluate the immunogenicity, potential toxicity, and local tolerance of the vaccine in animal models. In the immunogenicity study, three batches of the vaccine were intraperitoneally administered to National Institutes of Health (NIH)mice, resulting in 100% seropositivity for all three batches. Additionally, antibody levels notably increased as the immunization dosage increased. In acute toxicity study, no mortality was observed among the animals during the 14-day observation period, and no abnormalities in clinical signs were reported. Active systemic anaphylaxis assessment in guinea pigs showed no evidence of serious allergic reactions in the vaccine groups. In the repeat-dose toxicity study, where five intramuscular doses were administered every 2weeks, gross autopsy and histopathological examination revealed no vaccine-related systemic pathological changes in rats, with dose site irritant reactions mostly recovered at the end of recovery period. In conclusion, the vaccine demonstrated good local and systemic tolerance, supporting its clinical development.

Safety and tolerability of pooled human immune globulins after topical ophthalmic administration in New Zealand White rabbits

Purpose To evaluate the safety and tolerability of pooled human immune globulins, Flebogamma® 5% DIF and Flebogamma® 10% DIF, administered by topical ophthalmic instillation to New Zealand White (NZW) rabbits. Methods Male NZW rabbits were used in this study. In the acute single dose tolerability study, rabbits (n = 12) received a single topical dose of Flebogamma® 5% DIF. In the two-week repeated-dose tolerability study, rabbits (n = 5 for each group) were administered either Flebogamma® 5% DIF or Flebogamma® 10% DIF by topical bilateral administration four times daily (q.i.d.) between 8 am and 6 pm for a period of two weeks. Full ophthalmic examinations were conducted to evaluate ocular tolerability at baseline, Day 7, and Day 14. Results In the acute single dose study, mild hyperaemia was observed in 1 out of 4 eyes at each 4 h and 24 h post-instillation of Flebogamma® 5% DIF. In the repeated dose study, no ocular signs were detected after q.i.d. topical instillation of Flebogamma® 5% DIF, while Flebogamma® 10% DIF resulted in mild hyperaemia in 8 out of 10 eyes on Day 7, and 5 out of 10 eyes on Day 14. No positive corneal fluorescein staining was detected. Schirmer tear test results were unremarkable. No other ocular signs were observed. Administration of immune globulins had no effect on intraocular pressure. Conclusions Flebogamma® 5% DIF and Flebogamma® 10% DIF were well-tolerated by NZW rabbits following single and repeat dose topical ophthalmic administration, supporting the future development of topical pooled human immune globulins for the treatment of ocular surface disease.

Preliminary investigation of the safe dose of oleandrin when administered orally to Beagles.

The purpose of the study reported herein was to determine the dose of oleander extract and oleandrin (the key pharmacologically active constituent) that could be safely administered PO to dogs. 42 purebred Beagle dogs were used to study an extract of Nerium oleander. 3 studies were performed in 42 purebred young adult (ages 12 months or older) Beagle dogs using a supercritical fluid extract of N oleander leaves. The first study was an 8-day initial dose-ranging study in 2 dogs, a second 7-day repeat-dosing study was performed in 4 dogs, and the final study was performed in 32 dogs where test subjects were given extract or placebo once daily for 28 consecutive days via oral (gavage) administration followed by a 14-day recovery period. At 2.3 µg/kg of oleandrin, there were no observable adverse effects during the duration of the study. Adverse effects were not seen until doses exceeded 6.9 µg/kg of oleandrin, at which time mild, reversible clinical signs were noted. However, a dose > 460 µg of oleandrin/kg was fatal in 1 of 2 dogs in this study. The studies reported here, taken in totality, suggest that doses exceeding 6.9 µg/kg of oleandrin may be associated with cardiac abnormalities. An estimated no treatment effective adverse event oral dose of oleandrin appears to be 4.6 µg of oleandrin/kg. Higher doses may be tolerable but should be used with appropriate monitoring.

