Preclinical Safety Research Articles

Preclinical Efficacy And Safety Evaluation of AAV-OTOF in DFNB9 Mouse Model And Nonhuman Primate.

OTOF mutations are the principal causes of auditory neuropathy. There are reports on Otof-related gene therapy in mice, but there is no preclinical research on the drug evaluations. Here, Anc80L65 and the mouse hair cell-specific Myo15 promoter (mMyo15) are used to selectively and effectively deliver human OTOF to hair cells in mice and nonhuman primates to evaluate the efficacy and safety of OTOF gene therapy drugs. A new dual-AAV-OTOF-hybrid strategy to transfer full-length OTOF is generated, which can stably restore hearing in adult OTOFp.Q939*/Q939* mice with profound deafness, with the longest duration being at least 150 days, and the best therapeutic effect without difference in hearing from wild-type mice. An AAV microinjection method into the cochlea of cynomolgus monkeys without hearing impairment is further established and found the OTOF can be safely and effectively driven by the mMyo15 promoter in hair cells. In addition, the therapeutic dose of AAV drugs has no impact on normal hearing and does not cause significant systemic toxicity both in mouse and nonhuman primates. In summary, this study develops a potential gene therapy strategy for DFNB9 patients in the clinic and provides complete, standardized, and systematic research data for clinical research and application.

In Vitro and In Vivo Specificity and Biodistribution of a Novel CD8-Targeted Fusosome

Autologouschimeric antigen receptor (CAR) T cells represent a groundbreaking therapy for treating B-cell malignancies. However, the complexity, cost, time and toxicity associated with manufacturing and administering current CAR T cell therapies hinder their broader applications. To overcome CAR T limitations, Sana has developed a novel fusosome platform that can deliver a T cell targeted CAR gene therapy via systemic administration, without ablative pre-conditioning. The fusosome is a lentiviral vector pseudotyped with a targeted fusogen that specifically directs gene transfer into CD8+ cells. Our CD8-targeted CD19-directed CAR fusosome, SG299, transduces CD8+ cells in lymphoid tissues, showing preclinical safety and high selectivity, laying the groundwork for clinical evaluation of an in vivo CAR gene therapy. In vitro tissue cross reactivity (TCR) study was assessed to evaluate binding of the CD8 fusogen binder, a CD8-binding scFv, on tissues from Nemestrina macaques and compared these with tissues from human donors. SG299 specificity was also evaluated in vitro using panel of cell lines representing a breadth of receptor types. Cells were exposed to 8 IU/cell SG299 and vector integration was evaluated using ddPCR. Reverse transcriptase inhibitor nevirapine was used as control for the in vitro studies. To evaluate in vivo specificity and biodistribution, healthy nemestrina macaques were treated with a single dose of SG299 at 1 or 4 x 10 9 IU/kg (n=2 or 4) or saline (n=1 or 2) via intravenous infusion and evaluated for up to 35 days for vector biodistribution in CD8 enriched PBMCs and at terminal timepoint in panel of 17 tissues using ddPCR. In addition, we also demonstrated SG299 specificity with help of flow cytometry, in NALM6 tumor model in NSG mice. Briefly, NSG mice engrafted with human PBMCs were challenged with NALM6 tumors and treated with either SG299 or saline. The TCR study demonstrated that the CD8 fusogen binder is specific for the target tissues containing T cells and similar binding across multiple human and Nemestrina tissues. SG299 fusosome demonstrated CD8-specificity evaluated by transduction of cell types with CD8 receptor (SupT1 and PanT cells). There was no detectable off-target transduction in sets of primary as well as immortalized cell lines evaluated including pulmonary cells, renal epithelial cells, lymphoblast, endothelial cells and CD8KO cells. VCN analysis of nemestrina macaques PBMC and tissue samples following treatment with SG299 demonstrated dose dependent integrations per diploid genome through Day 35. All animals dosed 4 x 10 9 IU/kg showed quantifiable VCN signal through Day 34 in PBMC population enriched for CD8+ cells (0.0032 - 0.6544 vc/dg, vector copy per diploid genome). One out of 4 animals dosed at 1 x 10 9 IU/kg showed quantifiable VCN signal at Day 34 (0.0276 vc/dg). Tissues collected at necropsy on Day 35 demonstrated quantifiable VCN signal in spleen, lymph node, CD8+ splenocyte and lymphocyte, draining lymph node, heart ventricle, and injection site, for animals dosed at 4 x 10 9 IU/kg. One of 4 animals dosed 1 x 10 9 IU/kg showed quantifiable VCN in spleen, CD8+ splenocyte, and injection site. Flow cytometric analysis of peripheral blood of mice treated with SG299 showed a dose dependent increase in CAR T cells, and the CAR T cells were CD8+ T cells specific with no CAR T cell detection amongst the CD4+ T cells. Collectively, these studies demonstrate high on target specificity of SG299 fusosome to transduce CD8+ T cells in vitro and in vivo. Therefore, SG299 represents a novel therapeutic opportunity to generate CAR T cells in vivo and potentially overcome challenges associated with ex vivo CAR T therapies.

