Broad Dose Range Research Articles

Considering developmental neurotoxicity invitro data for human health risk assessment using physiologically-based kinetic modeling: deltamethrin case study.

Developmental neurotoxicity (DNT) is a potential hazard of chemicals. Recently, an in vitro testing battery (DNT IVB) was established to complement existing rodent in vivo approaches. Deltamethrin (DLT), a pyrethroid with a well-characterized neurotoxic mode of action, has been selected as a reference chemical to evaluate the performance of the DNT IVB. The present study provides context for evaluating the relevance of these DNT IVB results for the human health risk assessment of DLT by estimating potential human fetal brain concentrations after maternal exposure to DLT. We developed a physiologically based kinetic (PBK) model for rats which was then translated to humans considering realistic in vivo exposure conditions (acceptable daily intake [ADI] for DLT). To address existing uncertainties, we designed case studies considering the most relevant drivers of DLT uptake and distribution. Calculated human fetal brain concentrations were then compared with the lowest benchmark concentration achieved in the DNT IVB. The developed rat PBK model was validated on in vivo rat toxicokinetic data of DLT over a broad range of doses. The uncertainty based case study evaluation confirmed that repeated exposure to DLT at an ADI level would likely result in human fetal brain concentrations far below the in vitro benchmark. The presented results indicate that DLT concentrations in the human fetal brain are highly unlikely to reach concentrations associated with in vitro findings under realistic exposure conditions. Therefore, the new in vitro DNT results are considered to have no impact on the current risk assessment approach.

OCTOPOD: single-bunch tomography for angular-spectral characterization of laser-driven protons

Abstract Laser–plasma accelerated (LPA) proton bunches are now applied for research fields ranging from ultra-high-dose-rate radiobiology to material science. Yet, the capabilities to characterize the spectrally and angularly broad LPA bunches lag behind the rapidly evolving applications. The OCTOPOD translates the angularly resolved spectral characterization of LPA proton bunches into the spatially resolved detection of the volumetric dose distribution deposited in a liquid scintillator. Up to 24 multi-pinhole arrays record projections of the scintillation light distribution and allow for tomographic reconstruction of the volumetric dose deposition pattern, from which proton spectra may be retrieved. Applying the OCTOPOD at a cyclotron, we show the reliable retrieval of various spatial dose deposition patterns and detector sensitivity over a broad dose range. Moreover, the OCTOPOD was installed at an LPA proton source, providing real-time data on proton acceleration performance and attesting the system optimal performance in the harsh laser–plasma environment.

SPECT/CT-based dosimetry of salivary glands and iodine-avid lesions following 131I therapy

The purpose was to provideuptake and radiation dose estimates to salivary glands (SG) and pathologic lesions following radioiodine therapy (RIT)of differentiated thyroid cancer patients (DTC). A group of DTCpatients (n = 25) undergoing 131I therapy joined this study with varying amounts of therapeuticactivity. SequentialSPECT/CT scanswere acquiredat 4 ± 2, 24 ± 2, and 168 ± 3h following administration of 3497-9250MBq131I. An earlier experiment withAcrylic glass body phantom (PET Phantom NEMA 2012 / IEC 2008) was conducted for system calibration includingscatter, partial volume effect and count loss correction.Dose calculation was made via IDAC-Dose 2.1 code. The absorbed dose to parotid glands was 0.04-0.97Gy/GBq (median: 0.26Gy/GBq). The median absorbed dose to submandibular glands was 0.14Gy/GBq (0.05 to 0.56Gy/GBq). The absorbed dose to thyroid residues was from 0.55 to 399.5Gy/GBq (median: 21.8Gy/GBq), and that to distal lesions ranged from 0.78 to 28.0Gy/GBq (median: 3.12Gy/GBq). 41% of the thyroid residues received dose > 80Gy, 18% between 70-80Gy, 18% between 40-70Gy, and 23% has dose < 40Gy. In contrast, 18% of the metastases exhibited a dose > 80Gy, 9% between 40-60Gy, and the dose to the vast majority of lesions (64%) was < 40Gy. It was inferred that dose estimation after RIT with SPECT/CT is feasible to apply, together with good agreement with published 124I PET/CT dose estimates. A broad and sub-effective dose range was estimated for thyroid residues and distal lesions. Moreover, the current methodology might be useful for establishing a dose-effect relationship and radiation-induced salivary glands damage after RIT.