Oclacitinib (APOQUEL®) is a selective Janus kinase 1 inhibitor with efficacy in a canine model of flea allergic dermatitis.

Oclacitinib is a novel Janus kinase (JAK) inhibitor that potently inhibits JAK1-dependent cytokines involved in allergy, inflammation, and pruritus (IL-2, IL-4, IL-6, IL-13, and IL-31). Oclacitinib (Apoquel®, Zoetis Inc, Parsippany, NJ) is approved for the treatment/control of pruritus associated with allergic dermatitis and treatment/control of clinical manifestations of atopic dermatitis in dogs at least 12 months of age. To evaluate the effectiveness of oclacitinib in dogs with flea allergy dermatitis, the JAK1 selective inhibitor was tested in a placebo-controlled, masked, single-dose (0.4 mg/kg) or repeat-dose (0.4 mg/kg, twice daily for 2 weeks) study. Pruritic behaviors were quantitated by video recording, and erythema and skin lesions were assessed using a 10-cm visual analog scale (VAS). Results showed that oclacitinib reduced pruritus by 61% as early as 1.5 h after a single oral dose compared to placebo, with an average reduction (compared to placebo) of 85% 1-5 h after dosing (0.4 mg/kg; p < .0001). Oclacitinib also significantly reduced erythema (p < .0001) and skin lesion (p < .0005) VAS scores on Day 14 compared to placebo in a repeat dose study. No adverse events were noted during the conduct of these studies. IL-31 concentrations were elevated in the majority of dogs after flea infestation, suggesting JAK1-dependent cytokines may drive clinical signs of flea allergy dermatitis. These findings show that oclacitinib, an inhibitor of JAK1-dependent cytokines involved in allergy and inflammation can rapidly reduce clinical signs associated with flea allergic dermatitis in dogs.

Examination of in vitro and in vivo antitumor activity of PARP1 selective inhibitor D0112-005.

e15115 Background: The approved PARP inhibitors have demonstrated efficacy in ovarian, breast, prostate, and pancreatic cancers, particularly in homologous recombination repair (HRR)-deficient tumors, including BRCA mutant tumors. The severe hematological toxicity of first-generation PARPi in combination with chemotherapy is due to the lack of selectivity for PARP2. So, we developed selective PARP1 inhibitor D0112-005 with the aim to improve safety and efficacy. Methods: The selectivity of D0112-005 on PARP1, PARP2 was measured by chemiluminescent assay kit and PARP trap™ assay kit. The anti-proliferative ability of D0112-005 was evaluated by CCK-8 in BRCA mutant cell lines. In vivo anti-tumor effect of D0112-005 was evaluated in MDA-MB-436 BRCA1 mutant xenograft model. The potential for hematologic toxicity was evaluated in rats for 28 days repeat-dose and dogs for 14 days repeat-dose. Results: D0112-005 is a highly potent and selective inhibitor of PARP1 with 2000-fold selectivity for PARP1 over PARP2 and PARP3. D0112-005 trapped PARP1-DNA with an EC50 value of 6.49 nM. In contrast, the EC50 of PARP2-DNA trapping was 430μM. These data demonstrate that D0112-005 is a potent and selective PARP1 trapper. D0112-005 showed high anti-proliferation activity against BRCA mutant MDA-MB-436 and HCC1395 cell lines, with IC50 values of 2.06 nM and 2.446 nM, respectively. In the MDA-MB-436 BRCA1m xenograft model, a dose-dependent anti-tumor effect was observed after 21 days administration, with TGIs of 38.77%, 106.7% and 111.73% at the dose of 0.1, 0.3 or 1 mg/kg once daily by oral, respectively. In the 1 mg/kg group, the anti-tumor efficacy was maintained after 14 days of drug withdrawal. D0112-005 showed well tolerance without bodyweight loss. In rat 28 days repeat dose study, D0112-005 100mg/kg had less changes on hematologic parameters compared with Olaparib. The main toxic reaction of D0112-005 (10mg/kg) was reversible thrombocytopenia in dog 14 days repeated toxicity study. Conclusions: D0112-005 potently and selectively inhibits PARP1 resulting in excellent anti-tumor activity in vivo and in vitro, which also good safety profile in the repeated toxicity study in rats and dogs.

