Preclinical Safety Research Articles

Investigative safety strategies to improve success in drug development

Understanding and reducing attrition rate remains a key challenge in drug development. Preclinical and clinical safety issues still represent about 40% of drug discontinuation, of which cardiac and liver toxicities are the leading reasons. Reducing attrition rate can be achieved by various means, starting with a comprehensive evaluation of the potential safety issues associated to the primary target followed by an evaluation of undesirable secondary targets. To address these risks, a risk mitigation plan should be built at very early development stages, using a panel of in silico, in vitro, and in vivo models. While most pharmaceutical companies have developed robust safety strategies to de-risk genotoxicity and cardiotoxicity issues, partly driven by regulatory requirements; safety issues affecting other organs or systems, such as the central nervous system, liver, kidney, or gastro-intestinal system are less commonly addressed during early drug development. This paper proposes some de-risking strategies that can be applied to these target organ systems, including the use of novel biomarkers that can be easily integrated in both preclinical and clinical studies. Experiments to understand the mechanisms’ underlying toxicity are also important. Two examples are provided to demonstrate how such mechanistic studies can impact drug development. Novel trends in investigative safety are reviewed, such as computational modeling, mitochondrial toxicity assessment, and imaging technologies. Ultimately, understanding the predictive value of non-clinical safety testing and its translatability to humans will enable to optimize assays in order to address the key objectives of the drug discovery process, i.e., hazard identification, risk assessment, and mitigation

A BIVALENT TIM-3/PD-1 BISPECIFIC ANTIBODY FOR THE TREATMENT OF PD-1 ANTIBODY RESISTANT OR REFRACTORY SOLID TUMORS

Abstract Background Immune checkpoint inhibitors (ICI) PD-1/PD-L1 antibody are key drugs for the treatment of cancer. Bispecific antibody is one of the strategies aimed to meet the clinical needs for cancer patients who are resistant to or refractory from ICI treatment. TIM-3, one of the next generation of ICI targets, co-expressed on exhausted T cells with PD-1. It is also expressed by innate immune populations, including NK and DC. Dual blocking PD-1 and TIM-3 not only on T cells but also on DC, NK cells may achieve better clinical benefit for patients who are resistant to or refractory from ICI treatment. Method A bivalent TIM-3 and PD-1 bispecific antibody (Bis5) was developed, a series of in vitro and in vivo efficacy, preclinical pharmacokinetic and toxicity studies were conducted. A Phase I, multicenter, open-label study to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, immunogenicity and preliminary efficacy of Bis5 in patients with advanced and/or metastatic solid tumors is ongoing in China. Results Bis5 showed affinity of 5-8 nM to both TIM-3 and PD-1, with better cell activity than TIM-3 and PD-1 mAb combination to activated T cell as well as NK and DC, over the other clinical stage reference BsAb. In huPD-1/TIM-3 double knock in mice-CT26 tumor model, Bis5 showed significant tumor inhibition activity and doubled the survival rate, while neither PD-1 mAb, TIM-3 mAb nor PD-1 and TIM-3 antibody combination showed activity. The highest non-severe toxicity dose (HNSTD) was 200mg/kg in monkeys. Nine cohorts (0.001-15 mg/kg) are planned to be enrolled sequentially in the dose escalation part in the Phase I study, as of April 2023, seven cohorts enrollment have completed. No dose limiting toxicity was observed, and the optimal effective dose was not reached. No TRAE higher than grade 2 was observed. The TRAE with ≥10% Incidence was anemia. SD >4 or 2 months were shown in the suboptimal dose levels in NSCLC and CRC (0.3mg/kg, 1mg/kg). The Part 2 dose expansion will further characterize the safety profile and preliminary tumor response in several cohorts including NSCLC, CRC, ESCC etc. Conclusion Bis5 showed good preclinical efficacy and safety profile, its clinical performance is expected. Clinical trial information: NCT05357651.

