Preclinical Pharmacology Research Articles

Medical Student Perceptions of Integration and Effectiveness of Educational Methods Used in Analgesic Pharmacology Curricula

Despite the prevalence of pain, it's management complexity, and concerns about opioid over-prescription, literature does not make clear ideal ways to integrate analgesic pharmacotherapy education across medical education. While most institutions implement analgesic education during preclinical pharmacology, evidence suggests that impactful education must be integrated across the preclinical and clinical years. At our institution, opioid and analgesic pharmacology education is heavily taught in the second year (M2) and integrated across 4 years using interactive didactics, Team-based learning (TBL), Case-based learning (CBL), standardized patients, and simulations. Activities are housed in modules or clerkship, but also implemented through Interprofessional education (IPE) and Longitudinal Curricular Theme events (LCT) not coordinated with a pharmacology educator. Our goals in this poster are to share our analgesic curricula and outcomes evaluated by examining analgesic pharmacology knowledge and evaluating learner perspectives across time by surveying cohorts before they took pharmacology (CO2023, a baseline), in mid-clerkship (CO2022, had completed pharmacology) and nearing graduation (CO2021). Methods A retrospective analysis was performed for cohorts CO2022 and CO2021 on their M2 performance on opioid and analgesic pharmacology summative items. Students from 3 cohorts (N = 50, CO2023; 30, CO2022; 36, CO2021) volunteered to take a survey about analgesic educational themes related to comprehension, confidence in managing pain, educational methods, diversity within cases, and integration (basic and clinical; integration of IPE and LCT). Results Summative assessment showed M2's mastered opioid and pain concepts (Mean 86%) and retained knowledge through M3 & M4. Most agreed that education increased comprehension (25% CO2023, >80% for CO2022/CO2021) and confidence as they approached graduation (15% baseline, 67% CO2022; 80% CO2021) and most (>70%) agreed basic and clinical sciences were integrated effectively. However, less felt IPE or LCT analgesic education was integrated effectively (IPE: 21% CO2023; 36% CO2022; 50% CO2021: LCT: 13% CO2023; 44% CO2022, 50% CO2021), and few perceived that cases grew in complexity in M3 or M4 (60% CO2022; 57% CO2021) or represented diverse patients (16% CO2023; 55% CO2022; 56% CO2021). Conclusion Our analgesic curriculum is integrated across 4 years, and outcomes suggest that M2 and M3 educational activities promote learning and progressively increase perceptions of understanding and confidence in learners. However, our analysis indicates improvements are needed as many learners felt that IPE and LCT opioid events not coordinated with a pharmacology educator were not effectively integrated, cases did not increase in complexity across time, and educational experiences were not sufficiently diverse. Curricular details and suggestions for improvement will be presented in the poster.

The Marine Pharmacology and Pharmaceuticals Pipeline in 2020

Marine organisms continue to provide significant chemical biodiversity that contributes to both the global preclinical and clinical pharmaceutical pipelines. Thus, in December 2020, the clinical marine pharmaceutical pipeline consisted of 13 marine-derived drugs approved by the United States Food and Drug Administration (FDA) and 1 drug approved in Australia: for cancer, cytarabine (Cytosar-U®, Depocyt®, FDA-approved 1969); for herpes simplex virus, vidarabine (Vira-A®, FDA-approved 1976); for pain, ziconotide (Prialt®, FDA-approved 2004); for hypertriglyceridemia, omega-3-acid ethyl esters (Lovaza®, FDA-approved 2004, Vascepa®, FDA-approved 2012, Epanova®, FDA-approved 2014); for cancer, eribulin mesylate (Halaven®, FDA-approved 2010), brentuximab vedotin (Adcetris®, FDA-approved 2011), trabectedin (Yondelis®, FDA-approved 2015), Plitidepsin (Aplidine®, approved in Australia 2018), polatuzumab vedotin (Polivy™, FDA-approved 2019), enfortumab vedotin-ejfv (PACDEV™, FDA-approved 2019), lurbinectedin (Zepzelca™, FDA-approved 2020) and belantamab mafadotin-blmf (BlenrepTM, FDA-approved 2020). Both https://www.clinicaltrials.gov/ website as well as the pharmaceutical companies websites describing the development of marine-derived compounds currently in Phase I, Phase II and Phase III of clinical development were searched. We determined that there were 33 marine-derived compounds in active clinical trials in December 2020: 4 marine-derived compounds were in Phase III, 17 compounds in Phase II, and at least 12 compounds in Phase I, many of them auristatin-containing antibody drug conjugates. Regular updates on the clinical marine pharmaceutical pipeline will be posted on the dedicated website: https://www.midwestern.edu/departments/marinepharmacology/clinical-pipeline.xml. Providing novel leads for the marine pharmaceutical clinical pipeline is the global preclinical marine pharmacology pipeline that continues to generate considerable preclinical data on multiple pharmacological classes: Mayer, A.M.S., A.J. Guerrero, A. D. Rodríguez, O. Taglialatela-Scafati, F. Nakamura and N. Fusetani. Marine Pharmacology in 2014–2015: Marine Compounds with Antibacterial, Antidiabetic, Antifungal, Anti-Inflammatory, Antiprotozoal, Antituberculosis, and Antiviral Activities; Affecting the Immune and Nervous Systems, and other Miscellaneous Mechanisms of Action. Marine Drugs 18(5), 2019. PMID: 31861527. We thus conclude that as of December 2020, both the marine pharmacology preclinical and clinical pharmaceutical pipelines remained very active.