A phase Ia, dose-escalation study of IMB071703 injection in patients (pts) with recurrent or metastatic, advanced solid tumors.

e14502 Background: IMB071703(XFab-α4-1BB/CD40L), a 4-1BB Fab and triCD40L fusion protein that activates dendritic cell and primes tumor-reactive T cells, mediates immune response, and further inhibits tumor development. We confirmed the expansion of antigen-specific T cells in various mouse models and potent tumor regression administrated by IMB071703 alone or in combination with gemcitabine in vivo, concomitant with improved tumor-reactive T-cell infiltration. IMB071703 showed no signs of liver toxicity at the dose levels up to 51 mg/kg in a repeated-dose study in non-human primates. Here, we describe initial data from a phase Ia study (CTR20230326). Methods: Eligible patients with recurrent or metastatic, advanced solid tumors were enrolled in this study. IMB071703 was administrated at escalating doses of 0.05, 0.15, 0.5,1.0 and 1.5 mg/kg intratumorally Q1W in Cycle 1 (C1:21days), and Q2W from Cycle 2 and subsequent cycles (Cn+1:28 days). Enrolled pts would first undergo DLT observative assessment (21 days). After pts were assessed safely during DLT observation, they would continue the scheduled dosage of IMB071703 until disease progression, intolerable toxicity or other reasons specified in the clinical protocol. The study objectives included the safety, tolerability, PK, pharmacodynamics, immunogenicity, and preliminary efficacy. Results: 7 pts with recurrent or metastatic, advanced solid tumors (median age 60 years, range 42-71; 4 male, 3 female; prior lines of therapy with range 1-6) were treated. Three dose levels, 0.05 mg/kg(n=1), 0.15 mg/kg(n=3), and 0.5mg/kg(n=3), had been evaluated. 1.0mg/kg dose escalation is ongoing. On cutoff date of 18 Dec. 2023, 1 pt is on the study and 6 pts had discontinued treatment. The reasons for discontinuation included confirmed disease progression (n=5) and other reason(n=1). 7 pts (100%) experienced treatment-emergent adverse events (TEAEs). 5 pts (71%) experienced treatment-related adverse events (TRAEs≥10%): Injection reaction (pain, fever, chill) (28%), fatigue (11%). All TRAEs were ≤grade 2. No DLT was observed. One death due to disease progression, as only a SAE, was identified to be unrelated. No objective response was observed in 6 evaluable pts. 1 pt was SD and 5 pts were PD. Pharmacodynamic data showed CD40 was occupied ≥50% in the 0.5 mg/kg level. The percentage of CTL (CD3+CD8+) in peripheral blood was increased (0.15mg/kg and 0.5mg/kg) Pharmacokinetic analysis also showed the mean half-life of IMB071703 was 2.45 hours in three dose levels. Biomarker analysis indicated 4-1BB up-regulation and PD-1 down-regulation was associated with the favorable response. Conclusions: IMB071703 Showed a good safety profile at the dose levels of 0.05 mg/kg, 0.15 mg/kg, and 0.5mg/kg. Both favorable tolerability and preliminary antitumor activity warrant further evaluation in patients with advanced malignancies. Clinical trial information: CTR20230326 .

FOUR-WEEK ORAL ADMINISTRATION OF BALOXAVIR MARBOXIL AS AN ANTI-INFLUENZA VIRUS DRUG SHOWS NO TOXICITY IN CHICKENS.