Preclinical characterization of the efficacy and safety of biologic N-001 as a novel pain analgesic for post-operative acute pain treatment

Inhibition of actin remodeling in nerves modulates action potential propagation and therefore could be used to treat acute pain. N-001 is a novel protein analgesic engineered from several C. Botulinum toxins. N-001 targets sensory neurons through ganglioside GT1b binding and ADP-ribosylates G-actin reducing actin remodeling. The activity and efficacy of N-001 was evaluated previously in vitro and in a mouse inflammatory pain model. To assess the relevance of N-001 for treatment of acute post-surgical pain, the current study evaluated the efficacy of N-001 in a mouse hind-paw incision model by peri-incisional and popliteal nerve block administration combined with mechanical testing. N-001 provided relief of pain-like behavior over 3 days and 2 days longer than the conventional long-acting anesthetic bupivacaine. Preclinical safety studies of N-001 indicated the drug produced no toxic or adverse immunological reactions over multiple doses in mice. These results combined with past targeting results encourage further investigation of N-001 as an analgesic for post-operative pain management with the potential to function as a differential nociceptor-specific nerve block.

Comparison of Efavirenz and Doravirine Developmental Toxicity in an Embryo Animal Model.

In the past, one of the most widely used non-nucleoside reverse transcriptase inhibitors (NNRTI) in first-line antiretroviral therapy (ART) of HIV infection was efavirenz (EFV), which is already used as a cost-effective treatment in developing countries due to its efficacy, tolerability, and availability. However, EFV also demonstrates several adverse effects, like hepatotoxicity, altered lipid profile, neuropsychological symptoms, and behavioral effects in children after in utero exposure. In 2018, another NNRTI, doravirine (DOR), was approved due to its similar efficacy but better safety profile. Preclinical safety studies demonstrated that DOR is not genotoxic and exhibits no developmental toxicity or effects on fertility in rats. Zebrafish (Danio rerio) embryos have been widely accepted as a vertebrate model for pharmacological and developmental studies. We used zebrafish embryos as an in vivo model to investigate the developmental toxicity of DOR compared to EFV. After exposure of the embryos to the drugs from the gastrula stage up to different developmental stages (30 embryos for each arm, in three independent experiments), we assessed their survival, morphology, hatching rate, apoptosis in the developing head, locomotion behavior, vasculature development, and neutral lipid distribution. Overall, DOR showed a better safety profile than EFV in our model. Therapeutic and supra-therapeutic doses of DOR induced very low mortality [survival rates: 92, 90, 88, 88, and 81% at 1, 5, 10, 25, and 50 μM, respectively, at 24 h post fecundation (hpf), and 88, 85, 88, 89, and 75% at the same doses, respectively, at 48 hpf] and mild morphological alterations compared to EFV exposure also in the sub-therapeutic ranges (survival rates: 80, 77, 69, 63, and 44% at 1, 5, 10, 25, and 50 μM, respectively, at 24 hpf and 72, 70, 63, 52, and 0% at the same doses, respectively, at 48 hpf). Further, DOR only slightly affected the hatching rate at supra-therapeutic doses (97, 98, 96, 87, and 83% at 1, 5, 10, 25, and 50 μM, respectively, at 72 hpf), while EFV already strongly reduced hatching at sub-therapeutic doses (83, 49, 11, 0, and 0% at 1, 5, 10, 25, and 50 μM, respectively, at the same time endpoint). Both DOR at therapeutic doses and most severely EFV at sub-therapeutic doses enhanced apoptosis in the developing head during crucial phases of embryo neurodevelopment and perturbed the locomotor behavior. Furthermore, EFV strongly affected angiogenesis and disturbed neutral lipid homeostasis even at sub-therapeutic doses compared to DOR at therapeutic concentrations. Our findings in zebrafish embryos add further data confirming the higher safety of DOR with respect to EFV regarding embryo development, neurogenesis, angiogenesis, and lipid metabolism. Further studies are needed to explore the molecular mechanisms underlying the better pharmacological safety profile of DOR, and further human studies are required to confirm these results in the zebrafish animal model.

Preclinical evaluation of a novel single-shot pulsed field ablation system for pulmonary vein and atrial ablation.