Which QT Correction Method is Most Consistent?

An important aspect of the drug development is the preclinical determination of the effect a drug candidate has on the ECG QT-interval. The QT-interval is the period between the start of the Q-wave and end of the T-wave. This represents the time from the start of ventricular depolarization to the end of ventricular repolarization. A prolonged QT-interval is linked to the arrhythmia risk Torsade de Pointes and is used as a biomarker in preclinical safety pharmacology studies. Determining a compound's arrhythmia risk based on QT-interval prolongation is insufficient as the drug effect can be obscured by the physiological inverse relationship between heart rate and QT-interval. To better elucidate the drug-induced effect on QT-interval, it is standard practice to correct the QT-interval for changes in heart rate. The majority of safety pharmacology studies use telemetry techniques in conscious ambulatory animals. In these studies, a wide range of heart rates occur and correction methods are required regardless of test drug effects on heart rate. Many correction methods have been designed to account for the heart rate effect on QT. Adequacy of QT correction is dependent on the correction method, the species, sex, age, and even on environmental conditions. Historically fixed population QT correction methods are used, such as Bazett's and Fridericia's which rely on set formulas with effectiveness that varies by species. Individual animal correction methods are gaining popularity and are theoretically better to deal with variations in the individual relationship between heart rate and QT-interval. We aim to identify which correction methods are most broadly applicable and introduce the least variability in the corrected QT values. We hypothesize that an individual correction method, that accounts for multiple variables that affect the RR/QT relationship will be the most consistent across individuals, days, and species. To test this hypothesis, we utilized baseline data collected during numerous safety pharmacology assessments and calculated corrected QT values using various methods. The relationship between the corrected QT and heart rate were then compared. For each ECG recording, the correction method resulting in the lowest correlation between the corrected QT and heart rate was identified. A chi-squared test was used to determine which method was the best at reducing the correlation between heart rate and QT interval. We also determined which method produced the lowest variability in the corrected QT values. To do this, the corrected QT values were averaged for the entire recording for each method, and the resulting variabilities were compared. For each ECG recording, the correction method resulting in the lowest variability was determined and a chi-squared test was applied to determine which is most consistent. In conclusion our results demonstrate that individual correction methods are the most consistent, allowing for more accurate comparisons between subjects, species, and dose groups.

Inflammation as a Risk Factor in Cardiotoxicity: An Important Consideration for Screening During Drug Development.

Numerous commonly prescribed drugs, including antiarrhythmics, antihistamines, and antibiotics, carry a proarrhythmic risk and may induce dangerous arrhythmias, including the potentially fatal Torsades de Pointes. For this reason, cardiotoxicity testing has become essential in drug development and a required step in the approval of any medication for use in humans. Blockade of the hERG K+ channel and the consequent prolongation of the QT interval on the ECG have been considered the gold standard to predict the arrhythmogenic risk of drugs. In recent years, however, preclinical safety pharmacology has begun to adopt a more integrative approach that incorporates mathematical modeling and considers the effects of drugs on multiple ion channels. Despite these advances, early stage drug screening research only evaluates QT prolongation in experimental and computational models that represent healthy individuals. We suggest here that integrating disease modeling with cardiotoxicity testing can improve drug risk stratification by predicting how disease processes and additional comorbidities may influence the risks posed by specific drugs. In particular, chronic systemic inflammation, a condition associated with many diseases, affects heart function and can exacerbate medications’ cardiotoxic effects. We discuss emerging research implicating the role of inflammation in cardiac electrophysiology, and we offer a perspective on how in silico modeling of inflammation may lead to improved evaluation of the proarrhythmic risk of drugs at their early stage of development.