Endorectal contact radiation boosting: Making the case for dose AND volume reporting

IntroductionThe various rectal endoluminal radiation techniques all have steep, but different, dose gradients. In rectal contact brachytherapy (CXB) doses are typically prescribed and reported to the applicator surface and not to the gross tumor volume (GTV), clinical target volume (CTV) or organs at risk (OAR), which is crucial to understand tumor response and toxicity rates. To quantify the above-described problem, we performed a dose modeling study using a fixed prescription dose at the surface of the applicator and varied tumor response scenarios. MethodsEndorectal ultrasound-based 3D-volume-models of rectal tumors and the rectal wall were used to simulate the delivered dose to GTV, CTV and the rectal wall layers, assuming treatment with Maastro HDR contact applicator for rectal cancer with a fixed prescription dose to the applicator surface (equivalent to 3 × 30 Gy CXB) and various response scenarios. ResultsAn identical prescribed dose to the surface of the applicator resulted in a broad range of doses delivered to the GTV, CTV and the uninvolved intestinal wall. For example, the equieffective dose in 2 Gy per fraction (EQD2) D90% of the GTV varied between 63 and 231 Gy, whereas the EQD2 D2cc of the rectal wall varied between 97 and 165 Gy. ConclusionDoses prescribed at the surface are not representative of the dose received by the tumor and the bowel wall. This stresses the relevance of dose reporting and prescription to GTV and CTV volumes and OAR in order to gain insight between delivered dose, local control and toxicity and to optimize treatment protocols.

Development and External Validation of a Machine Learning Model for the Early Prediction of Doses of Harmful Intracranial Pressure in Patients with Severe Traumatic Brain Injury.

Treatment and prevention of elevated intracranial pressure (ICP) is crucial in patients with severe traumatic brain injury (TBI). Elevated ICP is associated with secondary brain injury, and both intensity and duration of an episode of intracranial hypertension, often referred to as "ICP dose," are associated with worse outcomes. Prediction of such harmful episodes of ICP dose could allow for a more proactive and preventive management of TBI, with potential implications on patients' outcomes. The goal of this study was to develop and validate a machine-learning (ML) model to predict potentially harmful ICP doses in patients with severe TBI. The prediction target was defined based on previous studies and included a broad range of doses of elevated ICP that have been associated with poor long-term neurological outcomes. The ML models were used, with minute-by-minute ICP and mean arterial blood pressure signals as inputs. Harmful ICP episodes were predicted with a 30 min forewarning. Models were developed in a multi-center dataset of 290 adult patients with severe TBI and externally validated on 264 patients from the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) dataset. The external validation of the prediction model on the CENTER-TBI dataset demonstrated good discrimination and calibration (area under the curve: 0.94, accuracy: 0.89, precision: 0.87, sensitivity: 0.78, specificity: 0.94, calibration-in-the-large: 0.03, calibration slope: 0.93). The proposed prediction model provides accurate and timely predictions of harmful doses of ICP on the development and external validation dataset. A future interventional study is needed to assess whether early intervention on the basis of ICP dose predictions will result in improved outcomes.

Epac2 in midbrain dopamine neurons contributes to cocaine reinforcement via enhancement of dopamine release.

Repeated exposure to drugs of abuse results in an upregulation of cAMP signaling in the mesolimbic dopamine system, a molecular adaptation thought to be critically involved in the development of drug dependence. Exchange protein directly activated by cAMP (Epac2) is a major cAMP effector abundantly expressed in the brain. However, it remains unknown whether Epac2 contributes to cocaine reinforcement. Here, we report that Epac2 in the mesolimbic dopamine system promotes cocaine reinforcement via enhancement of dopamine release. Conditional knockout of Epac2 from midbrain dopamine neurons (Epac2-cKO) and the selective Epac2 inhibitor ESI-05 decreased cocaine self-administration in mice under both fixed-ratio and progressive-ratio reinforcement schedules and across a broad range of cocaine doses. In addition, Epac2-cKO led to reduced evoked dopamine release, whereas Epac2 agonism robustly enhanced dopamine release in the nucleus accumbens in vitro. This mechanism is central to the behavioral effects of Epac2 disruption, as chemogenetic stimulation of ventral tegmental area (VTA) dopamine neurons via deschloroclozapine (DCZ)-induced activation of Gs-DREADD increased dopamine release and reversed the impairment of cocaine self-administration in Epac2-cKO mice. Conversely, chemogenetic inhibition of VTA dopamine neurons with Gi-DREADD reduced dopamine release and cocaine self-administration in wild-type mice. Epac2-mediated enhancement of dopamine release may therefore represent a novel and powerful mechanism that contributes to cocaine reinforcement.

Benchmark dose modeling of transcriptional data: a systematic approach to identify best practices for study designs used in radiation research