Pulsed field ablation (PFA) is a nonthermal ablative strategy that achieves cell death via electroporation. Herein, we investigated the preclinical safety and efficacy of PFA using two novel 8-French, 16-electrode spiral PFA/mapping catheters (ElePulse; CRC EP, Inc.). Bipolar PFA (>1.8 kV) was performed using 30 s, single-shot, QRS-gated applications. Altogether, 94 atrial structures were ablated in 23 swine, one canine, and one ovine, including right and left atria and atrial appendages, pulmonary veins, and superior and inferior (IVC) vena cavae. We also examined the impact of PFA on the phrenic nerve (14 swine) and on a deviated esophagus after delivery of PFA from inside the IVC (five swine). All applications were single-shot without catheter repositioning. Minimal microbubbling was observed without significant skeletal muscle twitching/activation (mean acceleration: 0.05 m/s2 ). There was a marked reduction in post-PFA versus pre-PFA atrial electrogram amplitude (0.17 ± 0.21 vs. 1.18 ± 1.08 mV; p < .0001). Lesion durability was demonstrated up to 3 months in all targeted tissues. Histologically, lesions were contiguous and transmural, except in the atrial appendage, and without any thermal effects. Magnetic resonance, gross, and histologic examinations of the brain, rete mirabile, and kidneys revealed no thromboembolism. No acute/long-term phrenic nerve dysfunction was encountered. Although within 2 h of ablation, histologic examinations of the esophagus revealed acute PFA-related changes in the muscular layer, these completely resolved by 21 ± 5 days. A novel, single-shot, spiral PFA system is capable of safely creating large, durable atrial lesions without significant adverse effects on the phrenic nerve or the esophagus.

LC-MS/MS Bioanalysis of Radioligand Therapeutic Drug Candidate for Preclinical Toxicokinetic Assessment.

Radioligand therapy (RLT) has gained significant momentum in recent years in the diagnosis, treatment, and monitoring of cancers. In preclinical development, the safety profile of RLT drug candidate(s) is investigated at relatively low dose levels using the cold (non-radioactive, e.g., 175Lu) ligand as a surrogate of the hot (radioactive, e.g., 177Lu) one in the "ligand-linker-chelator" complex. The formulation of the test article used in preclinical safety studies contains a mixture of free ligand (i.e., ligand-linker-chelator without metal) and cold ligand (i.e., ligand-linker-chelator with non-radioactive metal) in a similar molar ratio as seen under the manufacturing conditions for the RLT drug for clinical use, where only a fraction of free ligand molecules chelate the radioactive metal to form a hot ligand. In this very first report of LC-MS/MS bioanalysis of RLT molecules in support of a regulated preclinical safety assessment study, a highly selective and sensitive LC-MS/MS bioanalytical method was developed for the simultaneous determination of free ligand (NVS001) and cold ligand (175Lu-NVS001) in rat and dog plasma. Several unexpected technical challenges in relation to LC-MS/MS of RLT molecules were successfully addressed. The challenges include poor assay sensitivity of the free ligand NVS001, formation of the free ligand (NVS001) with endogenous metal (e.g., potassium), Ga loss from the Ga-chelated internal standard during sample extraction and analysis, "instability" of the analytes at low concentrations, and inconsistent IS response in the extracted plasma samples. The methods were validated according to the current regulatory requirements in a dynamic range of 0.5-250 ng/mL for both the free and cold ligands using a 25 μL sample volume. The validated method was successfully implemented in sample analysis in support of regulated safety studies, with very good results from incurred sample reanalysis. The current LC-MS/MS workflow can be expanded to quantitative analysis of other RLTs in support of preclinical RLT drug development.

Preclinical Safety Evaluation of Engineered Lactococcus lactis NZ3900 Biotherapeutics Using a 13-Week Repeated Dose Oral Toxicity Study in Rabbits

ABSTRACT Lactococcus lactis is one of the most important and safe microorganisms used in the dairy industry. Previously, we orally fed cholesterol-fed rabbits with recombinant Ling Zhi 8 (LZ8) protein from L. lactis NZ3900/pNZ8149 and showed anti-inflammatory effects during the development of fatty liver and atheroma. In order to generate a clinically effective oral immunotherapy for the treatment of allergy, L. lactis clones for Der p 2, Per a 2, and Cla c 14 were introduced into L. lactis NZ3900/pNZ8149 to obtain recombinant allergen-based vaccines. In this study, we developed a triple-antigen vaccine formulation, Lactis- IL-1Ra, Lactis-LZ8, Lactis-Triple, and evaluated their preclinical safety in rabbits by oral administration with repeated doses for 90 days. All of the rabbits survived the challenge without any adverse clinical signs or abnormal changes. A notable advantage of this vaccine is that it can be given orally, and thus self-administration at home would be feasible.