Basmisanil, a highly selective GABAA-\u03b15 negative allosteric modulator: preclinical pharmacology and demonstration of functional target engagement in man

GABAA-α5 subunit-containing receptors have been shown to play a key modulatory role in cognition and represent a promising drug target for cognitive dysfunction, as well as other disorders. Here we report on the preclinical and early clinical profile of a novel GABAA-α5 selective negative allosteric modulator (NAM), basmisanil, which progressed into Phase II trials for intellectual disability in Down syndrome and cognitive impairment associated with schizophrenia. Preclinical pharmacology studies showed that basmisanil is the most selective GABAA-α5 receptor NAM described so far. Basmisanil bound to recombinant human GABAA-α5 receptors with 5 nM affinity and more than 90-fold selectivity versus α1, α2, and α3 subunit-containing receptors. Moreover, basmisanil inhibited GABA-induced currents at GABAA-α5 yet had little or no effect at the other receptor subtypes. An in vivo occupancy study in rats showed dose-dependent target engagement and was utilized to establish the plasma exposure to receptor occupancy relationship. At estimated receptor occupancies between 30 and 65% basmisanil attenuated diazepam-induced spatial learning impairment in rats (Morris water maze), improved executive function in non-human primates (object retrieval), without showing anxiogenic or proconvulsant effects in rats. During the Phase I open-label studies, basmisanil showed good safety and tolerability in healthy volunteers at maximum GABAA-α5 receptor occupancy as confirmed by PET analysis with the tracer [11C]-Ro 15-4513. An exploratory EEG study provided evidence for functional activity of basmisanil in human brain. Therefore, these preclinical and early clinical studies show that basmisanil has an ideal profile to investigate potential clinical benefits of GABAA-α5 receptor negative modulation.

Pharmacokinetic, Pharmacodynamic, and Drug-Interaction Profile of Remdesivir, a SARS-CoV-2 Replication Inhibitor.

Remdesivir (RDV, Veklury®) is a once-daily, nucleoside ribonucleic acid polymerase inhibitor of severe acute respiratory syndrome coronavirus 2 replication. Remdesivir has been granted approvals in several countries for use in adults and children hospitalized with severe coronavirus disease 2019 (COVID-19). Inside the cell, remdesivir undergoes metabolic activation to form the intracellular active triphosphate metabolite, GS-443902 (detected in peripheral blood mononuclear cells), and ultimately, the renally eliminated plasma metabolite GS-441524. This review discusses the pre-clinical pharmacology of RDV, clinical pharmacokinetics, pharmacodynamics/concentration-QT analysis, rationale for dose selection for treatment of patients with COVID-19, and drug–drug interaction potential based on available in vitro and clinical data in healthy volunteers. Following single-dose intravenous administration over 2 h of an RDV solution formulation across the dose range of 3–225 mg in healthy participants, RDV and its metabolites (GS-704277and GS-441524) exhibit linear pharmacokinetics. Following multiple doses of RDV 150 mg once daily for 7 or 14 days, major metabolite GS-441524 accumulates approximately 1.9-fold in plasma. Based on pharmacokinetic bridging from animal data and available human data in healthy volunteers, the RDV clinical dose regimen of a 200-mg loading dose on day 1 followed by 100-mg maintenance doses for 4 or 9 days was selected for further evaluation of pharmacokinetics and safety. Results showed high intracellular concentrations of GS-443902 suggestive of efficient conversion from RDV into the triphosphate form, and further supporting this clinical dosing regimen for the treatment of COVID-19. Mathematical drug–drug interaction liability predictions, based on in vitro and phase I data, suggest RDV has low potential for drug–drug interactions, as the impact of inducers or inhibitors on RDV disposition is minimized by the parenteral route of administration and extensive extraction. Using physiologically based pharmacokinetic modeling, RDV is not predicted to be a clinically significant inhibitor of drug-metabolizing enzymes or transporters in patients infected with COVID-19 at therapeutic RDV doses.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40262-021-00984-5.

Pharmacological Insights Into Safety and Efficacy Determinants for the Development of Adenosine Receptor Biased Agonists in the Treatment of Heart Failure.

Adenosine A1 receptors (A1R) are a potential target for cardiac injury treatment due to their cardioprotective/antihypertrophic actions, but drug development has been hampered by on-target side effects such as bradycardia and altered renal hemodynamics. Biased agonism has emerged as an attractive mechanism for A1R-mediated cardioprotection that is haemodynamically safe. Here we investigate the pre-clinical pharmacology, efficacy and side-effect profile of the A1R agonist neladenoson, shown to be safe but ineffective in phase IIb trials for the treatment of heart failure. We compare this agent with the well-characterized, pan-adenosine receptor (AR) agonist NECA, capadenoson, and the A1R biased agonist VCP746, previously shown to be safe and cardioprotective in pre-clinical models of heart failure. We show that like VCP746, neladenoson is biased away from Ca2+ influx relative to NECA and the cAMP pathway at the A1R, a profile predictive of a lack of adenosine-like side effects. Additionally, neladenoson was also biased away from the MAPK pathway at the A1R. In contrast to VCP746, which displays more ‘adenosine-like’ signaling at the A2BR, neladenoson was a highly selective A1R agonist, with biased, weak agonism at the A2BR. Together these results show that unwanted hemodynamic effects of A1R agonists can be avoided by compounds biased away from Ca2+ influx relative to cAMP, relative to NECA. The failure of neladenoson to reach primary endpoints in clinical trials suggests that A1R-mediated cAMP inhibition may be a poor indicator of effectiveness in chronic heart failure. This study provides additional information that can aid future screening and/or design of improved AR agonists that are safe and efficacious in treating heart failure in patients.