Purpose Benchmark dose (BMD) modeling is a method commonly used in chemical toxicology to identify the point of departure (POD) from a dose-response curve linked to a health-related outcome. Recently, its application in the analysis of transcriptional data for quantitative adverse outcome pathway (AOP) development is being explored. As AOPs are informed by diverse data types, it is important to understand the impact of study parameters such as dose selection, the number of replicates and dose range on BMD outputs for radiation-induced genes and pathways. Materials and methods Data were selected from the Gene Expression Omnibus (GSE52403) that featured gene expression profiles of peripheral blood samples from C57BL/6 mice 6 hours post-exposure to 137Cs gamma-radiation at 0, 1, 2, 3, 4.5, 6, 8 and 10.5 Gy. The dataset comprised a broad dose range over multiple dose points with consistent dose spacing and multiple biological replicates. This dataset was ideal for systematically transforming across three categories: (1) dose range, (2) dose-spacing and (3) number of controls/replicates. Across these categories, 29 transformed datasets were compared to the original dataset to determine the impact of each transformation on the BMD outputs. Results Most of the experimental changes did not impact the BMD outputs. The transformed datasets were largely consistent with the original dataset in terms of the number of reproduced genes modeled and absolute BMD values for genes and pathways. Variations in dose selection identified the importance of the absolute value of the lowest and second dose. It was determined that dose selection should include at least two doses <1 Gy and two >5 Gy to achieve meaningful BMD outputs. Changes to the number of biological replicates in the control and non-zero dose groups impacted the overall accuracy and precision of the BMD outputs as well as the ability to fit dose-response models consistent with the original dataset. Conclusion Successful application of transcriptomic BMD modeling for radiation datasets requires considerations of the exposure dose and the number of biological replicates. Most important is the selection of the lowest doses and dose spacing. Reflections on these parameters in experimental design will provide meaningful BMD outputs that could correlate well to apical endpoints of relevance to radiation exposure assessment.

Retention of brivaracetam in adults with drug-resistant epilepsy at a single tertiary care center

IntroductionBrivaracetam (BRV) is licensed as an adjunctive treatment for focal epilepsy. We describe our clinical experience with BRV at a large UK tertiary center. MethodsAdults initiated on BRV between July 2015 and July 2020 were followed up until they discontinued BRV or September 2021. Data on epilepsy syndrome, duration, seizure types, concomitant and previous antiseizure medication (ASM) use, BRV dosing, efficacy, and side effects were recorded. Efficacy was categorized as temporary (minimum three months) or ongoing (at last follow-up) seizure freedom, ≥50% seizure reduction, or other benefits (e.g., no convulsions or daytime seizures). Brivaracetam retention was estimated using Kaplan–Meier survival analysis. ResultsTwo-hundred people were treated with BRV, of whom 81% had focal epilepsy. The mean (interquartile range [IQR]) follow-up time was 707 (688) days, and the dose range was 50–600 mg daily. The mean (IQR) of the previous number of used ASMs was 6.9 (6.0), and concomitant use was 2.2 (1.0). One-hundred and eighty-eight people (94%) had previously discontinued levetiracetam (LEV), mainly due to side effects. 13/200 (6.5%) were seizure free for a minimum of six months during treatment, and 46/200 (23%) had a ≥50% reduction in seizure frequency for six months or more. Retention rates were 83% at six months, 71% at 12 months, and 57% at 36 months. Brivaracetam was mostly discontinued due to side effects (38/75, 51%) or lack of efficacy (28/75, 37%). Concomitant use of carbamazepine significantly increased the hazard ratio of discontinuing BRV due to side effects (p = 0.006). The most commonly reported side effects were low mood (20.5%), fatigue (18%) and aggressive behavior (8.5%). These side effects were less prevalent than when the same individuals took LEV (low mood, 59%; aggressive behavior, 43%). Intellectual disability was a risk factor for behavioral side effects (p = 0.004), and a pre-existing mood disorder significantly increased the likelihood of further episodes of low mood (p = 0.019). ConclusionsBrivaracetam was effective at a broad range of doses in managing drug-resistant epilepsy across various phenotypes, but less effective than LEV in those who switched due to poor tolerability on LEV. There were no new tolerability issues, but 77% of the individuals experiencing side effects on BRV also experienced similar side effects on LEV.

Abstract 3382: Detection of cancer in dogs using a novel genetic-based synthetic biomarker platform