Discovery of YK-029A, a novel mutant EGFR inhibitor targeting both T790 M and exon 20 insertion mutations, as a treatment for NSCLC

Although traditional EGFR-TKIs have advanced the treatment landscape of NSCLC with sensitive driver mutations (del19 or L858R), some NSCLC patients with EGFR exon 20 insertion mutations have been left with few effective therapies. The development of novel TKIs is still in progress. Herein, we describe the structure-guided design of a novel selective and orally bioavailable inhibitor, YK-029A, which could overcome both the T790 M mutations and exon 20 insertion of EGFR. YK-029A inhibited EGFR signaling, suppressed sensitive mutations and ex20ins of EGFR-driven cell proliferation, and was largely effective with oral administration in vivo. Furthermore, YK-029A exhibited significant antitumor activity in EGFRex20ins-driven patients-derived xenograft (PDX) models, preventing tumor progression or causing tumor regression at well-tolerated dosages. Based on the outcomes of preclinical efficacy and safety studies, YK-029A will enter phase Ⅲ clinical trials for the treatment of EGFRex20ins NSCLC.

Chemo-radiotherapy with 177Lu-PLGA(RGF)-CXCR4L for the targeted treatment of colorectal cancer.

More than 1.9 million new cases of colorectal cancer and 935,000 deaths were estimated to have occurred worldwide in 2020. Therapies for metastatic colorectal cancer include cytotoxic chemotherapy and targeted therapies in multiple lines of treatment. Nevertheless, the optimal use of these agents has not yet been resolved. Regorafenib (RGF) is an Food and Drug Administration (FDA)-authorized multikinase inhibitor indicated for patients with metastatic colorectal cancer, non-responding to priority lines of chemotherapy and immunotherapy. Nanoparticles have been used in specific applications, such as site-specific drug delivery systems, cancer therapy, and clinical bioanalytical diagnostics. C-X-C Chemokine receptor type 4 (CXCR4) is the most widely-expressed chemokine receptor in more than 23 human cancer types, including colorectal cancer. This research aimed to synthesize and preclinically evaluate a targeted nanosystem for colorectal cancer chemo-radiotherapy using RGF encapsulated in Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles coated with a CXCR4 ligand (CXCR4L) and 177Lu as a therapeutic β-emitter. Empty PLGA and PLGA(RGF) nanoparticles were prepared using the microfluidic method, followed by the DOTA and CXCR4L functionalization and nanoparticle radiolabeling with 177Lu. The final nanosystem gave a particle size of 280 nm with a polydispersity index of 0.347. In vitro and in vivo toxicity effects were assessed using the HCT116 colorectal cancer cell line. 177Lu-PLGA(RGF)-CXCR4L nanoparticles decreased cell viability and proliferation by inhibiting Erk and Akt phosphorylation and promoting apoptosis. Moreover, in vivo administration of 177Lu-PLGA(RGF)-CXCR4L significantly reduced tumor growth in an HCT116 colorectal cancer xenograft model. The biokinetic profile showed hepatic and renal elimination. Data obtained in this research justify additional preclinical safety trials and the clinical evaluation of 177Lu-PLGA(RGF)-CXCR4L as a potential combined treatment of colorectal cancer.

Next Generation Sequencing Workshop at the Royal Society of Medicine (London, May 2022): how genomics is on the path to modernizing genetic toxicology.