Preclinical and clinical pharmacology of EPI-7386, an androgen receptor N-terminal domain inhibitor for castration-resistant prostate cancer.

119 Background: EPI-7386 is the newest of the “anitens”, a new class of compounds designed to inhibit androgen receptor activity by binding to the N-terminal domain (NTD) of the AR. Through this novel method of AR inhibition, anitens can block AR transcription even in the presence of AR ligand-binding domain (LBD) resistance mechanisms including point mutations and splice variants. Compared to the first generation aniten, EPI-506, which showed poor pharmacokinetic properties in patients, EPI-7386 is metabolically stable in vitro and in vivo. A Phase 1 clinical trial of EPI-7386 in metastatic castration-resistant prostate cancer patients failing standard of care therapies is ongoing and the pharmacokinetic properties of the drug in preclinical models as well as in the initial cohort of patients are presented. Methods: The metabolic stability of EPI-7386 was evaluated in vitro in mouse, rat, dog, monkey, and human hepatocytes. Projected PK parameters in humans were estimated from in vitro and in vivo clearance correlation (IVIVC). Induction of CYP isoforms was evaluated in human hepatocyte cultures. In patients, plasma concentrations of EPI-7386 were determined by LC-MS-MS, and 4-beta-hydroxycholesterol levels in plasma were followed over time as an indirect indicator of CYP3A induction. Results: In vitro hepatocyte studies demonstrated good metabolic stability for EPI-7386 with an in vitro half-life > 360 min. In animal PK studies, the terminal half-life of EPI-7386 was approximately 5.8 hours in mouse, 4.9 hours in rat, 13.4 hours in dog and the plasma clearance was low across species. The oral bioavailability of EPI-7386 ranged from 33–112% in mouse to > 100% in rat and dog. Using IVIVC, a predicted human clearance of 0.16–0.39 mL/min/kg was calculated for EPI-7386, which was in line with allometric scaling from animal PK parameters. Human PK profiles of different doses of EPI-7386 were simulated using predicted oral bioavailability, clearance, and volume of distribution. Cmax and AUC0–24h for the Phase 1 first-in-human study (NCT04421222) starting dose of 200 mg dose were predicted to be 6,915 ng/mL and 137,278 ng•h/mL respectively. A comparison between estimated PK parameters and actual values observed in the first patient cohort will be presented. Human hepatocyte CYP induction studies showed that EPI-7386 is not an inducer of CYP1A2 but may have the potential to induce CYP2B6 and CYP3A4. A comparison of 4-beta-hydroxy cholesterol levels measured during the phase 1 will be presented along with a comparison drawn from in vitro models. Conclusions: Pre-clinical characterization predicts that EPI-7386 has the appropriate PK and metabolic properties to afford exposure in patients at potentially efficacious levels following once-daily oral administration. PK measurements in the initial cohort of patients treated in the Phase 1 study will be presented. Clinical trial information: NCT04421222.

Marine Pharmacology in 2016-2017: Marine Compounds with Antibacterial, Antidiabetic, Antifungal, Anti-Inflammatory, Antiprotozoal, Antituberculosis and Antiviral Activities; Affecting the Immune and Nervous Systems, and Other Miscellaneous Mechanisms of Action.

The review of the 2016–2017 marine pharmacology literature was prepared in a manner similar as the 10 prior reviews of this series. Preclinical marine pharmacology research during 2016–2017 assessed 313 marine compounds with novel pharmacology reported by a growing number of investigators from 54 countries. The peer-reviewed literature reported antibacterial, antifungal, antiprotozoal, antituberculosis, and antiviral activities for 123 marine natural products, 111 marine compounds with antidiabetic and anti-inflammatory activities as well as affecting the immune and nervous system, while in contrast 79 marine compounds displayed miscellaneous mechanisms of action which upon further investigation may contribute to several pharmacological classes. Therefore, in 2016–2017, the preclinical marine natural product pharmacology pipeline generated both novel pharmacology as well as potentially new lead compounds for the growing clinical marine pharmaceutical pipeline, and thus sustained with its contributions the global research for novel and effective therapeutic strategies for multiple disease categories.