Discovery of BT-114143, a Novel and Potent Small-Molecule Plasminogen Inhibitor for Hyperfibrinolysis

Blood coagulation and fibrinolysis are essential physiological functions, whose dysregulation is a life-threatening emergent situation requiring immediate medical intervention. In particular, hyperfibrinolysis-triggered bleeding occurs in certain medical conditions such as postpartum hemorrhage (PPH) or during perioperative procedures, natural disasters, traumatic accidents as well as battlefield situations, and is one of the major causes of mortalities worldwide during peacetime and war. Tranexamic acid (TXA) has been widely used as an antifibrinolytic drug since 1960s, however, the low potency of TXA has limited its clinical applications. Here, we have developed BT-114143, a novel and potent small-molecule plasminogen inhibitor. BT-114143 exhibited over four-fold higher affinity for recombinant human plasminogen than TXA in surface plasmon resonance (SPR) analysis. Similarly, affinity of BT-114143 was at least one magnitude higher than that of either epsilon-aminocaproic acid (EACA) or p-aminomethylbenzoic acid (PAMBA) (Table 1). In vivo efficacy studies were conducted in recombinant tissue plasminogen activator (rt-PA)-triggered rat tail bleeding model and rabbit liver bleeding model by hepatectomy. BT-114143 reduced bleeding time dose-dependently in both animal models, but also exhibited similar potency as compared to TXA. Similar potency between BT-114143 and TXA was further confirmed by rt-PA triggered fibrinolysis in either rat or rabbit whole blood in thromboelastography (TEG) assay, suggesting interspecies difference in response to bleeding. However, ex vivo studies on human plasma clot lysis assay and human whole blood TEG assay (Figure 1) demonstrated nearly ten-fold antifibrinolytic potency as compared to TXA, suggesting superior efficacy for treatment of hyperfibrinolysis-related bleedings. In addition, a panel of GLP toxicology studies has confirmed the preclinical safety of BT-114143. Moreover, electrophysiological analysis has demonstrated comparable inhibitory effect of BT-114143 and TXA on GABA A receptor and Glycine receptor. At present, BT-114143 is undergoing phase I clinical study in China (CTR20222910) and is expected to apply for FDA approval for clinical trial in 2024. Furthermore, BT-114143 is being developed for intramuscular (i.m.) injection, making it more suitable for emergency situations including PPH, traumatic injuries, natural disasters, and combat injuries.

Safety profile of herbal medicines submitted for marketing authorization in Tanzania: a cross-sectional retrospective study

BackgroundThe popular use of herbal medicines necessitates national regulatory authorities to have efficient mechanisms for the control of these products including marketing authorization (MA) and safety follow-up. Herbal medicines like conventional medicines require assessment of efficacy, safety and quality information before MA can be granted. However, the complete proof of safety is mainly based on the history of the long-term traditional use. Herbal medicines can cause adverse reactions due to various factors and thus require clinical trials to ensure their safety. Herbal medicines treatment practices involve combinations of different plants to achieve the desired effect while multiple herbal components have been known to cause herbal–herbal toxicity and interactions due to variety of complex active ingredients in plants. Compliance with regulatory requirements on herbal medicines has been shown to be difficult for manufacturers since different countries have different regulatory requirements with wide variations which results in the MA of very few herbal medicines. Limited studies on dossiers of marketing authorization of herbal medicines have been performed in other countries, with no studies in African regulatory system settings. The aim of this study is to determine the type of safety documentation that is submitted on herbal medicines application dossiers to support MA in Tanzania.MethodsA cross-sectional retrospective study of herbal medicines dossiers submitted at the Tanzania Medicines and Medical Devices Authority from 2009 to 2020 was conducted.ResultsAs many as 75% of the herbal products applications were combination products made by more than one herbal substance or plant. Out of 84 dossiers subjected to analysis the majority did not provide evidence of preclinical (55%) and clinical safety data (68%). Evidence of safety data in humans was mostly from the literature (70%) and not manufacturers’ clinical studies. Quality parameters with safety implications were not included in 48% and 23% of the active herbal substance and finished product specifications, respectively.ConclusionAnalysis of the herbal medicine dossiers submitted showed major deficiencies of safety data to support MA. Manufactures need to provide evidence to support the safety of their products for evidence-based regulatory decisions and to avoid multiple reviews of the applications.

Preclinical safety evaluation of continuous UV-A lighting in an operative setting.