The use of error-corrected Next Generation Sequencing (ecNG) to determine mutagenicity has been a subject of growing interest and potentially a disruptive technology that could supplement, and in time, replace current testing paradigms in preclinical safety assessment. Considering this, a Next Generation Sequencing Workshop was held at the Royal Society of Medicine in London in May 2022, supported by the United Kingdom Environmental Mutagen Society (UKEMS) and TwinStrand Biosciences (WA, USA), to discuss progress and future applications of this technology. In this meeting report, the invited speakers provide an overview of the Workshop topics covered and identify future directions for research. In the area of somatic mutagenesis, several speakers reviewed recent progress made with correlating ecNGS to classic in vivo transgenic rodent mutation assays as well as exploring the use of this technology directly in humans and animals, and in complex organoid models. Additionally, ecNGS has been used for detecting off-target effects of gene editing tools and emerging data suggest ecNGS potential to measure clonal expansion of cells carrying mutations in cancer driver genes as an early marker of carcinogenic potential and for direct human biomonitoring. As such, the workshop demonstrated the importance of raising awareness and support for advancing the science of ecNGS for mutagenesis, gene editing, and carcinogenesis research. Furthermore, the potential of this new technology to contribute to advances in drug and product development and improve safety assessment was extensively explored.

T cell redirecting and TLR7/8 agonist encapsulated liposomes targeting HER2+ tumors: Preclinical efficacy, pharmacokinetics and safety evaluations.

e14590 Background: T-cell dependent bispecific antibodies (TDBs) have shown limited success in solid tumor treatments. We hypothesized that it may be due to TDBs limited distribution in solid tumor tissues. We therefore propose to take advantage of the EPR (enhanced permeability and retention) effect of the liposome system to improve the PKPD properties of TDBs. The liposomes are named TRAFsomes (T cell Redirecting Antibody Fragment-anchored Liposomes). HF5050 is a TRAFsome containing on average 6 anti-CD3 and 10 anti-Her2 on each liposome surface and the toll-like TLR-7/8 agonist Resiquimod encapsulated inside at specific lipid to drug ratios. It preclinical efficacy, pharmacokinetics and safety evaluation studies are conducted to support the IND application. Methods: The T cell redirecting and target specific cytotoxic effect of HF5050 were evaluated in five PBMC co-cultured cancer cell models with different Her2 expression. Tumor cells derived from adenoid cystic carcinoma tumor tissues was also included. T cell redirection and activation are examined by T cell surface markers CD25/CD69 and secretion of IFN gamma, Perforin, and granzymes B in the absence and presence of cancer cells. For the incorporation of TLR7/8 agonist Resiquimod, we evaluated the effect of drug dose by varying the drug loading ratios in order to achieve a synergistic effect. For the in vivo efficacy studies, the N87 gastric cancer CDX model and an adenoid cystic carcinoma tumor PDX model were set on PBMC reconstructed humanized NCG-B2M mice. Pharmacokinetic and tissue distribution studies are carried out in tumor bearing mouse models as well. Results: HF5050 has shown to be a highly effective anti-cancer agent in both in vitro and in vivo studies. It mediates T cells-tumor cells redirection and cytotoxicity in a dose-dependent manner, especially in Her-2 highly expressing cell lines. HF5050 also effectively stimulates T cell activation and displays high efficacy in PBMC reconstruct humanized NCG-B2M tumor-bearing mice. Pharmacokinetic research confirms liposome successfully maintains drug levels in the bloodstream, and improves drug accumulation within tumor tissues. Conclusions: Overall, these data demonstrate that HF5050 processing a novel cell bridging function between T cells and tumor cells, and with the capability of activating immune cells. Based on the encouraging preclinical results, HF5050 exhibits promising development potential.

SAT-178 - AHB-137, a novel and potent hepatitis B virus antisense oligonucleotide with a favorable preclinical pharmacokinetics and safety profile

SAT-178 - AHB-137, a novel and potent hepatitis B virus antisense oligonucleotide with a favorable preclinical pharmacokinetics and safety profile

Preclinical development of RAG1-40-31L: A novel small activating RNA-lipid conjugate targeting tumor suppressor gene p21 for treatment of non-muscle invasive bladder cancer.