Abstract Early detection of cancer is an important driver of increased survival, quality of life and reduced healthcare costs. Although liquid biopsy provides a new option for early cancer detection, the limitations of detecting a few molecules of blood-based biomarkers naturally shed by the cancer remain. Earli is developing a highly sensitive, orthogonal approach that uses a genetic construct that usurps dysregulated pathways and actively forces cancer cells to drive the expression of a detectable ‘synthetic’ biomarker. We previously used murine xenograft models to validate the tumor detection properties of EARLI-001, a circular DNA containing a human derived cancer-activated promoter to drive the transient expression of secreted embryonic alkaline phosphatase (SEAP), a protein normally expressed only during fetal development. Biodistribution following intravenous (IV) administration of EARLI-001 shows a broad tissue tropism, enabling the potential to monitor multiple tissues for malignant cells. A single dose of EARLI-001 administered in a pulmonary metastatic cancer mouse model resulted in a 95-fold increase in serum SEAP compared to healthy controls. Recognizing the inherent limitations of murine models, we evaluated EARLI-001 in canines diagnosed with cancer. Dog cancers share many features with human malignancies including spontaneous tumor formation, heterogeneity, growth kinetics, histology, and comparable dysregulated genetic pathways. Furthermore, the use of dogs allows the study of EARLI-001 in an immune competent setting and with substantially increased size (up to 65 kg) comparable to scaling into human subjects. EARLI-001 was first tested for safety and biodistribution in 54 purpose-bred beagles in MTD and GLP toxicology studies which reveal that it is safe and well tolerated across a broad range of clinical doses. Biodistribution analysis in 12 tissues showed nanoplasmid levels peaking in the first days post dose and rapidly decreased to background levels by 60 days. These non-tumor bearing dogs showed no serum SEAP signal at all doses. Following IACUC approval and informed owner consent, EARLI-001 was administered to companion dogs in a standard 3 x 3 dose escalation study, with an expanded mid-range cohort. These companion dogs harbored a wide variety of spontaneous tumors in different tissues of origin. In tumor bearing dogs, EARLI-001 elicited a clear and discernable cancer-activated SEAP signal in 1 of 3 dogs dosed at 0.01 mg/kg, 3 of 6 dosed at 0.03 mg/kg, 2 of 2 at 0.07 mg/kg and 8 of 8 dogs dosed at 0.1 mg/kg. Furthermore, the MTD was not reached, and no significant dose-limiting toxicity was observed in these older, disease-burdened animals. These results show the utility of the platform to interrogate tumor biology and detect the presence of malignancies in relevant in vivo models and provided the critical safety and efficacy data towards a first-in-human clinical study in lung cancer initiated in 2021. Citation Format: Regina Nieu, Emily Phenix, Jennifer Hauss, Hadley Hanson, Nga Ho, Alex Harwig, Shireen Rudina, Badri Ananthanarayanan, Hong Chang, Bijee George, Maggie C. Louie, Julie Bulman-Fleming, Michael Kent, David Suhy. Detection of cancer in dogs using a novel genetic-based synthetic biomarker platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3382.

Soybean dose-response to 2,4-D and dicamba at vegetative and reproductive growth stages.

Field experiments were conducted across multiple sites in 2012 and 2013 to describe sensitivity of soybean to 2,4-D (six doses) and dicamba (seven doses) at V3 and R1 growth stages. Further experiments were conducted under greenhouse conditions in 2017 and 2018 to compare soybean response to several dicamba herbicides across a broader range of doses than those tested in the field. Soybean yield loss was 6.1-fold greater from 2,4-D exposure at V3 compared to R1 and 1.4 times greater from dicamba exposure at R1 than at V3. In V3 exposures, soybean was 15.4 times more sensitive to dicamba than 2,4-D and 134.4-fold more sensitive to dicamba when exposed at R1. Plant injury and height correlations to grain yield resulted in coefficients ranging from 0.65 to 0.91. In greenhouse experiments, five dicamba products were tested at up to 19 doses and as low as 0.002 g ae ha-1 (3.6 × 10-6 % of maximum single use-rate); however, no differences were observed among formulations used in dicamba-resistant crops versus traditional formulations. A no observable effects dose was not identified due to responses observed even at the lowest doses tested, although hormesis effects were observed in plant height. These data suggest that the sensitivity of soybean to dicamba is much greater than what has previously been reported. However, as has been indicated by previous work, that injury does not always result in yield loss. © 2022 Society of Chemical Industry.

Biphasic reward effects are characteristic of both lorcaserin and drugs of abuse: implications for treatment of substance use disorders.

Lorcaserin is a modestly selective agonist for 2C serotonin receptors (5-HT2CR). Despite early promising data, it recently failed to facilitate cocaine abstinence in patients and has been compared with dopamine antagonist medications (antipsychotics). Here, we review the effects of both classes on drug reinforcement. In addition to not being effective treatments for cocaine use disorder, both dopamine antagonists and lorcaserin can have biphasic effects on dopamine and reward behavior. Lower doses can cause enhanced drug taking with higher doses causing reductions. This biphasic pattern is shared with certain stimulants, opioids, and sedative-hypnotics, as well as compounds without abuse potential that include agonists for muscarinic and melatonin receptors. Additional factors associated with decreased drug taking include intermittent dosing for dopamine antagonists and use of progressive-ratio schedules for lorcaserin. Clinically relevant doses of lorcaserin were much lower than those that inhibited cocaine-reinforced behavior and can also augment this same behavior in different species. Diminished drug-reinforced behavior only occurred in animals after higher doses that are not suitable for use in patients. In conclusion, drugs of abuse and related compounds often act as biphasic modifiers of reward behavior, especially when evaluated over a broad range of doses. This property may reflect the underlying physiology of the reward system, allowing homeostatic influences on behavior.

Sex- and Dose-Dependent Differences in the Development of an Addiction-Like Phenotype Following Extended-Access Fentanyl Self-Administration.