High pathogenicity avian influenza is an acute zoonotic disease with high mortality in birds caused by a high pathogenicity avian influenza virus (HPAIV). Recently, HPAIV has rapidly spread worldwide and has killed many wild birds, including endangered species. Baloxavir marboxil (BXM), an anti-influenza agent used for humans, was reported to reduce mortality and virus secretion from HPAIV-infected chickens (Gallus domesticus, order Galliformes) at a dosage of ≥2.5 mg/kg when administered simultaneously with viral challenge. Application of this treatment to endangered birds requires further information on potential avian-specific toxicity caused by repeated exposure to BXM over the long term. To obtain information of potential avian-specific toxicity, a 4-wk oral repeated-dose study of BXM was conducted in chickens (n = 6 or 7 per group), which are commonly used as laboratory avian species. The study was conducted in reference to the human pharmaceutical guidelines for nonclinical repeated-dose drug toxicity studies to evaluate systemic toxicity and exposure. No adverse changes were observed in any organs examined, and dose proportional increases in systemic exposure to active pharmaceutical ingredients were noted from 12.5 to 62.5 mg/kg per day. BXM showed no toxicity to chickens at doses of up to 62.5 mg/kg per day, at which systemic exposure was approximately 71 times higher than systemic exposure at 2.5 mg/kg, the reported efficacious dosage amount, in HPAIV-infected chickens. These results also suggest that BXM could be considered safe for treating HPAIV-infected endangered birds due to its high safety margin compared with the efficacy dose. The data in this study could contribute to the preservation of endangered birds by using BXM as a means of protecting biodiversity.

Implications of chronic moderate protein-deficiency malnutrition on doxorubicin pharmacokinetics and cardiotoxicity in early post-weaning stage

BackgroundMalnutrition is a common problem in developing countries, and the impact of severe malnutrition on optimal treatment outcomes of chemotherapy in pediatric cancer patients is well documented. However, despite being a more prevalent and distinct entity, moderate malnutrition is until now unexplored for its effects on treatment outcomes. AimsIn this study we aimed to investigate the molecular basis of altered pharmacokinetics and cardiotoxicity of doxorubicin observed in early-life chronic moderate protein deficiency malnutrition. Materials and methodsWe developed an animal model of early-life moderate protein-deficiency malnutrition and validated it using clinical samples. This model was used to study pharmacokinetic and toxicity changes and was further utilized to study the molecular changes in liver and heart to get mechanistic insights. Key findingsHere we show that moderate protein-deficiency malnutrition in weanling rats causes changes in drug disposition in the liver by modification of hepatic ABCC3 and MRP2 transporters through the TNFα signalling axis. Furthermore, malnourished rats in repeat-dose doxorubicin toxicity study showed higher toxicity and mortality. A higher accumulation of doxorubicin in the heart was observed which was associated with alterations in cardiac metabolic pathways and increased cardiotoxicity. SignificanceOur findings indicate that moderate malnutrition causes increased susceptibility towards toxic side effects of chemotherapy. These results may necessitate further investigations and new guidelines on the dosing of chemotherapy in moderately malnourished pediatric cancer patients.

A novel B7-H3 x CD3 bispecific T-cell engager for the treatment of B7-H3+ tumors.