e16620 Background: Loss-of-function of tumor suppressor genes are the dominant driving force in tumorigenesis. Restoration in function and/or expression of these genes holds tremendous therapeutic potential in cancer treatment. Bladder cancer is the 9th leading cause of cancer-related deaths in the United States in which ~75% of all newly diagnosed cases are non-muscle invasive bladder cancer (NMIBC). Standard-of-care includes intravesical installation of Bacillus Calmette-Guerin following tumor resection with an expected failure rate of ~50% within 6 months for patients with high-grade tumors. RNA activation (RNAa) is a mechanism in which short RNA duplexes termed small activating RNA (saRNA) induce gene transcription by targeting non-coding regulatory sequence ( i.e., promoters) embedded in chromatin. Thus, saRNA-induced overexpression of p21WAF1/CIP1 (p21) – a tumor suppressor of several anti-growth pathways – may hold therapeutic potential in bladder cancer. Through conjugation of a novel lipid ( i.e., C5X5), we have created a p21-targeting saRNA drug candidate referred to as RAG01-40-31L for delivery to bladder cancer cells in vivo. The aim of this work was to characterize the intravesical administration of RAG01-40-31L on p21 induction and its anti-tumor potential in NMBIC models. Methods: In vitro expression analyses were performed to investigate p21 induction via saRNA in KU7 and T24 bladder cancer cell lines by RT-qPCR and immunoblot analysis. Cell cycle analysis and tumorigenicity assays examined impact of RAG1-40-31L on cancer cell growth. Orthotopic human bladder tumors were established in female BALB/c nude mice using the recombinant cell line KU7 engineered to overexpress luciferase reporter. Tumor growth was monitored in live animals via luciferase bioluminescence using an in vivo imaging system. Plasma and tissue were harvested for drug exposure and expression analytics. Results: In vitro analyses indicated RAG1-40-31L activated p21 expression by approximately 6-fold and inhibited bladder cancer cell growth ( i.e., cell cycle arrest, apoptosis, and senescence) in a dose-dependent manner with low nanomolar potency. Intravesical administration of RAG1-40-31L (3 times per week for 2 weeks) inhibited growth of orthotopic tumors by 65% in mouse bladder models. Single-dose pharmacokinetics demonstrated a half-life (t1/2) of ~114 h with minimal systemic exposure (~0.05% of that of bladder tissue). Conclusions: These data constitute a preclinical proof-of-concept for saRNA as a novel p21-targeting modality in the treatment of cancer. The C5X5 lipid conjugate appears to be a viable approach to enhance oligonucleotide delivery and activity in bladder cancer. Preclinical safety assessment studies to enable clinical trials of RAG01-40-31L are currently underway.

Upgrading the non-gene-editing, allogeneic ThisCART platform for extending persistence in vivo.

e14569 Background: Lack of in vivo persistence is the major challenge in allogeneic CAR-T field. Various HLA-related gene-editing strategies are being explored to prevent rejection by host T and NK cells (Table 1). This study reports a platform upgrade on ThisCART (TCR and HLA-I intracellular sequestered), represented by ThisCART19C cells. Methods: ThisCART19C cells were produced with a single lentivirus vector, encoding a CD19-targeting CAR, a KDEL-tagged anti-CD3 scFv, and the HCMV US11 protein. The surface expression of CAR19, TCRαβ and HLA-A/B/E was measured by flow cytometer. Specific tumor cytotoxicity was evaluated both in vitro and in vivo. Furthermore, the safety of donor derived ThisCART19C cells were tested in a xenogeneic GvHD model. Results: Similar to our ThisCART19A product currently in multi-center trials, ThisCART19C cells were readily produced to a quantity and quality supporting clinical investigation. ThisCART19C cells demonstrated an anticipated and stable surface immunotype: CAR19+, TCRαβ-, HLA-A-, HLA-B-，HLA-E+，with great potential for simultaneous evasion of both host T and NK cells. Similar to autologous CD19-directed CAR-T, ThisCART19C exhibited potent CD19-specific cytotoxicity in vitro and in vivo. No xenogeneic GvHD reaction was observed in murine model over an extended period. Conclusions: To our knowledge, ThisCART19C represents the first application of viral evading strategy in promoting the clinical persistence of allogeneic CAR-T cells. Its efficient processing, potent cytotoxicity and favored preclinical safety profile warrant clinical investigation. [Table: see text]

Standardizing the strategy in glioblastoma trials to promote success in cell and gene therapy clinical development.