Synthesis and structure-activity relationships of 3,4,5-trisubstituted-1,2,4-triazoles: high affinity and selective somatostatin receptor-4 agonists for Alzheimer's disease treatment.

Somatostatin receptor-4 (SST4) is highly expressed in brain regions affiliated with learning and memory. SST4 agonist treatment may act to mitigate Alzheimer's disease (AD) pathology. An integrated approach to SST4 agonist lead optimization is presented herein. High affinity and selective agonists with biological efficacy were identified through iterative cycles of a structure-based design strategy encompassing computational methods, chemistry, and preclinical pharmacology. 1,2,4-Triazole derivatives of our previously reported hit (4) showed enhanced SST4 binding affinity, activity, and selectivity. Thirty-five compounds showed low nanomolar range SST4 binding affinity, 12 having a K i < 1 nM. These compounds showed >500-fold affinity for SST4 as compared to SST2A. SST4 activities were consistent with the respective SST4 binding affinities (EC50 < 10 nM for 34 compounds). Compound 208 (SST4 K i = 0.7 nM; EC50 = 2.5 nM; >600-fold selectivity over SST2A) display a favorable physiochemical profile, and was advanced to learning and memory behavior evaluations in the senescence accelerated mouse-prone 8 model of AD-related cognitive decline. Chronic administration enhanced learning with i.p. dosing (1 mg kg-1) compared to vehicle. Chronic administration enhanced memory with both i.p. (0.01, 0.1, 1 mg kg-1) and oral (0.01, 10 mg kg-1) dosing compared to vehicle. This study identified a novel series of SST4 agonists with high affinity, selectivity, and biological activity that may be useful in the treatment of AD.

Inebilizumab in AQP4-Ab-positive neuromyelitis optica spectrum disorder.

Until recently, the use of preventative immunotherapy in neuromyelitis optica spectrum disorders (NMOSD) was based on observational studies and clinical experiences. Meanwhile, the first drugs, among others the monoclonal antibody inebilizumab, were approved for the treatment of aquaporin-4 (AQP4) antibody-positive NMOSD. Inebilizumab binds to the CD19 antigen on B cells and leads to B-cell depletion. The first two dosages of 300 mg inebilizumab are administered intravenously at an interval of 2 weeks followed by further infusions every 6 months. In the placebo-controlled pivotal phase II/III study N-MOmentum, inebilizumab significantly prolonged the time to a first adjudicated relapse in AQP4 antibody-positive patients compared with placebo. The most frequent side effects were infusion reactions, urinary and respiratory tract infections, and arthralgia. This review presents data on clinical and preclinical pharmacology, administration, safety aspects and clinical trials of inebilizumab.

Delgocitinib in atopic dermatitis.

Delgocitinib ointment is a newly approved topical medication for the treatment of atopic dermatitis (AD). AD usually has onset in early childhood and is a common, often chronic relapsing inflammatory skin condition that affects all age groups. AD has a large impact on patients' quality of life including physical health and emotional impacts. Conventional management of AD consists of phototherapy and treatments that comprise corticosteroids (both topically and systemically), topical calcineurin inhibitors (tacrolimus and pimecrolimus) or systemic immunosuppressants like ciclosporin. New targeted therapies for AD, including interleukin (IL)-4/13 inhibitors, Janus kinase (JAK) inhibitors and IL-13 inhibitors have been introduced for both topical and systemic use. In this review, we give an overview of the use of delgocitinib, the first topical JAK inhibitor to be approved for AD, including its preclinical pharmacology, pharmacokinetics and metabolism, safety, existing clinical trials and future directions.

Lasmiditan for acute treatment of migraine.

Lasmiditan, a selective 5-HT1F receptor agonist, is a harbinger for the novel ditan class of medications for acute migraine treatment. Lasmiditan activates 5-HT1F receptors on presynaptic trigeminal nerve terminals, which impedes the release of calcitonin gene-related peptide (CGRP) from trigeminal nerve endings and thereby suppresses activation of the trigeminovascular system. Notably, lasmiditan does not result in vasoconstriction, making lasmiditan a migraine-specific acute treatment option for those with cardiovascular risk factors. However, lasmiditan does have centrally mediated side effects. This review will discuss the background of lasmiditan development, its preclinical pharmacology, safety, drug interactions and indications for use.

Mitapivat, a novel pyruvate kinase activator, for the treatment of hereditary hemolytic anemias.