Germicidal ultraviolet (UV-C) light has been shown as an effective modality for disinfection in laboratory settings and in the operative room. Traditionally, short-wavelength UV-C devices, which have previously been shown to cause DNA damage, are utilized only for disinfection in pre- and post-operative settings and are not continuously active during operations. Continuous use of intraoperative UV light has potential to decrease pathogens and subsequent surgical site infections (SSIs), which arise in approximately 5-15% of operative cases. SSIs are a significant determinant of patient morbidity, readmission rates, and overall cost. Therefore, a method of UV light disinfection with a low risk of DNA damage is needed so that greater antimicrobial protection can be afforded to patients during the entirety of their surgical procedures. A new disinfection device that harnesses longer-wavelength UV-A light to disinfect the surgical field throughout the entirety of the procedure, including pre- and post-operation has been developed. This study aimed to determine if UV-A light administered intraoperatively was safe, as defined by the minimal presence of DNA damage and safe amounts of reflection upon medical personnel. Using in vitro models, we examined the differential impacts of UV-C and UV-A light on DNA damage and repair pathways. In a murine model, we looked at the production of DNA damage photoproduction in relation to UV-A versus UV-C exposure. Our results show UV-A light does not induce a significant amount of DNA damage at the cellular or tissue level. Furthermore, a preclinical porcine study showed that surgical personnel were exposed to safe levels of UV-A irradiance from an overhead UV-A light used during an operation. The amount of UV-A transmitted through surgical personal protective equipment (PPE) also remained within safe levels. In conclusion, we found that UV-A may be safe for intraoperative use.

RBD5044 - a novel anti-ApoC3 GalNAc-siRNA drug resulted in sustained and profound reduction of triglycerides in mice and Rhesus monkeys

Abstract Introduction Hypertriglyceridemia leads to endothelial dysfunction and contributes to the onset and progression of atherosclerosis as well as an increased risk of acute pancreatitis. Inhibition of Apolipoprotein-CIII (APOC3) via the RNA interference mechanism is supposed to reduce plasma Triglycerides (TG) levels with durable and high efficacy. RBD5044, which targets APOC3 mRNA in the liver, is a chemically-modified double-stranded small interfering RNA (siRNA) conjugated with N-acetylgalactosamine (Ribo-GalSTAR). Purpose To investigate the preclinical efficacy and safety profile of RBD5044. Methods Rhesus monkeys with spontaneously developed hypertriglyceridemia were given a single subcutaneous (sc) injection of RBD5044. Humanized APOC3 transgenic mice were treated with a single dose or repeated doses (once every two weeks for three doses, Q2W×3) of RBD5044. Plasma was collected to quantify APOC3 and lipid biomarkers to evaluate preclinical efficacy of RBD5044. SD rats and cynomolgus monkeys were used to evaluate the preclinical toxicology for subcutaneously injected RBD5044, including single-dose toxicity studies and 4-week repeat-dose pivotal toxicology studies. Results In the rhesus monkeys with spontaneous hypertriglyceridemia, the maximum level of inhibition reached 61.2 % in APOC3 and resulted in a TG reduction by 53.3%. The levels of both APOC3 and TG were recovered to baseline till Day 85 after a single dose of 3 mg/kg RBD5044. In humanized APOC3 transgenic mice, RBD5044 achieved an average maximum inhibition level of over 90% for APOC3 and around 90% for TG after both single and repeated doses. The APOC3 and TG levels were recovered on Day 63 after a single dose of 3 mg/kg. On Day 63 after the last repeated dose at 3 mg/kg, the inhibition level remained at 41.6% for APOC3 and 49.4% for TG, hence did not recover to baseline within the study period. RBD5044 also showed reductions in total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels and elevations in high-density lipoprotein cholesterol (HDL-C) levels. Toxicology studies showed there is a sufficient therapeutic margin that supports the clinical development of RBD5044 with NOAEL of 40 mg/kg in rats and 300 mg/kg in monkeys. Conclusions RBD5044 shows high efficacy, long duration and a good safety profile in preclinical studies. The Phase I trial of RBD5044 injection in healthy volunteers has been initiated in Australia.Figure 1Figure 2

Inhibition of Factor XI using a GalNAc-siRNA RBD5049: a novel antithrombotic drug with high potency and long duration

Abstract Introduction Bleeding risk remains a major concern of the existing anticoagulants. Inhibition Factor XI (FXI) using small molecule inhibitors, antibodies or antisense oligonucleotides (ASOs) to inactive intrinsic pathway of coagulation have been developed as a safer treatment option over the last decade. siRNA is a nucleic acid drug with class advantages of high selectivity, long duration and benign safety profiles. The current abstract summarizes key preclinical efficacy and safety data supporting the first-in-human study. Purpose To evaluate efficacy and safety, of a GalNAc-siRNA molecule RBD4059, for development of a novel antithrombotic treatment. Methods In vitro potency of several siRNAs were assessed and screened in HepG2 cells. Upon selection of the lead candidate, the siRNA was conjugated with a GalNAc molecule, using RIBO-GalSTAR liver targeting system. In vivo efficacy of RBD4059 was tested in C57BL/6J mice and cynomolgus non-human primates (NHPs). The antithrombotic effect of RBD4059 was determined in arterial and venous thrombosis models induced by FeCl3. Enoxaparin sodium (Lovenox) with clinical equivalent dose (4 mg/kg) was used as a control. Single- and repeated- dose GLP toxicology studies were performed in both CD-1 mice and NHPs. Results IC50 values of RBD4059 was 0.56 nM HepG2 cells. Dose-dependent and long-lasting effects of RBD4059 on the inhibition of plasma FXI activity (FXIa) were observed in mice and in NHPs. RBD4059 at a single dose of 9 mg/kg achieved a maximum of 89% plasma FXIa reduction and 1.38-fold APTT prolongation in mice, and the inhibitory effects could be maintained until Day 64. RBD4059 at 3 mg/kg or 9 mg/kg significantly prevented flow reduction due to FeCl3-induced thrombosis in both arterial and venous thrombosis models, and efficacy was superior than enoxaparin. In NHPs, the inhibitory effects of FXIa were maintained until Day 71 with the maximum effect of 77% FXIa reduction and 1.34-fold APTT prolongation. GLP toxicology studies showed good safety profile without any signs of increased bleeding despite supra-physiological doses and the NOAEL was considered 600 mg/kg in mice and 400 mg/kg in NHPs. Conclusions RBD4059 has demonstrated high potency/efficacy and long duration regarding FXI inhibition and with large safety margin. As the first siRNA based molecule targeting FXI, RBD4059 has now entered Phase 1 trial in human healthy volunteers.Figure 1