e13604 Background: Glioblastoma is the most common and aggressive brain tumor, with a high recurrence rate and low survival. We are increasing our understanding of its biology, and more targeted therapeutical agents are available. However, the GBM prognosis has not improved over the last decades. Multiple clinical trials on Cell and Gene Therapies are ongoing but, there is a huge need to define standardized and innovative strategies to promote better patient selection, implement adaptative designs and specific laboratory assays, and tailor the clinical endpoint assessments. Methods: Medical experts panel agreed on the following insights and gave advice to improve the design and conduct of GBM trials. Results: The criteria for selecting patients are not uniform among studies. The 2021 WHO Classification of central nervous system tumors has yet to be universally adopted, and different tumor entities are being treated as GBM according to the prior 2016 classification. Furthermore, the required genetic testing panel is only available in some countries as part of routine testing. A wide range of CGT therapies are being studied with different mechanisms of action and are well suited to be administered as combinatory regimens and multiple-dosing schedules to boost the antitumor effect. The implementation of master protocols offers the possibility to test different schedules to identify the most promising for further investigation during early phase development. There is a lack of biomarkers for the patient- or cancer-specific sensitivity and the efficacy of CGT. This is important not only to target clinical treatments to the appropriate patients and identify responding/non-responding patients early during treatment but also to improve the preclinical models and identify targets for further therapeutic development. Investigators should use standard endpoint criteria when reporting clinical outcomes. It has been common to refrain from introducing any preliminary-effect objective in the early stages. This tendency changes after guidance from experts and regulatory agencies about maximizing early-phase data. Another vital endpoint in the early phases is assessing patient immunogenicity against CG therapy based on humoral and cell virus-specific and tumor-specific immune responses. This is especially important as there are significant concerns in translating preclinical safety results to the clinic due to the species-specificity of different viruses and critical differences between immune systems. Conclusions: Today, the purpose of treatment for GBM is to extend the survival period and to manage the disease while preserving the patient’s quality of life. The goal of CGT is a durable complete response, but to achieve this target, we will need a deep analysis of how we perform clinical trials and move to an expert and tailored research environment.

P014 Safety and toxicity profile of SPL84, an inhaled antisense oligonucleotide cystic fibrosis therapeutic

The ASO field is an emerging area of drug development that targets the disease source at the RNA level, offering a promising alternative to therapies targeting downstream processes. SpliSense is developing SPL84, an inhaled ASO for the treatment of CF patients carrying the 3849 +10 kb C->T mutation. In order to move into the clinic, we performed a full battery of preclinical safety and toxicology studies with SPL84.

Abstract ND04: BAY 2965501: A highly selective DGK- ζ inhibitor for cancer immuno-therapy with first-in-class potential

Abstract The second messenger diacyl glycerol (DAG) plays a key role in T cell receptor downstream signaling and thereby in T-cell activation. Diacylglycerol kinase zeta (DGK-ζ) is a lipid kinase that can down-modulate T-cell activation by catalyzing the conversion of DAG to phosphatidic acid, thereby acting as a ligand independent, intracellular immune checkpoint. Inhibition of DGK- ζ offers the potential to enhance T cell priming against suboptimal tumor antigens and to overcome multiple immune-suppressive mechanisms in the tumor microenvironment. Bayer AG in collaboration with the German Cancer Research Center (DKFZ) have developed BAY 2965501, a highly selective, orally available DGK-ζ inhibitor which shows a unique binding mode and good DMPK profile. The compound enhances the reactivity of human and mouse T-cells, both in the priming and the effector phase, and overcomes the inhibitory impact of suppressive factors, such as adenosine and prostaglandin E2. Oral dosing of BAY 2965501 shows T cell dependent efficacy in syngeneic, murine tumor models. Furthermore, this results in reactivation of exhausted T cells in vivo. Preclinical safety studies showed only mild findings, so that good clinical tolerability is expected. Collectively, the preclinical data support first in human (FiH) phase I testing of therapeutic potential. A FiH trial with BAY 2965501 in patients with advanced solid tumors including NSCLC, gastric/GEJ AdCa, ccRCC, and melanoma patients, is currently enrolling patients (NCT05614102). This study will evaluate the safety, tolerability, maximum tolerated or administered dose, pharmacokinetics, pharmacodynamics, and tumor response profile of BAY 2965501. Citation Format: Rienk Offringa, Catherine Olesch, Frederik Cichon, Mareike Grees, Norbert Schmees, Ulrike Roehn, Florian Prinz, Judith Guenther, Detlef Stoeckigt, Michael Erkelenz, Thi Thanh Uyen Nguyen, Ulf Boemer, Nicolas Werbeck, Marc Kunze, Kirstin Petersen, Dennis Kirchhoff. BAY 2965501: A highly selective DGK- ζ inhibitor for cancer immuno-therapy with first-in-class potential [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 ND04.