Opioid use disorder (OUD) is a major epidemic in the United States, and fentanyl is a major culprit. The National Institute on Drug Abuse has highlighted an urgent need for research on the risks and outcomes of OUD with fentanyl; a better understanding of sex/gender differences is also critically needed given that the opioid epidemic has been particularly impactful on women. In response to this need, we developed a rat model of OUD with fentanyl and showed that sex impacts relapse vulnerability following extended-access self-administration under a low fentanyl dose. Here, our goal was to determine sex differences across a broad dose range, including high doses expected to maximize the expression of addiction-like features (e.g., vulnerability to relapse and physical dependence). Male and female rats were assigned to self-administer one of four fentanyl doses (0.25, 0.75, 1.5, and 3.0 µg/kg/infusion), and once they acquired, they were given extended (24-h/day), intermittent access (2, 5 min trials/h, fixed-ratio 1) to fentanyl for 10 days. Physical dependence (spontaneous weight loss) was assessed during early withdrawal, and relapse vulnerability was assessed on withdrawal day 15 using an extinction/cue-induced reinstatement procedure. Despite markedly higher intake in the high- versus low-dose groups, each group responded similarly during relapse testing (extinction and cue-induced reinstatement). However, number of infusions, or frequency of use, during extended access was predictive of later vulnerability to relapse, whereas total intake impacted physical dependence given that weight loss only occurred following the discontinuation of fentanyl self-administration at the three highest doses. Females self-administered more fentanyl each day and within each binge (active trial), and had longer lasting weight loss during withdrawal than males. Relapse vulnerability was also higher in females than males and highest in females tested during estrus. These findings indicate that sex is an important risk factor for patterns and levels of fentanyl intake, relapse, and physical dependence, and while fentanyl intake predicts physical dependence, frequency of use predicts relapse.

Inhaled mosliciguat (BAY 1237592): targeting pulmonary vasculature via activating apo-sGC

BackgroundOxidative stress associated with severe cardiopulmonary diseases leads to impairment in the nitric oxide/soluble guanylate cyclase signaling pathway, shifting native soluble guanylate cyclase toward heme-free apo-soluble guanylate cyclase. Here we describe a new inhaled soluble guanylate cyclase activator to target apo-soluble guanylate cyclase and outline its therapeutic potential.MethodsWe aimed to generate a novel soluble guanylate cyclase activator, specifically designed for local inhaled application in the lung. We report the discovery and in vitro and in vivo characterization of the soluble guanylate cyclase activator mosliciguat (BAY 1237592).ResultsMosliciguat specifically activates apo-soluble guanylate cyclase leading to improved cardiopulmonary circulation. Lung-selective effects, e.g., reduced pulmonary artery pressure without reduced systemic artery pressure, were seen after inhaled but not after intravenous administration in a thromboxane-induced pulmonary hypertension minipig model. These effects were observed over a broad dose range with a long duration of action and were further enhanced under experimental oxidative stress conditions. In a unilateral broncho-occlusion minipig model, inhaled mosliciguat decreased pulmonary arterial pressure without ventilation/perfusion mismatch. With respect to airway resistance, mosliciguat showed additional beneficial bronchodilatory effects in an acetylcholine-induced rat model.ConclusionInhaled mosliciguat may overcome treatment limitations in patients with pulmonary hypertension by improving pulmonary circulation and airway resistance without systemic exposure or ventilation/perfusion mismatch. Mosliciguat has the potential to become a new therapeutic paradigm, exhibiting a unique mode of action and route of application, and is currently under clinical development in phase Ib for pulmonary hypertension.

ADAR1 Splicing Modulation As a Mechanism to Eradicate Immunologically Silent Leukemia Stem Cells