A novel FOXP3 knockout-humanized mouse model for pre-clinical safety and efficacy evaluation of Treg-like cell products

Forkhead box P3 (FOXP3) is an essential transcription factor for regulatory T cell (Treg) function. Defects in Tregs mediate many immune diseases including the monogenic autoimmune disease immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), which is caused by FOXP3 mutations. Treg-cell products are a promising modality to induce allograft tolerance or reduce the use of immunosuppressive drugs to prevent rejection, as well as in the treatment of acquired autoimmune diseases. We have recently opened a Phase I clinical trial for IPEX patients using autologous engineered Treg-like cells, CD4LVFOXP3. To facilitate the preclinical studies, a novel humanized-mouse (hu-mouse) model was developed whereby immune-deficient mice were transplanted with human hematopoietic stem progenitor cells (HSPCs) in which the FOXP3 gene was knocked out (FOXP3KO) using CRISPR/Cas9. Mice transplanted with FOXP3KO HSPCs had impaired survival, developed lymphoproliferation 10-12 weeks post-transplant and T-cell infiltration of the gut, resembling human IPEX. Strikingly, injection of CD4LVFOXP3 into the FOXP3KO hu-mice restored in vivo regulatory functions including control of lymphoproliferation and inhibition of T cell infiltration in the colon. This humanized-mouse disease model can be reproducibly established and constitutes an ideal model to assess preclinical efficacy of human Treg cell investigational products.

Ibrutinib combination therapy for advanced gastrointestinal and genitourinary tumours: results from a phase 1b/2 study

BackgroundIbrutinib, a first-in-class inhibitor of Bruton’s tyrosine kinase, is approved for the treatment of various B-cell malignancies and chronic graft-versus-host disease. Based on encouraging preclinical data, safety and efficacy of ibrutinib combined with companion drugs for advanced renal cell carcinoma (RCC), gastric/gastroesophageal junctional adenocarcinoma (GC), and colorectal adenocarcinoma (CRC) were evaluated.MethodsIbrutinib 560 mg or 840 mg once daily was administered with standard doses of everolimus for RCC, docetaxel for GC, and cetuximab for CRC. Endpoints included determination of the recommended phase 2 dose (RP2D) of ibrutinib in phase 1b and efficacy (overall response rate [ORR] for GC and CRC; progression-free survival [PFS] for CRC) in phase 2.ResultsA total of 39 (RCC), 46 (GC), and 50 (RCC) patients were enrolled and received the RP2D. Safety profiles were consistent with the individual agents used in the study. Confirmed ORRs were 3% (RCC), 21% (GC), and 19% (CRC). Median (90% CI) PFS was 5.6 (3.9–7.5) months in RCC, 4.0 (2.7–4.2) months in GC, and 5.4 (4.1–5.8) months in CRC.ConclusionsClinically meaningful increases in efficacy were not observed compared to historical controls; however, the data may warrant further evaluation of ibrutinib combinations in other solid tumours.Trial registrationClinicalTrials.gov, NCT02599324.

CLL-1-Targeted Allogeneic CAR-T Cells Exhibit High on-Target Specificity and Potent Cytotoxicity in Preclinical Models of Acute Myeloid Leukemia