Preclinical safety and biodistribution assessment of Ad-KCNH2-G628S administered via atrial painting in New Zealand white rabbits.

Post-operative atrial fibrillation (POAF) is the most common complication after cardiac surgery. Despite implementation of several pharmacological strategies, incidence of POAF remains at approximately 30%. An adenovirus vector encoding KCNH2-G628S has proven efficacious in a porcine model of AF. In this preclinical study, 1.5 × 1010 or 1.5 × 1012 Ad-KCNH2-G628S vector particles (vp) were applied to the atrial epicardium or 1.5 × 1012 vp were applied to the whole epicardial surface of New Zealand White rabbits. Saline and vector vehicle served as procedure controls. Animals were followed for up to 42 days. Vector genomes persisted in the atria up to 42 days, with no distribution to extra-thoracic organs. There were no adverse effects attributable to test article on standard toxicological endpoints or on blood pressure, left atrial or ventricular ejection fractions, electrocardiographic parameters, or serum IL-6 or troponin concentrations. Mononuclear infiltration of the myocardium of the atrial free walls of low-dose, but not high-dose animals was observed at 7 and 21 days, but these changes did not persist or affect cardiac function. After scaling for heart size, results indicate the test article is safe at doses up to 25 times the maximum proposed for the human clinical trial.

A Review on Polyherbal Formulation Used in the Treatment of Gastric Acidity

Acidity is a medical condition caused by an excess of acid production. This acid is produced by the stomach glands. Acidity causes symptoms such as stomach ulcers, gastric inflammation, heartburn, and dyspepsia. Acidity is a global issue caused by an imbalance between the acid-secreting mechanisms of the stomach and the proximal intestine. It causes a lot of problems in the lives of many people. An antacid reduces acidity and provides relief. This review article discusses different polyherbal formulations (PHF) used to treat gastric acidity. Due to its role in medicine and therapy, PHF has been used all over the world. The current study discusses a variety of commercial and non-commercial polyherbal formulations used worldwide to treat gastric acidity, as well as their therapeutic potential, clinical and preclinical outcomes, and safety and efficacy. This review will make it easier to learn everything about the previous scientific research as well as the crucial details about the antacid properties of polyherbal preparations, which will encourage young researchers to conduct additional studies in the future to protect people from gastric acidity. Natural herbs and their extracts are used in herbal treatment as an alternative to conventional medication to treat various conditions. It has been demonstrated that several medications contain medicinal components useful for treating ulcers or stomach acidity. The subject of the current work is the claim that a polyherbal formulation can be utilised as an alternative to currently available antacid formulations on the market. As a result, those plants that can reduce stomach acid are chosen, and a formulation is created.

Study of acute toxicity of GIZh-298

Relevance. Assay of acute toxicity is a mandatory step in a preclinical safety study of medicines and pharmaceutical substances. The purpose of this study was to determine the parameters of acute toxicity of the substance GIZH-298. Methods. GIZH-298 was administered once orally to outbred mice of both sexes at doses of 350–550 mg/kg. Control mice received 0.5 ml of 1 % starch solution under the same conditions. During the experiment, the death of animals, signs of intoxication were observed, and the clinical picture was recorded. Pathological anatomical autopsy of the dead mice was performed as they died. Surviving mice were autopsied 14 days after the start of the experiment, immediately after their euthanasia. Results. The average lethal doses of the GIZh-298 substance when administered orally to mice were determined: LD50 in females was 356 mg/kg, LD50 in males was 438 mg/kg. Conclusion. Compound GIZH-298 for oral administration according to the classification of Sidorov K.K. (1973) is a moderately toxic substance and can be assigned to the 3rd class of toxicity, and in accordance with GOST 12.1.007-76 — to the 3rd hazard class.