Secondary acute myeloid leukemia (sAML) is the most therapeutically recalcitrant form of AML with a life expectancy of less than 12 months. Secondary AML evolves from relatively prevalent myeloproliferative neoplasms (MPNs), myelodysplastic syndrome (MDS), or after chemotherapy, radiation therapy, or hematopoietic cell transplantation (HCT) that together confer a 14% risk of sAML at 15 years. Cumulative sequencing studies show that human splicing factor mutations, epigenetic spliceosome deregulation, RNA editing-induced splicing alterations, and pro-survival splice isoform switching drive dormant leukemia stem cell (LSC) generation and sAML resistance to chemotherapy and molecularly targeted agents resulting in high rates of relapse. LSC are immunologically silent in part because they activate adenosine deaminase acting on dsRNA (ADAR1), which attenuates the innate immune response. In addition, therapeutic splicing modulation has the potential to induce neoepitope formation and augment checkpoint inhibitor therapy. Thus, there is a pressing need for clinical development of splicing modulatory agents that eradicate therapy resistant LSC and reduce sAML drug resistance and relapse. Rebecsinib (17 S-FD-895) is a pharmacologically stable, potent, and selective small molecule splicing modulator that targets the SF3B core of the spliceosome at the interface of SF3B1, SF3B3 and PHF5A. We previously showed that Rebecsinib inhibits human LSC maintenance in sAML models at doses that spare normal hematopoietic stem and progenitor cells (HSPCs). In IND-enabling studies, we now demonstrate that splicing modulation with this potent agent is a pre-clinical tox-proven strategy to eradicate LSC with the potential to overcome immune checkpoint resistance via inhibition of ADAR1 splicing and activity. We further describe targeted LSC eradication that correlates with detection of unique intron-retained and exon-skipped transcripts that can be quantified by splice isoform-specific qRT-PCR and RNA-sequencing analyses and can be used as predictive biomarkers to monitor molecular responses to Rebecsinib treatment. Mechanistically, the therapeutic effects were accompanied by on-target splicing modulatory effects, including reductions in pro-survival MCL1L transcripts and splicing factor gene products such as SF3B1 and SF3B3, which form part of the splicing modulator binding pocket as well as alterations in self-renewal promoting ADAR1 and STAT3beta transcripts. In multi-species toxicology and pharmacokinetic/pharmacodynamic studies, Rebecsinib induced splicing modulation and was well-tolerated over a broad range of doses. Because of disrupted spliceosome function, SF3B1 overexpression and increased dependence on pro-survival splice isoform expression, Rebecsinib-mediated induction of pro-survival to pro-apoptotic splice isoform switching inhibits sAML LSC survival and self-renewal at doses that spare normal HSPCs in vitro and in humanized mouse models commensurate with dose-dependent changes in splicing reporter exon skipping and SF3B1, MCL1, BCL2 and CD44 isoform levels. Together, this potent and selective agent along with biomarkers of response to splicing modulation provide a sensitive method of detecting activity and mechanism of action of Rebecsinib, and demonstrate its LSC selectivity in humanized stromal co-cultures and humanized mouse models, which will have utility in future clinical development of this novel therapeutic agent. DisclosuresCrews: Ionis Pharmaceuticals: Research Funding. Burkart: Algenesis: Other: Co-founder. Jamieson: Forty Seven Inc.: Patents & Royalties.

CD47-Blocker TTI-622 Shows Single-Agent Activity in Patients with Advanced Relapsed or Refractory Lymphoma: Update from the Ongoing First-in-Human Dose Escalation Study

CD47-Blocker TTI-622 Shows Single-Agent Activity in Patients with Advanced Relapsed or Refractory Lymphoma: Update from the Ongoing First-in-Human Dose Escalation Study

Biguanide drugs enhance cytotoxic effects of cisplatin by depleting aspartate and NAD+ in sensitive cancer cells

ABSTRACT Biguanide drugs (metformin and phenformin) have drawn interest for potential cancer treatments, and laboratory studies show that some cancer cells are selectively sensitive to growth-inhibitory effects of biguanides. Examining metabolic pathways affected by biguanide treatments in cancer cells that are highly sensitive to biguanides, we found that biguanide treatment depletes cellular levels of both aspartate and NAD+. Experiments to replenish these metabolites or block steps of the aspartate-malate shuttle suggest that depletion of both metabolites, rather than either aspartate of NAD+ individually, is critical for growth-inhibitory effects of biguanide exposure. Even in sensitive cancer cells, though, biguanide treatment alone over a broad range of doses only inhibits cell replication without significantly affecting cell viability. Noting that clinical observations of biguanide efficacy have used combinations of agents that typically include cisplatin, we found that biguanide treatment at a cytostatic level substantially decreases survival of lung cancer and breast cancer cells when co-treated with cisplatin at doses that alone are also non-cytotoxic. This striking enhancement of cisplatin toxicity by biguanides depends on reductions of levels of NAD+ and aspartate, since addition of either of these metabolites prevented this potentiation of cisplatin cytotoxicity. Thus, biguanide drugs can have cytotoxic effects when used in combination with other cancer drugs, such as cisplatin, and depleting cellular levels of NAD+ and aspartate is critical for enhancing the cytotoxicity of cisplatin by biguanide drugs in sensitive cancer cells.

Abstract 1518: Dose optimization and radiation dosimetry of CD45-targeting 225Actinium-armed antibody as a conditioning agent for adoptive cell therapy