Background: CLL-1 is a compelling therapeutic target for acute myeloid leukemia (AML) as it is highly expressed on AML tumor cells and leukemic stem cells, but is not expressed on hematopoietic stem cells. An allogeneic anti-CLL-1 CAR-T cell therapy (CB-012) is in development for relapsed or refractory (r/r) AML. The CAR was generated with a fully human scFv targeting CLL-1 that was selected from a panel of scFvs. CB-012 was engineered with a next-generation Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology to leverage both checkpoint disruption and immune cloaking for potentially improved antitumor activity. Methods: Cas12a chRDNA guides were implemented to generate five genome edits in the manufacture of CB-012. A fully human anti-CLL-1 CAR transgene was site-specifically inserted into the TRAC gene, thereby eliminating TCR expression to reduce graft-versus-host disease. A B2M-HLA-E fusion transgene was inserted into the native B2M gene, preventing expression of all HLA class I antigens except HLA-E, to blunt both T and NK cell-mediated allograft rejection of the CAR-T cells. A knockout of the PDCD1 gene prevented PD-1 receptor expression and thus PD-L1 ligand binding to prolong antitumor activity. This multiplex genome-editing strategy was designed to enhance the antitumor activity of CB-012. In vitro and in vivo studies evaluated specificity for antigen binding, antigen-dependent activity, and preclinical safety assessments. Results: CB-012 CAR-T cells express a fully human anti-CLL-1 scFv-containing CAR construct and demonstrate potent antigen-dependent cytotoxic activity in human AML cell line co-cultures. In AML xenograft models, a single-dose administration of CB-012 CAR-T cells exerted robust tumor control, leading to significant prolongation of survival. Cell binding studies suggested that the anti-CLL-1 scFv does not exhibit tissue cross-reactivity when examined in the context of a collection of cell types representing vital tissues. In an unbiased cell surface protein microarray, the anti-CLL-1 scFv demonstrated specific interaction with human CLL-1, without detectable non-specific interactions. CB-012 CAR-T cells exhibited limited tissue infiltration and expansion in treatment naïve, immunocompromised mouse models. Conclusion: CB-012 demonstrated highly specific and potent CLL-1-targeted cytolytic activity in vitro and in vivo. Specificity of the anti-CLL-1 scFv was further demonstrated by screening in an unbiased protein-binding study and no adverse safety signals were observed in murine models. These data support advancing the development of CB-012 into a first-in-human clinical trial for patients with r/r AML.

Novel CD123xCD3 Bispecific Igm Antibody, Igm-2537, Potently Induces T-Cell Mediated Cytotoxicity of Acute Myeloid Leukemia Cells In Vivo and in Vitro with Minimal Cytokine Release

Introduction: New therapeutics are needed for the effective treatment of acute myeloid leukemia (AML), as standard chemotherapeutics are poorly tolerated and ~50% of patients relapse primarily due to the incomplete elimination of leukemia stem cells (LSCs). CD123, the IL-3Rα chain, is highly expressed on leukemic blasts and LSCs, and is further increased in patients with poor prognostic factors. Bispecific T-cell engager (TCE) antibodies and CAR-T cells targeting CD123 have shown promising clinical efficacy in AML patients, but cytokine release syndrome limits their therapeutic window and remains a major safety concern. IGM-2537 is a novel pentameric IgM bispecific TCE antibody engineered with ten anti-CD123 binding sites, and an anti-CD3ε single chain Fv domain fused to the joining chain to engage T-cells. Here, we report that IGM-2537 not only potently induces T-cell mediated cytolytic killing of AML cells in vitro and in vivo but also demonstrates minimal cytokine release, and little to no effect on normal hematopoietic progenitor cells and human vascular endothelial cells (HUVECs) with in vitro and ex vivo studies. Additionally, an improved tolerability, excellent pharmacodynamic (PD) response and lower cytokine release was observed with a cynomolgus cross-reactive IgM TCE in monkeys. Materials and Methods:Surface plasma resonance (SPR), cell membrane protein array, and alanine scanning were utilized to characterize human CD123 binding affinity, specificity and epitope mapping, respectively. T-cell dependent cellular cytotoxicity (TDCC) assays were employed to evaluate the potency of IGM-2537 target cytotoxicity, T cell activation and cytokine release. Ex vivo AML colony formation assays were used to assess the activity on AML blasts. Human MV-4-11 and MOLM-13 xenograft tumor models in humanized NSG dKO mice were utilized to determine in vivo anti-tumor activity of IGM-2537. Healthy human PBMCs and bone marrow (BM) samples as well as HUVECs were used in ex vivo assays to address potential safety concerns of IGM-2537 mediated effects on hematopoietic progenitor cells and HUVECs, respectively. Lastly, to confirm that a CD123 directed IgM-based TCE molecule can potentially provide better safety profile in vivo, a cynomolgus-cross reactive IgM TCE comparator was assessed in monkeys for tolerability and PK/PD responses. Results and Conclusions: IGM-2537 bound with high affinity and avidity to CD123. In vitro, IGM-2537 engaged both CD123 and CD3 to induce potent T-cell activation and T-cell mediated cytotoxicity of CD123 + AML cells, and autologous CD123 + basophils and plasmacytoid dendritic cells (pDCs). Though IGM-2537 demonstrated comparable maximal killing activity to a comparator IgG TCE, IGM-2537 demonstrated minimal cytokine release. In ex vivo patient-derived AML or normal BM colony formation assays, IGM-2537 eliminated AML colony forming cells at physiologically relevant effector/target (E/T) ratios but had little or no effect on normal hematopoietic progenitors. In addition, IGM-2537 demonstrated no cytotoxicity against HUVECs regardless of their CD123 expression at basal levels or at maximal levels upregulated by TNF-a and IFN-g, in contrast to a comparator IgG TCE. In vivo, IGM-2537 completely inhibited tumor growth in two AML xenograft tumor models, a subcutaneous MV-4-11 model, and a systemic MOLM-13 model in humanized NSG dKO mice. To further evaluate the potency and safety of the CD123xCD3 IgM bispecific TCE format in vivo, a cynomolgus cross-reactive CD123xCD3 IgM bispecific TCE was evaluated for tolerability and PD responses in cynomolgus monkeys using single doses. All animals tolerated the cross-reactive bispecific IgM TCE well at doses up to 10 mg/kg (the maximal dose level evaluated), a dose 100-fold greater than published doses of a comparator IgG TCE. Complete depletion of CD123 + basophils and substantial reductions of pDCs were seen in blood and BM with minimal to no cytokine induction. In summary, IGM-2537 demonstrated potent in vitro and in vivo T-cell mediated cytotoxicity of AML cell lines with minimal cytokine induction. The encouraging preclinical safety profiles were further supported by little/no effect on normal hematopoietic progenitor cells and HUVECs. These preclinical data support the clinical development of IGM-2537 for the treatment of AML and other hematological malignancies.