Mitapivat (AG-348) is a novel, first-in-class oral small molecule allosteric activator of the pyruvate kinase enzyme. Mitapivat has been shown to significantly upregulate both wild-type and numerous mutant forms of erythrocyte pyruvate kinase (PKR), increasing adenosine triphosphate (ATP) production and reducing levels of 2,3-diphosphoglycerate. Given this mechanism, mitapivat has been evaluated in clinical trials in a wide range of hereditary hemolytic anemias, including pyruvate kinase deficiency (PKD), sickle cell disease, and the thalassemias. The clinical development of mitapivat in adults with PKD is nearly complete, with the completion of two successful phase III clinical trials demonstrating its safety and efficacy. Given these findings, mitapivat has the potential to be the first approved therapeutic for PKD. Mitapivat has additionally been evaluated in a phase II trial of patients with alpha- and beta-thalassemia and a phase I trial of patients with sickle cell disease, with findings suggesting safety and efficacy in these more common hereditary anemias. Following these successful early-phase trials, two phase III trials of mitapivat in thalassemia and a phase II/III trial of mitapivat in sickle cell disease are beginning worldwide. Promising preclinical studies have additionally been done evaluating mitapivat in hereditary spherocytosis, suggesting potential efficacy in erythrocyte membranopathies as well. With convenient oral dosing and a safety profile comparable with placebo in adults with PKD, mitapivat is a promising new therapeutic for several hereditary hemolytic anemias, including those without any currently US Food and Drug Administration (FDA) or European Medicines Agency (EMA)–approved drug therapies. This review discusses the preclinical studies, pharmacology, and clinical trials of mitapivat.

Guidelines for FAIR sharing of preclinical safety and off-target pharmacology data

Pre-competitive data sharing can offer the pharmaceutical industry significant benefits in terms of reducing the time and costs involved in getting a new drug to market through more informed testing strategies and knowledge gained by pooling data. If sufficient data is shared and can be co-analyzed, then it can also offer the potential for reduced animal usage and improvements in the in silico prediction of toxicological effects. Data sharing benefits can be further enhanced by applying the FAIR Guiding Principles, reducing time spent curating, transforming and aggregating datasets and allowing more time for data mining and analysis. We hope to facilitate data sharing by other organizations and initiatives by describing lessons learned as part of the Enhancing TRANslational SAFEty Assessment through Integrative Knowledge Management (eTRANSAFE) project, an Innovative Medicines Initiative (IMI) partnership which aims to integrate publicly available data sources with proprietary preclinical and clinical data donated by pharmaceutical organizations. Methods to foster trust and overcome non-technical barriers to data sharing such as legal and IPR (intellectual property rights) are described, including the security requirements that pharmaceutical organizations generally expect to be met. We share the consensus achieved among pharmaceutical partners on decision criteria to be included in internal clearance procedures used to decide if data can be shared. We also report on the consensus achieved on specific data fields to be excluded from sharing for sensitive preclinical safety and pharmacology data that could otherwise not be shared.

U-47700 and Its Analogs: Non-Fentanyl Synthetic Opioids Impacting the Recreational Drug Market.

The recreational use of opioid drugs is a global threat to public health and safety. In particular, an epidemic of opioid overdose fatalities is being driven by illicitly manufactured fentanyl, while novel synthetic opioids (NSOs) are appearing on recreational drug markets as standalone products, adulterants in heroin, or ingredients in counterfeit drug preparations. Trans-3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide (U-47700) is a prime example of a non-fentanyl NSO that is associated with numerous intoxications and fatalities. Here, we review the medicinal chemistry, preclinical pharmacology, clandestine availability, methods for detection, and forensic toxicology of U-47700 and its analogs. An up-to-date summary of the human cases involving U-47700 intoxication and death are described. The evidence demonstrates that U-47700 is a potent μ-opioid receptor agonist, which poses a serious risk for overdosing and death. However, most analogs of U-47700 appear to be less potent and have been detected infrequently in forensic specimens. U-47700 represents a classic example of how chemical entities from the medicinal chemistry or patent literature can be diverted for use in recreational drug markets. Lessons learned from the experiences with U-47700 can inform scientists, clinicians, and policymakers who are involved with responding to the spread and impact of NSOs.