e14509 Background: B7-H3 (CD276) is a transmembrane protein highly expressed in solid tumors but with limited expression in normal tissues. Its presence is strongly associated with the inhibition of T-cell activity, cancer progression, and invasion. Furthermore, B7-H3 is strongly associated with poor prognosis and reduced patient survival. Given its crucial role in tumor biology, B7-H3 has emerged as an attractive therapeutic target for drug development. We utilized ExMab heterodimer platform and common light chain technology to develop EX105, a next-generation B7-H3×CD3 bispecific antibody. This innovative T-cell engager exhibits excellent anti-tumor activity in mouse models and good safety profile in NHP tox study. Methods: EX105 was generated using ExMab heterodimer platform and common light chain technology to form a heterodimeric IgG1 structure. Preclinical pharmacology studies, efficacy, and safety were evaluated. Anti-tumor activity study of EX105 includes in vitro T-cell cytotoxicity assays and in vivo PBMC reconstituted xenograft mice models. Preclinical safety evaluation of EX105 was performed through GLP single-dose and repeat-dose tox studies in non-human primates (NHPs). Results: Based on ELISA and FACS analyses, EX105 can bind to both human and cynomolgus monkey B7-H3 and CD3 antigens. Furthermore, SPR data suggest that EX105 exhibits a high affinity to B7-H3 and a much lower affinity to CD3. In consistent with that, EX105 only induced relatively low cytokine release. Nevertheless, EX105 showed a potent in vitro T cell cytotoxicity against B7-H3+ tumor cell lines. The in vivo anti-tumor activity of EX105 was further determined in several tumor xenograft models. Treatment with EX105 resulted in remarkable tumor regression in both “hot” tumor model and “cold” tumor model, indicating its effectiveness across a range of tumor types. Tox study in NHPs showed minimal adverse effects and no liver impairment after EX105 administration, even at high doses. The cytokine release data observed in this study suggests a good safety profile of EX105, with much lower cytokine release compared to other T cell engagers. Conclusions: These findings collectively indicate that EX105 has the potential to become a promising therapeutic agent for the treatment of various B7-H3 positive cancers. The first-in-human clinical trial for patients with advanced B7-H3 positive cancers is due to start.

Biodistribution and preclinical safety profile of legubicin: A novel conjugate of doxorubicin and a legumain-cleavable peptide linker.

Legubicin is a novel conjugate of doxorubicin and a legumain-cleavable peptide linker. It has been developed to ameliorate the side effects of doxorubicin. Biodistribution in tumor-bearing mice, acute tolerance, and potential systemic toxic effects in Sprague-Dawley rats and beagle dogs of legubicin were assessed. Legubicin exists mainly as a protein complex in plasma after entering the circulation. Compared with conventional doxorubicin at an equal molar dose in mice, we found higher exposure to doxorubicin in tumor (approximately 1.7-fold increase) while lower exposure in normal tissues (an ~3.26-, 3.46-, and 1.29-fold reduction in heart, kidney, and plasma, respectively) in tumor-bearing mice after intravenous injection of legubicin. The acute maximum tolerance dose (MTD) of legubicin was >16 mg/kg doxorubicin equivalent in female rats, 11 mg/kg doxorubicin equivalent in male rats (LD50 of conventional doxorubicin is 10.51 mg/kg), and >8mg/kg doxorubicin equivalent in dogs (MTD of conventional doxorubicin is 1.5mg/kg). Four-week repeat-dose toxicity studies of intravenous legubicin were conducted in rats (5, 10, and 25 mg/kg/dose once weekly) and dogs (3/1.5, 10/5, and 20/10mg/kg/dose once weekly); the dose levels were reduced from the second dose due to intolerable legubicin-associated toxicity at 20 mg/kg. Major organs of toxicity included the gastrointestinal tract, lymphoid and hematopoietic organs, kidney, skin, liver, reproductive organs, and peripheral nerves, which are all associated with doxorubicin. However, cardiotoxicity was only noted at MTD dose levels. Altogether, our results confirm an improved safety profile of legubicin over conventional doxorubicin and support its clinical benefit for treating cancer.

Towards a natural treatment for mania: red onion husk extract modulates neuronal resilience, redox signalling, and glial activation