The CathPilot: Performance Validation and Preclinical Safety and Feasibility Assessment.

Peripheral endovascular revascularization procedures often fail due to technical limitations of guidewire support, steering, and visualization. The novel CathPilot catheter aims to address these challenges. This study assesses the safety and feasibility of the CathPilot and compares its performance to conventional catheters for peripheral vascular interventions. The study compared the CathPilot to non-steerable and steerable catheters. The success rates and access times for a relevant target inside a tortuous vessel phantom model were assessed. The reachable workspace within the vessel and the guidewire's force delivery capabilities were also evaluated. To validate the technology, chronic total occlusion tissue samples were used ex vivo to compare crossing success rates with conventional catheters. Finally, in vivo experiments in a porcine aorta were conducted to evaluate safety and feasibility. The success rates for reaching the set targets were 31%, 69%, and 100% with the non-steerable catheter, the steerable catheter, and the CathPilot, respectively. CathPilot had a significantly larger reachable workspace, and allowed for up to four times higher force delivery and pushability. In crossing of chronic total occlusion samples, the CathPilot achieved a success rate of 83% and 100%, for fresh and fixed lesions respectively, which was also significantly higher than conventional catheters. The device was fully functional in the in vivo study, and there were no signs of coagulation or damage to the vessel wall. This study shows the safety and feasibility of the CathPilot system and its potential to reduce failure and complication rates in peripheral vascular interventions. The novel catheter outperformed conventional catheters in all defined metrics. This technology can potentially improve the success rate and outcome of peripheral endovascular revascularization procedures.

Preclinical long-term safety of intraspinal transplantation of human dorsal spinal GABA neural progenitor cells

Human pluripotent stem cell (hPSC)-derived neurons have shown promise in treating spinal cord injury (SCI). We previously showed that hPSC-derived dorsal spinal γ-aminobutyric acid (GABA) neurons can alleviate spasticity and promote locomotion in rats with SCI, but their long-term safety remains elusive. Here, we characterized the long-term fate and safety of human dorsal spinal GABA neural progenitor cells (NPCs) in naive rats over one year. All grafted NPCs had undergone differentiation, yielding mainly neurons and astrocytes. Fully mature human neurons grew many axons and formed numerous synapses with rat neural circuits, together with mature human astrocytes that structurally integrated into the rat spinal cord. The sensorimotor function of rats was not impaired by intraspinal transplantation, even when human neurons were activated or inhibited by designer receptors exclusively activated by designer drugs (DREADDs). These findings represent a significant step toward the clinical translation of human spinal neuron transplantation for treating SCI.

Discovery of MK-1468: A Potent, Kinome-Selective, Brain-Penetrant Amidoisoquinoline LRRK2 Inhibitor for the Potential Treatment of Parkinson's Disease.

Genetic mutation of the leucine-rich repeat kinase 2 (LRRK2) protein has been associated with Parkinson's disease (PD), a disabling and progressive neurodegenerative disorder that is devoid of efficacious disease-modifying therapies. Herein, we describe the invention of an amidoisoquinoline (IQ)-derived LRRK2 inhibitor lead chemical series. Knowledge-, structure-, and property-based drug design in concert with rigorous application of in silico calculations and presynthesis predictions enabled the prioritization of molecules with favorable CNS "drug-like" physicochemical properties. This resulted in the discovery of compound 8, which was profiled extensively before human ether-a-go-go (hERG) ion channel inhibition halted its progression. Strategic reduction of lipophilicity and basicity resulted in attenuation of hERG ion channel inhibition while maintaining a favorable CNS efflux transporter profile. Further structure- and property-based optimizations resulted in the discovery of preclinical candidate MK-1468. This exquisitely selective LRRK2 inhibitor has a projected human dose of 48 mg BID and a preclinical safety profile that supported advancement toward GLP toxicology studies.

In Vitro Wear of a Novel Vitamin E Crosslinked Polyethylene Lumbar Total Joint Replacement.