Abstract 4556: Preclinical pharmacology and safety of RO7297089, a novel anti-BCMA/CD16a bispecific antibody for the treatment of multiple myeloma

Abstract RO7297089 is a bispecific antibody (IgG-scFv) targeting Bcell maturation antigen (BCMA) and CD16a (FcγRIIIA) that is being developed for the treatment of multiple myeloma (MM). BCMA is exclusively expressed on plasmablasts and differentiated plasma cells (PCs), and is overexpressed on malignant PCs in MM patients. CD16a is expressed on natural killer (NK) cells, monocytes, mast cells, and macrophages. Herein, we characterized the mode of action and safety profile of RO7297089 in vitro and in vivo. RO7297089 showed potent cell killing when using BCMA+ MM tumor cell lines as target cells and human peripheral blood mononuclear cells, NK cells or macrophages as effector cells. Minimal increases in TNFα (2x) and IFNγ (4x), but not other cytokines/chemokines, were observed compared to the vehicle control treatment only in the presence of the BCMA+ MM tumor cell line up to the concentration tested. This suggests that, unlike T-cell engagers, the risk of cytokine release syndrome in patients receiving RO7297089 is low. Cynomolgus monkey is the only relevant nonclinical species for RO7297089 as it showed binding to both recombinant CD16 and BCMA with comparable affinity to human antigens. Following five weekly intravenous administrations to monkeys at 0, 15, and 50 mg/kg, RO7297089 was well tolerated. In line with the mechanism of action, there were no test article-related cytokine increases or adverse findings observed in both dose levels. Systemic exposure of RO7297089 was approximately dose proportional from 15 to 50 mg/kg. Anti-drug antibodies (ADA) were observed in some animals at both dose levels, and ADA-related decreases in concentrations were observed at only 15 mg/kg. To evaluate in vivo activity, RO7297089-related effects on total plasma sBCMA and PCs were assessed. Elevations of sBCMA levels (100x) were observed post dose at both dose levels, and these effects returned to predose levels in animals that did not maintain concentrations at 15 mg/kg, suggesting that RO7297089 bound to and stabilized circulating cynomolgus sBCMA. Time- and dose-dependent reductions in serum IgM levels were observed at both dose levels. Changes in PC numbers were not detected by immunophenotyping; however, gene expression analysis of PC markers was included and demonstrated clear reductions in mRNA expression levels of PC markers including BCMA and J-chain in blood at both dose levels, suggesting reductions in BCMA+ cells. Collectively, these studies suggest that RO7297089 selectively kills BCMA+ cells by engaging CD16a-positive immune cells and has a favorable safety profile. Citation Format: Satoko Kakiuchi-Kiyota, Melissa M. Schutten, Adeyemi O. Adedeji, Hao Cai, Robert Hendricks, Luna Liu, Sivan Cohen, Aaron M. Fullerton, Nicholas Corr, Lanlan Yu, Denise de Almeida Nagata, Shelly Zhong, Michael Dillon, Christoph Spiess, Steve R. Leong, Bing Zheng, Susanne Wingert, Uwe Reusch, Stefan Knackmuss, Thorsten Ross, Andrew Polson, Ayse M. Ovacik. Preclinical pharmacology and safety of RO7297089, a novel anti-BCMA/CD16a bispecific antibody for the treatment of multiple myeloma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4556.

Abstract 4518: Pre-clinical development of NL-201: A de novo α-independent IL-2/IL-15 agonist

Abstract Interleukin-2 (IL-2) and interleukin-15 (IL-15) promote anti-tumor immune activity by stimulating effector T and NK cells expressing the β and γ subunits of the IL-2 and IL-15 receptors. However, preferential activation of off-target cell populations expressing the high-affinity α subunit (including regulatory T cells, eosinophils, and endothelial cells) has limited the safety, therapeutic benefit, and widespread clinical use of these cytokines. Recently, we used sophisticated computational methods to create Neoleukin-2/15 (Neo-2/15), an entirely de novo α-independent agonist of both the IL-2 and IL-15 receptors. Unlike strategies that attempt to sterically inhibit or mutate the α-binding region of IL-2 or IL-15, Neo-2/15 was computationally designed to have no α-binding interface, while also having increased affinity for the β and γ signaling subunits. As a result, Neo-2/15 stimulates effector T and NK cells more potently and selectively than IL-2 or IL-15. Here, we report on the preclinical development and pharmacology of NL-201, a long-circulating variant of Neo-2/15 that is intended for clinical applications. NL-201 was derived from Neo-2/15 via the site-specific conjugation of a single PEG molecule and is manufactured using a simple and scalable process. NL-201 has a prolonged blood half-life and a sustained pharmacodynamic effect while retaining the high potency, selectivity, and thermodynamic stability of Neo-2/15. Preclinical studies demonstrate the robust antitumor activity of NL-201 as a single agent across a wide range of syngeneic murine tumor models, including those that do not respond well to checkpoint inhibitors. Due to its stability, high potency, and selectivity, NL-201 may overcome the long-standing challenges associated with the clinical use of IL-2 and IL-15 for cancer immunotherapy. Citation Format: Carl D. Walkey, Paul Hara, Laurie Tatalick, Umut Ulge, Jonathan Drachman, Daniel-Adriano Silva. Pre-clinical development of NL-201: A de novo α-independent IL-2/IL-15 agonist [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4518.