BackgroundRed onion husk, a readily available agricultural waste material, contains diverse bioactive compounds with potential health benefits. This study aimed to assess the safety and therapeutic potential of red onion husk extract in managing manic-like symptoms and associated neurochemical dysfunctions.MethodsAcute and repeated oral dose studies were conducted in mice and rats to evaluate the safety profile of the extract. FT-IR analysis identified functional groups in the extract, while GC-MS analysis identified specific bioactive compounds in the flavonoid-rich fraction. A ketamine-induced manic behaviour model in Wistar rats was employed to assess the extract’s efficacy in attenuating manic-like symptoms. Behavioural and neurochemical analyses were performed to further investigate the extract’s effects.ResultsThe extract demonstrated a favourable safety profile in both acute and repeated dose studies. FT-IR analysis revealed a complex mixture of organic compounds, including hydroxyl groups, alkynes/nitriles, aromatic and non-aromatic C = C bonds, amines, and polysaccharides. GC-MS analysis identified 17 bioactive compounds, including five-methyl-2-phenylindolizine, methadone N-oxide, and 3-phenylthiane, S-oxide. Ketamine administration significantly increased oxidative stress markers, TBARS, and suppressed antioxidant enzyme activities (SOD, GPx, CAT) in both the cerebral cortex and hippocampus, alongside elevated acetylcholinesterase (AchE) activity, indicating enhanced neuronal excitability. Pre-treatment with FRF (25 mg/kg) effectively mitigated ketamine-induced oxidative stress, as evidenced by reduced TBARS levels and partially restored SOD and GPx activities. Interestingly, FRF significantly increased CAT activity (p < 0.001), potentially suggesting an additional compensatory mechanism. Notably, FRF pre-treatment also counteracted ketamine-upregulated AchE activity, offering neuroprotection against heightened neuronal excitability.ConclusionRed onion husk extract exhibits a favourable safety profile and exerts potent antioxidant and neuroprotective effects, possibly through modulating Nrf2 signalling pathways. Its ability to counteract ketamine-induced oxidative stress and neuronal hyperactivity highlights its potential as a complementary therapeutic strategy for managing manic episodes in bipolar disorder. Further research is warranted to elucidate the precise molecular mechanisms underlying FRF’s action and explore its clinical efficacy in human studies.

Preclinical characterization of a novel investigational monoclonal antibody CM313 with potent CD38-positive cell killing activity.

CM313 is currently under clinical investigation for treatments of multiple myeloma, systemic lupus erythematosus, and immune thrombocytopenia. We aimed to report the preclinical profile of the novel therapeutic anti-CD38 monoclonal antibody (mAb) CM313, with an emphasis on the difference with other CD38-targeting mAb. The binding of CM313 to CD38 recombinant protein across species was assessed using ELISA. The binding of CM313 to CD38-positive (CD38+) cells was detected using flow cytometry assays. CM313-induced complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and apoptosis on different CD38+ cells were assessed by LDH release assays or flow cytometry assays. The effect of CM313 on CD38 enzymatic activity was measured using fluorescence spectroscopy. CM313 immunotoxicity in human blood was assessed using flow cytometry assays, ELISA, and LDH release assays. Anti-tumor activity of CM313 was assessed in multiple mouse xenograft models. Safety profile of CM313 were evaluated in cynomolgus monkeys and human CD38 transgenic (B-hCD38) mice. There exist unique sequences at complementarity-determining regions (CDR) of CM313, which facilitates its affinity to CD38 is consistently higher across a spectrum of CD38+ cell lines than daratumumab. In vitro studies showed that CM313 induces comparable killing activity than daratumumab, including ADCC, CDC, ADCP, apoptosis induced by Fc-mediated cross-linking, and effectively inhibited the enzymatic activity of CD38. However, CM313 showed more potent CDC than isatuximab. In vivo, CM313 dose-dependently inhibited xenograft tumor growth, both as a monotherapy and in combination with dexamethasone or lenalidomide. Furthermore, CM313 was well tolerated with no drug-related clinical signs or off-target risks, as evidenced by 4-week repeat-dose toxicology studies in cynomolgus monkeys and B-hCD38 mice, with the later study showing no observed adverse effect level (NOAEL) of 300mg/kg once weekly. CM313 is a novel investigational humanized mAb with a distinct CDR sequence, showing comparable killing effects with daratumumab and stronger CDC activity than isatuximab, which supports its clinical development.