A novel, lumbar total joint replacement (TJR) design has been developed to treat degeneration across all three columns of the lumbar spine (anterior, middle, and posterior columns). Thus far, there has been no in vitro studies that establish the preclinical safety profile of the vitamin E-stabilized highly crosslinked polyethylene (VE-HXLPE) lumbar TJR relative to historical lumbar anterior disc replacement for the known risks of wear and impingement faced by all motion preserving designs for the lumbar spine. In this study we asked, (1) what is the wear performance of the VE-HXLPE lumbar TJR under ideal, clean conditions? (2) Is the wear performance of VE-HXLPE in lumbar TJR sensitive to more aggressive, abrasive conditions? (3) How does the VE-HXLPE lumbar TJR perform under impingement conditions? A lumbar TJR with bilateral VE-HXLPE superior bearings and CoCr inferior bearings was evaluated under clean, impingement, and abrasive conditions. Clean and abrasive testing were guided by ISO 18192-1 and impingement was assessed as per ASTM F3295. For abrasive testing, CoCr components were scratched to simulate in vivo abrasion. The devices were tested for 10 million cycles (MC) under clean conditions, 5 MC under abrasion, and 1 MC under impingement. Wear rates under clean and abrasive conditions were 1.2 ± 0.5 and 1.1 ± 0.6 mg/MC, respectively. The VE-HXLPE components demonstrated evidence of burnishing and multidirectional microscratching consistent with microabrasive conditions with the cobalt chromium spherical counterfaces. Under impingement, the wear rates ranged between 1.7 ± 1.1 (smallest size) and 3.9 ± 1.1 mg/MC (largest size). No functional or mechanical failure was observed across any of the wear modes. Overall, we found that that a VE-HXLPE-on-CoCr lumbar total joint replacement design met or exceeded the benchmarks established by traditional anterior disc replacements, with wear rates previously reported in the literature ranging between 1 and 15 mg/MC. The potential clinical benefits of this novel TJR design, which avoids long-term facet complications through facet removal with a posterior approach, were found to be balanced by the in vitro tribological performance of the VE-HXLPE bearings. Our encouraging in vitro findings have supported initiating an FDA-regulated clinical trial for the design which is currently under way.

Physiological oxygen measurements in vitro-Schrödinger's cat in 3D cell biology.

After the development of 3D cell culture methods in the middle of the last century and the plethora of data generated with this culture configuration up to date, it could be shown that a three-dimensional arrangement of cells in most of the cases leads to a more physiological behavior of the generated tissue. However, a major determinant for an organotypic function, namely, the dissolved oxygen concentration in the used in vitro-system, has been neglected in most of the studies. This is due to the fact that the oxygen measurement in the beginning was simply not feasible and, if so, disturbed the measurement and/or the in vitro-system itself. This is especially true for the meanwhile more widespread use of 3D culture systems. Therefore, the tissues analyzed by these techniques can be considered as the Schrödinger's cat in 3D cell biology. In this perspective paper we will outline how the measurement and, moreover, the regulation of the dissolved oxygen concentration in vitro-3D culture systems could be established at all and how it may be possible to determine the oxygen concentration in organoid cultures and the respiratory capacity via mito stress tests, especially in spheroids in the size range of a few hundred micrometers, under physiological culture conditions, without disturbances or stress induction in the system and in a high-throughput fashion. By this, such systems will help to more efficiently translate tissue engineering approaches into new in vitro-platforms for fundamental and applied research as well as preclinical safety testing and clinical applications.

Development of a Single-Neurosphere Culture to Assess Radiation Toxicity and Pre-Clinical Cancer Combination Therapy Safety.

Radiation therapy (RT) is a crucial treatment modality for central nervous system (CNS) tumors but toxicity to healthy CNS tissues remains a challenge. Additionally, environmental exposure to radiation during nuclear catastrophes or space travel presents a risk of CNS toxicity. However, the underlying mechanisms of radiation-induced CNS toxicity are not fully understood. Neural progenitor cells (NPCs) are highly radiosensitive, resulting in decreased neurogenesis in the hippocampus. This study aimed to characterize a novel platform utilizing rat NPCs cultured as 3D neurospheres (NSps) to screen the safety and efficacy of experimental drugs with and without radiation exposure. The effect of radiation on NSp growth and differentiation was assessed by measuring sphere volume and the expression of neuronal differentiation markers Nestin and GFAP and proliferation marker Ki67. Radiation exposure inhibited NSp growth, decreased proliferation, and increased GFAP expression, indicating astrocytic differentiation. RNA sequencing analysis supported these findings, showing upregulation of Notch, BMP2/4, S100b, and GFAP gene expression during astrogenesis. By recapitulating radiation-induced toxicity and astrocytic differentiation, this single-NSp culture system provides a high-throughput preclinical model for assessing the effects of various radiation modalities and evaluates the safety and efficacy of potential therapeutic interventions in combination with radiation.