Abstract 1899: Extracellular lipid starvation modulates the effects of BRAF inhibitors in melanoma

Abstract Melanoma is the most aggressive of all skin cancers, being the leading cause of death for cutaneous malignancy, with elevated metastatic spread and drug resistance. Because the deregulation of components of lipid metabolism may contribute to melanoma cell resistance, the aim of this study was to examine the dependence of melanoma cell growth on extracellular lipids, the associated lipid metabolism gene expression profiles and lipid impact on sensitivity to BRAF inhibitors. We applied preclinical pharmacology approaches including western blot analyses, growth inhibition assays quantitative Real-time PCR (qRT-PCR) in melanoma cell lines including variants with acquired resistance to BRAF inhibitors. Two matched pairs of sensitive and PLX4032-resistant cell lines (LM16 and LM36) were used. Both pairs carried V600E BRAF gene mutation and BRAF gene amplification, and LM36R displayed mutant NRAS. When compared to the parental sensitive cells, LM16R cells showed increased activation of MAPK-ERK and PI3K-Akt-mTOR pathways, while LM36R cells displayed increased activation of IGF1R and LKB1. Under standard culture conditions, when compared to the parental sensitive cells, both the resistant variants showed reduced expression of DHCR24 (24-Dehydrocholesterol Reductase), and LM16R showed reduced protein level of the lipogenic enzyme FASN (Fatty Acid Synthase) while LM36R cells displayed similar FASN protein levels. When grown in lipid-free medium, LM16 and LM16R cells increased the expression of both FASN and DHCR24 proteins. These changes were particularly evident for DHCR24 in LM16R cells after 48 h of growth in the absence of lipids. Lipid starvation markedly reduced cell growth of LM16-LM16R as well as LM36-LM36R pairs over time, albeit with a lower impact at higher cell density. Growth inhibition assays indicated that all the cells cultured in lipid-free medium were more sensitive to PLX4032 than those cultured in standard medium. An analysis of the expression of lipid metabolism genes by qRT-PCR showed that after 48 hours under lipid-free culture conditions the levels of FASN were slightly increased in LM16 and LM16R cells but not in the LM36 pair. Conversely, the mRNA levels of DHCR24 were enhanced upon lipid starvation only in LM16 and LM16R cell lines. Besides, under these conditions, HMGCR (3-Hydroxy-3-Methylglutaryl-CoA Reductase) mRNA levels increased in LM16, LM16R and LM36R cell lines. ACSS2 (Acetyl-coenzyme A synthetase 2) was increased particularly in the LM16 cells. These findings suggest that lipid starvation redirects lipid pathways toward cholesterol synthesis (acetate-cholesterol) by increasing ACSS2, HMGCR as well as DHCR24 levels. Thus, extracellular lipid availability may influence tumor cell response to treatment, a finding to be considered in the frame of a personalized therapy tailored to the specific characteristics of each patient. Citation Format: Giovanni L. Beretta, Elisabetta Vergani, Cristina Corno, Stella Tinelli, Monica Rodolfo, Laura Gatti, Paola Perego. Extracellular lipid starvation modulates the effects of BRAF inhibitors in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1899.

Abstract 684: Gastrointestinal/liver-targeted TLR7 agonist for treatment of colorectal and liver cancers

Abstract Anti-PD1/L1 has become the backbone of immune therapy for many cancer types. However, the majority of patients do not respond due to a myriad of mechanisms affecting the tumor microenvironment, including low tumor mutational burden and low immune cell infiltrate. Innate immune agonists can potentially address both issues by increasing tumor antigen presentation and tumor microenvironment inflammation, thereby turning cold tumors hot. As clinical proof-of-concept, many intratumoral TLR/STING agonists have shown promising anti-tumor responses as single agents and in combination with checkpoint inhibitors; however, successful clinical applications have been limited to cutaneous-accessible tumor types. APR003 is an orally-administered GI/liver-targeted small molecule TLR7 agonist designed for the treatment of visceral colorectal and liver cancers. Studies in mice and monkeys demonstrate robust Type-1 Interferon pathway activation with good tolerability. APR003 is efficacious in several syngeneic orthotopic models of liver and colon cancer, both as single agent and in combination with anti-PDL1. Mechanistic studies show enhancement of CD103+ dendritic cells in tumor draining lymph nodes and tumor antigen-specific T cells in the tumor microenvironment. Pre-clinical pharmacology data will be presented. Citation Format: Andrew T. Miller, Evelyn Rodrigo, Manny Corpuz, David Plouffe, Tom Y.-H. Wu. Gastrointestinal/liver-targeted TLR7 agonist for treatment of colorectal and liver cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 684.