Acute, subacute, repeat dose toxicity and safety studies of BiosimCovex (COVID-19 Nosode) in animal model

Introduction: BiosimCovex, a coronavirus nosode’s biological preparation was sourced from preclinical samples. The study needs to investigate the acute, subacute oral toxicity of different homeopathic drugs. The acute, subacute, repeat dose toxicity and safety testing of this newly developed nosode were conducted as per the OECD and New Drug and Clinical Trial (NDCT 19) rules. Material and methods In acute, subacute, repeat dose toxicity study, the homeopathic drug was administered orally at body weight, and animals were observed for toxic symptoms for 14 days. The study design consisted of 10 animals (05 males and 05 females) in acute mice and rats, 48 animals (24 males and 24 females) in subacute/repeat dose rats, and 24 (12 males/12 females) in repeat dose rabbit studies. Results Results indicate that in the acute and subacute studies of the rat and mice, no mortalities or clinical signs were found. The body weight and body weight gain in rats were normal and decreased in mice in the acute study. The feed consumption was normal for rats and mice. In male rabbits’ body weight was decreased and in other groups body weight gain observed was normal. The clinical signs were found to be normal in rats and rabbits. Histopathology results reveal that the BiosimCovex has not produced any major reactive and toxic changes in all the systemic organs when administered by an oral route up to a high dose. Conclusion No toxicity was documented using acute, subacute, and repeat dose studies using BiosimCovex, a coronavirus nosode.

The association between histopathologic effects and liver weight changes induced in mice and rats by chemical exposures: an analysis of the data from Toxicity Reference Database (ToxRefDB).

Absolute (ALW) and relative (RLW) liver weight changes are sensitive endpoints in repeat-dose rodent toxicity studies, and their changes are often used for quantitative assessment of health effects induced by hepatotoxic chemicals using the benchmark dose-response modeling (BMD) approach. To find biologically relevant liver weight changes to chemical exposures, we evaluated all data available for liver weight changes and associated liver histopathologic findings from the Toxicity Reference Database (ToxRefDB). Our analysis of 389 subchronic mouse and rat studies for 273 chemicals found significant differences in treatment-related ALW and RLW changes between dose groups with and without liver histopathologic changes. In addition, we demonstrate that chemical treatment-induced ALW and RLW changes can predict the presence of histopathologic findings and inform the selection of biologically relevant liver weight changes for BMD modeling and derivation of toxicity values.

Abstract 3122: Preclinical development of a novel camptothecin-based antibody-drug conjugate targeting solid tumors expressing Claudin-6