Abstract Background: Bone marrow transplantation, ex vivo gene therapies targeting hematological disorders, and other forms of adoptive cell therapies (ACT) require complete or partial removal of the host immune cells by a process called conditioning. This is usually accomplished with total body irradiation (TBI) or chemotherapy, but both modalities can result in significant toxicities, especially in elderly patients. As a result, more effective and safe alternatives to TBI and chemotherapy for conditioning prior to ACT are therefore needed. We have recently demonstrated that the murine CD45-targeting antibody 30F11 armed with the beta emitters 131Iodine or 177Lutetium successfully eliminated various cohorts of immune cells in murine models and ensured effective tumor control during adoptive T cell therapy (Dawicki W., et al. Oncotarget 2020). Here we report the results of a dose optimization study and radiation dosimetry results for 30F11 antibody labeled with the powerful alpha emitter 225Actinium (225Ac). Methods: Female C57BL/6 mice were treated with a broad dose range of 225Ac-30F11 antibody (100-500 nCi per mouse). Survival of mice was observed for 25 days post treatment, and white blood cells (WBC), red blood cells (RBC) and hematopoietic stem cells (HSC) were enumerated by flow cytometry. The cellularity of bone marrow in surviving mice was assessed histologically. Radiation dosimetry calculations were performed to yield the doses delivered to the various organs including those with significant numbers of immune cells. Results: The survival study demonstrated that all mice receiving 100 and 250 nCi doses of 225Ac-30F11 survived, 40% survived with 300 nCi, and 0% with the 400 and 500 nCi doses. While the 500 nCi group showed signs of radiation toxicity, the 100-400 nCi doses had no effect on RBC and preserved liver and kidney function. Flow cytometry and histology revealed that up to 30% of WBC and HSC were lost in the 100-250 nCi group, up to 70% in the 300 nCi group and almost complete acellularity of bone marrow was observed in 400 nCi group. Radiation dosimetry calculations determined the absorbed doses to immune organs such as bone marrow and spleen were comparable to those for beta emitters, while the doses to other organs were lower than for beta emitters. Conclusions: This study demonstrates that low doses of an 225Ac-armed CD45-targeting antibody can safely and efficiently reduce the numbers of host immune cells which would be sufficient for gene therapy or adoptive T cell therapy while high doses can completely eliminate host immune cells, thus providing the conditions for bone marrow transplant engraftment and expansion. Citation Format: Ravendra Garg, Qing Liang, Eileen M. Geoghegan, Kevin J. Allen, Wojciech Dawicki, Dale L. Ludwig, Ekaterina Dadachova. Dose optimization and radiation dosimetry of CD45-targeting 225Actinium-armed antibody as a conditioning agent for adoptive cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1518.

Targetting CD38 in T Cell Acute Lymphoblastic Leukemia

Targeted immunotherapy has become a promising emerging therapeutic strategy for many cancers. No safe and effective immunotherapies have been developed for T-cell acute lymphoblastic leukemia (T-ALL). CD38 is a transmembrane glycoprotein found on the cell surface of activated T cells, terminally differentiated B cells, but relatively low levels on normal lymphoid and myeloid cells. Daratumumab (Janssen Biotech, Inc.) is a human monoclonal antibody that binds to CD38 and is used in the treatment of refractory multiple myeloma (MM).To ensure CD38 is a relevant target in T-ALL, we evaluated CD38 expression by flow cytometry from 21 patients with T-ALL (10 early T-cell precursor (ETP) and 11 non-ETP) at diagnosis and after induction chemotherapy. CD38 was expressed in all samples and surface expression did not change after induction (mean CD38 mean fluorescence intensity (MFI) at diagnosis vs end-of-induction: 3.27 log vs 3.19 (p = 0.25). Similarly, we evaluated CD38 expression of 15 primary T-ALL xenograft samples (7 ETP and 8 non-ETP T-ALL), and only 1 sample demonstrated low CD38 expression. In contrast, we evaluated CD38 expression in 10 patients with de novo B-ALL at diagnosis and found CD38 expression was significantly decreased after induction therapy (5351 vs. 723, p-0.002). Therefore we hypothesized that targeting CD38 with daratumumab would be effective against T-ALL.We xenografted primary ALL blasts from 15 different patients, including 7 with ETP-ALL and 8 with non-ETP T-ALL. Mice were randomized to daratumumab (200 μg /mouse intraperitoneally weekly) vs isotype control (200 μg/mouse; 5 mice per arm for each sample) after they developed >1% peripheral blood (pb) blasts by FACS. Disease burden was assessed by FACS enumeration of pb blasts weekly and splenic blasts at sacrifice.We demonstrate striking efficacy of daratumumab monotherapy in 6 of 7 ETP-ALL samples with significant reduction of pb and splenic blasts. Mice treated with high disease burden from 5 of the 8 non-ETP T-ALL samples were moribund immediately after daratumumab injection, presumably from tumor lysis. Autopsy performed on moribund compared to isotype-treated animals did not reveal embolism. We repeated the experiments in the 8 non-ETP T-ALL samples and the one ETP sample that did not respond when treated at high disease burden. For these experiments, we treated the mice after injection but before detectable pb blasts (termed low disease burden). Using this approach, daratumumab was effective in 7 of 8 non-ETP ALL samples and the one ETP sample that was non-responsive when treated at high disease burden. In summary, we found dara monotherapy was effective in patient-derived xneograft models developed from 14 of 15 patients with T-ALL.The 1 T-ALL sample that did not respond to daratumumab, was the only sample with low CD38 expression. Therefore we hypothesize that the clinical benefit from daratumumab reflects the level of CD38 expression. In myeloid and MM cells, low dose all-trans retinoic acid (ATRA) significantly upregulates CD38 expression. We have similarly tested T-ALL cell lines across a broad range of ATRA doses, however ATRA has no effect on CD38 expression on T-ALL cells. Experiments are ongoing to evaluate alternative agents such as interferon to upregulate CD38 and CRISPR/Cas9 to knock-out CD38.In summary, we found daratumumab is a highly effective novel monotherapy for T-ALL in preclinical models. Based on this work, we are planning to open a multi-institutional early phase clinical trial for children and young adults with relapsed and refractory T-ALL. DisclosuresMaude:Novartis Pharmaceuticals: Consultancy, Other: Medical Advisory Boards. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; Adaptimmune: Consultancy; Jazz Pharmaceuticals: Consultancy; University of Pennsylvania: Patents & Royalties.