Safety, immunogenicity and efficacy of Relcovax®, a dual receptor binding domain (RBD) and nucleocapsid (N) subunit protein vaccine candidate against SARS-CoV-2 virus

SARS-CoV-2, severe acute respiratory syndrome coronavirus-2, causes coronavirus disease- 2019 (COVID-19). Mostly, COVID-19 causes respiratory symptoms that can resemble those of a cold, the flu, or pneumonia. COVID-19 may harm more than just lungs and respiratory systems. It may also have an impact on other parts of the body and debilitating effects on humans, necessitating the development of vaccines at an unprecedented rate in order to protect humans from infections. In response to SARS-CoV-2 infection, mRNA, viral vector-based carrier and inactivated virus-based vaccines, as well as subunit vaccines, have recently been developed. We developed Relcovax®, a dual antigen (Receptor binding domain (RBD) and Nucleocapsid (N) proteins) subunit protein vaccine candidate. Preliminary mouse preclinical studies revealed that Relcovax® stimulates cell-mediated immunity and provides broader protection against two SARS-CoV-2 variants, including the delta strain. Before conducting human studies, detailed preclinical safety assessments are required, so Relcovax® was tested for safety, and immunogenicity in 28-day repeated dose toxicity studies in rats and rabbits. In the toxicity studies, there were no mortality or morbidity, abnormal clinical signs, abnormalities in a battery of neurobehavioral observations, abnormalities in detailed clinical and ophthalmological examinations, or changes in body weights or feed consumption. In any of the studies, no abnormal changes in organ weights, haematology, clinical chemistry, urinalysis parameters, or pathological findings were observed. Immunogenicity tests on rats and rabbits revealed 100 % seroconversion. Relcovax® was therefore found to be safe in animals, with a No Observed Adverse Effect Level (NOAEL) of 20 µg/protein in rats and rabbits. In efficacy studies, Relcovax® immunised hamsters demonstrated dose-dependent protection against SARS-CoV-2 infection, with a high dose (20 µg/protein) being the most protective, while in cynomolgus macaque monkey study, lowest dose 5 µg/protein had the highest efficacy. In conclusion, Relcovax® was found to be safe, immunogenic, and efficacious in in vivo studies.

Safety Profile of a New Dimethylaminoethanol Derivative by Oral Administration to Laboratory Animals

A new derivative of dimethylaminoethanol, butanedioic and trans-butenedioic acids (laboratory code ADK-17) was synthesized at the Department of Organic Chemistry (Professor I.P. Yakovlev is the Head of the Department) of St. Petersburg State Chemical and Pharmaceutical University (SPCPU). This is a promising compound planned for use as an oral dosage form. In this work, we aim to evaluate manifestations of the general and specific toxicity of the new drug. Laboratory animals (white mice, rats, rabbits, guinea pigs) were used as test systems for the preclinical safety study of the new compound. Manifestations of general toxicity (acute and chronic), local irritant action, allergenic properties, immunotoxic action were studied. The reproductive toxicity of the ADK-17 drug when administered intragastrically was studied. The studied drug was found to exhibit low toxicity, cause no changes in the biochemical and morphological parameters of rats and rabbits under the conditions of course use, and have no negative effects on internal organs. When administered intragastrically in maximum doses, the drug causes no irritating effects. The use of the drug did not lead to the development of a local allergic reaction of an immediate type. Carrying out sensitization in the DTH reaction showed the absence of a sensitizing effect. The study of the immunotoxic effect of the ADK-17 drug showed that its prolonged intragastric administration for 30 days at doses of 93 and 930 mg/kg did not lead to a violation of the humoral immune response and a decrease in antibody production. Setting a delayed-type hypersensitivity reaction also confirms that the drug does not affect the development of cellular immunity. The conducted experimental studies of the drug’s reproductive toxicity in rats showed that its repeated intragastric administration at doses of 50 and 250 mg/kg to pregnant females did not have a negative effect on the reproductive system of laboratory animals, embryo- and fetotoxic, as well as teratogenic effects, having no effect on the antenatal and postnatal development of offspring.

Preclinical In-Vivo Safety and Toxicokinetics of D-PLEX100 in an Abdominal Surgery Incision Model in Juvenile Miniature Swine.

D-PLEX100 (D-PLEX) is a novel product candidate made of a polymer-lipid-based matrix (PLEX platform) which contains doxycycline that is being released at a constant rate for 30days. D-PLEX was developed to prevent surgical site infections, which are a major global health challenge. Previous studies have shown its safety in adult humans, adult swine, and adult rabbits. The aim of this study was to assess the toxicity and safety of D-PLEX also in juvenile animals to support future clinical trials in pediatric patients. Yucatan miniature swine were selected as a model, primarily due to their relatively larger mass. D-PLEX or placebo (formulation without doxycycline) was administered locally to abdominal incisions, and the animal's safety parameters were followed for 9months and compared to sham-control swine. There was no evidence of any systemic safety concern or local toxicity at the incision site in D-PLEX-treated animals. D-PLEX was detected after 1month and was fully resorbed at the 3-month time point. The surgical incision sites were fully healed at the 6-month time point in all D-PLEX-treated animals. Toxicokinetic (TK) assessments revealed that doxycycline exhibited low Cmax and therefore minimal systemic exposure following a single dose of local administration. This study provides evidence for the safety of D-PLEX and PLEX-based formulation in juvenile miniature swine and supports its further testing in clinical pediatric population. In addition, it can be used as a reference for future preclinical studies aiming to evaluate the safety of other PLEX-based product candidates for the pediatric population.