Abstract Claudin-6 is a member of the claudin family and consists of four transmembrane helices, two extracellular loops, and an amino- and carboxyl-terminal tail with a PDZ-binding motif in the cytoplasm. It is involved in the formation of tight junctions and is expressed in developing human epithelial structures during early-to-mid gestation while expression in iadult tissues is mostly absent. Claudin-6 expression is upregulated in ovarian cancer, testicular cancer, endometrial cancer, non-small cell lung cancer (NSCLC) and gastric cancer. Here, the generation and preclinical characterization of a novel Claudin-6 specific antibody-drug conjugate (ADC) is described, composed of a humanized IgG1 directed against human Claudin-6 site-specifically conjugated a novel payload containing a valine-alanine cleavable linker and a camptothecin warhead with a drug to antibody ratio of 6. FcγR-mediated effector function of the ADC was abrogated via mutations in the Fc portion of the antibody. The purpose of this study was to evaluate the expression of Claudin-6 in different tumour indications and characterise the in vitro and in vivo anti-tumour activity in human cancer cell lines and patient-derived xenograft (PDX) models. In addition, the tolerability of the ADC was evaluated in a repeat-dose toxicity study cynomolgus monkeys. Cell binding studies using Fabs showed high affinity binding to Claudin-6, while affinity for Claudin-9 was 16-fold lower with no binding to Claudin-3 and Claudin-4. Cross-reactivity with cynomolgus monkey and mouse Claudin-6 was also demonstrated. Fcgamma receptor binding studies confirmed the strongly reduced binding of the antibody to FcγI, FcγI,IIa, FcγIIb and FcγIIa, consistent with the introduction of mutations in the Fc domain of the antibody. In vitro, the Claudin-6 ADC showed potent and specific cytotoxicity against various Claudin-6-expressing solid cancer cell lines, including OVCAR3, PA-1, OV-90, FU-97, BeWo, NTERA-2 and NUGC3 in comparison to a non-binding control ADC conjugated to the same payload, and it also showed potent bystander killing. In vivo, a single dose intravenous (IV) of the Claudin-6 ADC demonstrated potent anti-tumour activity in multiple solid tumour xenograft models, including OVCAR3 and PA-1. Furthermore, administration of the Claudin-6 ADC to cynomolgus monkeys was well tolerated up to 40 mg/kg IV when given on Day 1 and 22. Cell membrane expression of Claudin-6 was confirmed by immunohistochemistry (IHC) in ovarian cancer (70%), testicular cancer (98%), endometrial cancer (30%), NSCLC (28%) and gastric cancer (24%), confirming expression of Claudin-6 in indications with high unmet need. In summary, this novel Claudin-6 ADC demonstrated potent, specific anti-tumour activity in Claudin-6 expressing cancer-derived models and was well tolerated in cynomolgus monkey, supporting future clinical development of this ADC. Citation Format: Nicola Tsang, Nicolas Veillard, Elizabeth Horsley, Karin Havenith, Narinder Janghra, Kristina Zaitseva, Cecile Oblette, Ian Kirby, Paul W. Hogg, Francesca Zammarchi, Lolke de Haan, Patrick H. van Berkel. Preclinical development of a novel camptothecin-based antibody-drug conjugate targeting solid tumors expressing Claudin-6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3122.

Abstract 2371: BGB-B167, a first-in-class 4-1BB/CEACAM5 bispecific antibody, exhibits potent in vitro and &amp;lt; for vivo antitumor activity and &amp;lt; for superior safety profile in preclinical models

Abstract 4-1BB (CD137) is a key costimulatory immunoreceptor and a promising therapeutic target in cancer. CEACAM5 (CEA) is a well-established tumor associated antigen overexpressed in many cancers, including colorectal, gastric, lung, pancreatic cancer, liver, breast and thyroid cancers. BGB-B167 is a novel immunoglobulin G (IgG)-based bispecific antibody targeting 4-1BB and CEA and is under clinical development for the treatment of advanced or metastatic solid tumors in humans. BGB-B167 binds to its target proteins with high specificity and affinity. Potent and CEA-dependent functional activities were demonstrated using peripheral blood mononuclear cell (PBMC)-based immune cell activation and cytotoxicity assays. In humanized 4-1BB knock-in mice bearing human CEA-expressing tumors, BGB-B167 exhibited potent, dose-associated single-agent efficacy as well as synergistic antitumor activity in combination with anti-PD-1 antibody. BGB-B167 was well tolerated in 1-month repeat-dose toxicology study in cynomolgus monkeys. Here, we describe the characterization of BGB-B167 with regard to preclinical proof-of-concept and basic drug-like properties. The combined dataset provides an overview on the design, mode of action, preclinical pharmacology and safety profile of BGB-B167. Citation Format: Zhuo Li, Liang Qu, Xin Chen, Liu Xue, Jie Li, Qi Liu, Jian Sun, Hanzi Sun, Yun Chen, Yuanyuan Xie, Wanyi Wang, Lin Zhu, Penghao Wang, Xiaosui Zhou, Hongjia Hou, Jie Chen, Xinyi Liu, Yilu Zhang, Ning Liu, Xinyi Liang, Shuo Zhang, Xiaoyan Tang, Jing Song, Tong Zhang, Xiaomin Song, Xuesong Liu, Kang Li, Chichi Huang. BGB-B167, a first-in-class 4-1BB/CEACAM5 bispecific antibody, exhibits potent in vitro and &amp;lt; for vivo antitumor activity and &amp;lt; for superior safety profile in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2371.