High spatial resolution dosimetry with uncertainty analysis using Raman micro-spectroscopy readout of radiochromic films.

The purpose of this work is to develop a new approach for high spatial resolution dosimetry based on Raman micro-spectroscopy scanning of radiochromic film (RCF). The goal is to generate dose calibration curves over an extended dose range from 0 to 50Gy and with improved sensitivity to low (<2Gy) doses, in addition to evaluating the uncertainties in dose estimation associated with the calibration curves. Samples of RCF (EBT3) were irradiated at a broad dose range of 0.03-50Gy using an Elekta Synergy clinical linear accelerator. Raman spectra were acquired with a custom-built Raman micro-spectroscopy setup involving a 500 mW, multimode 785 nm laser focused to a lateral spot diameter of 30µm on the RCF. The depth of focus of 34µm enabled the concurrent collection of Raman spectra from the RCF active layer and the polyester laminate. The preprocessed Raman spectra were normalized to the intensity of the 1614cm-1 Raman peak from the polyester laminate that was unaltered by radiation. The mean intensities and the corresponding standard deviation of the active layer Raman peaks at 696, 1445, and 2060cm-1 were determined for the 150×100µm2 scan area per dose value. This was used to generate three calibration curves that enabled the conversion of the measured Raman intensity to dose values. The experimental, fitting, and total dose uncertainty was determined across the entire dose range for the dosimetry system of Raman micro-spectroscopy and RCF. In contrast to previous work that investigated the Raman response of RCFs using different methods, high resolution in the dose response of the RCF, even down to 0.03Gy, was obtained in this study. The dynamic range of the calibration curves based on all three Raman peaks in the RCF extended up to 50Gy with no saturation. At a spatial resolution of 30×30µm2 , the total uncertainty in estimating dose in the 0.5-50Gy dose range was [6-9]% for all three Raman calibration curves. This consisted of the experimental uncertainty of [5-8]%, and the fitting uncertainty of [2.5-4.5]%. The main contribution to the experimental uncertainty was determined to be from the scan area inhomogeneity which can be readily reduced in future experiments. The fitting uncertainty could be reduced by performing Raman measurements on RCF samples at further intermediate dose values in the high and low dose range. The high spatial resolution experimental dosimetry technique based on Raman micro-spectroscopy and RCF presented here, could become potentially useful for applications in microdosimetry to produce meaningful dose estimates in cellular targets, as well as for applications based on small field dosimetry that involve high dose gradients.

The long-term efficacy of cannabidiol in the treatment of refractory epilepsy.

Cannabidiol (CBD) has been shown to reduce seizures among patients with refractory epilepsies of various etiologies in recent clinical trials and an expanded access program (EAP). Most studies report efficacy over short time periods (<1year), with little published on longer term efficacy. Here, we investigate the efficacy of CBD for a treatment period of up to 60months (median = 45.5months). We conducted a retrospective review of patient-reported seizure logs and medical records for 54 subjects with refractory epilepsy who enrolled in the Massachusetts General Hospital's open-label EAP for CBD as a new treatment for epilepsy. We analyzed the effect of CBD on seizure frequencies and concomitant antiepileptic drug (AED) use at 1year after starting treatment and the most recent study visit. Our results indicate that CBD maintains its efficacy for controlling seizures from Year 1 to the most recent study visit. The percentage of seizure responders remained similar at these time points (41.7%-42.6%), and the seizure response rate was also maintained (p=.12). Efficacy was also seen over a broad dose range, and up to 50mg/kg/day. CBD was particularly effective for controlling seizures in the setting of tuberous sclerosis complex and for reducing epileptic spasms and absence seizures. Although CBD use did not lead to an overall decrease in concomitant AEDs, most subjects reduced the dose of at least one concomitant AED compared to baseline. CBD was generally well tolerated, with drowsiness and diarrhea as the primary adverse reactions. This study demonstrates CBD does not lose its efficacy in controlling seizures over a treatment period of up to 60months. Taken alongside other results on the efficacy and tolerability of CBD in the treatment of refractory epilepsies, our results provide evidence that CBD is an effective, safe, and well-tolerated AED for long-term use.