Post Drug Treatment Research Articles

In vivo pharmacokinetic, pharmacodynamic and brain concentration comparison of fentanyl and para-fluorofentanyl in rats.

In 2022, para-fluorofentanyl (pFF) rose to the 6th most reported drug and the most reported fentanyl analog in the United States according to the Drug Enforcement Administration (DEA). pFF differs from fentanyl by the addition of a single fluorine group. To date, pFF has not been extensively evaluated in vivo and is frequently seen in combination with fentanyl. In the present study, the pharmacodynamic (PD) and pharmacokinetic (PK) properties and brain region-specific concentrations of pFF were evaluated in male Sprague-Dawley rats and compared to fentanyl. A 300μg/kg subcutaneous dose of fentanyl or pFF was administered to assess PD and PK parameters as well as brain region concentrations. PD parameters were evaluated via a tail flick test to evaluate analgesia and core body temperature to measure hypothermia, a surrogate marker of overall opioid toxicity. Fentanyl and pFF were found to be equally active at the tested dose in terms of tail flick response with both compounds producing an analgesic response that lasted up to 240min post-drug treatment. pFF induced a significantly greater hypothermic effect compared to fentanyl with a maximum temperature decrease of -5.6 ℃. Plasma PK parameters (T1/2, AUC, etc.) did not differ between fentanyl and pFF. However, pFF concentrations in the medulla, hippocampus, frontal cortex and striatum were more than two times the fentanyl concentrations. The increase in brain concentrations and greater hypothermic effect suggests that pFF is potentially more dangerous than fentanyl.

PERSISTENCE POSITIVITY OF LACTATE DEHYDROGENASE RAPID DIAGNOSTIC TESTS ANTIGENS IN PREGNANT WOMEN AFTER ANTIMALARIA TREATMENT IN SOME HOSPITALS IN KATSINA

Malaria is a life-threatening disease caused by parasites transmitted to people through the bites of infected female Anopheles mosquitoes. It is preventable and curable. This study investigates the persistent positivity of Lactate Dehydrogenase (pLDH) Rapid diagnostic tests antigens in pregnant women after antimalarial treatment. The study was conducted in Turai Yaradua Maternity and Children’s Hospital Katsina and Federal Medical Centre Katsina. Samples were collected with the help of trained physicians. The result from this study shows that lactate dehydrogenase antigen persists in the blood after treatment with fensider (antimalaria drug). After administering the drug to the pregnant women found to be positive for malaria, it was observed that in all the age groups, day 3, day 7, and day 14 post-drug treatment were all still positive for malaria using the pLDH malaria rapid diagnostic tool. It was also observed that after 21 days post-treatment, all the different age groups were still positive for malaria except those aged 45 to 50 (95.0%). There is no statistically significant difference between the age of pregnancy p>0.05. The percentage positivity of pLDH started changing day 21 in 3rd trimester and the change peak at day 28 post-drug treatment in all three trimesters. There is no statistical significance difference between the age of pregnancy p>0.05. However, the first trimester shows the highest negative result (66.7%). It also shows that age, tribe, trimester of pregnancy, and occupation were statistically not associated with the persistence positivity of the pLDH. The parasite density might be responsible..

DIPG-61. ARGININE DEPRIVATION USING ADI-PEG20 IMPROVES SURVIVAL IN A SYNGENEIC DIFFUSE MIDLINE GLIOMA MOUSE MODEL

Abstract BACKGROUND Diffuse midline glioma (DMG-H3K27) presents a significant challenge in paediatric oncology, with a <10% two-year survival rate despite standard palliative radiotherapy. Tumour infiltration behind the blood-brain barrier hampers drug delivery, contributing to clinical trial failures. ADI-PEG20 exploits tumour metabolic vulnerabilities: lack of argininosuccinate synthetase and reliability on extracellular arginine for proliferation. This study investigates ADI-PEG20’s efficacy in a syngeneic DMG mouse model. METHODS Twenty C57BL/6J mice were intracranially injected with a DMG cell line (H1H3BK27M, DNp53, PDGFRAD642V, Acvr1G328V) and monitored via bioluminescence imaging from day 7. Weekly treatments began on day 10, comprising ADI-PEG20 (5 I.U.) alone, ADI-PEG20 with concurrent radiation (2 Gy), radiation alone, or saline (0.9%), with each group comprising n=5. Radiation was administered 24 hours post-drug treatment. Bioluminescence imaging was conducted twice weekly, with mice culled upon symptom onset. Immunohistochemistry was performed on brain tissue. RESULTS The combination and ADI-PEG20 monotherapy cohorts exhibited comparable median survival rates, both surpassing those of the radiation-only and saline groups, with the latter exhibiting the lowest median survival. A significant disparity in survival curves was observed overall (p<0.0001), confirmed by log-rank (Mantel-Cox) test. Pairwise comparisons revealed significant differences in survival curves between the combination and radiation-only (p=0.0066), ADI-PEG20 monotherapy and radiation-only (p=0.0018), and ADI-PEG20 monotherapy and saline (p=0.0062) groups. Minimal divergence in IVIS signal intensity was noted across groups. Histological examination via H&E staining demonstrated marked tumour regression in mice treated with ADI-PEG20 ± radiation, contrasting with the persistent tumour mass evident in the radiation-only and saline cohorts, alongside discernible heterogeneity in cellular phenotypes within the ADI-PEG20 ± groups. CONCLUSIONS In conclusion, ADI-PEG20 shows promise as a therapeutic strategy for DMG, offering a significant survival advantage over radiation and saline. Ongoing efforts will elucidate its mechanistic underpinnings, explore its efficacy across diverse DMG models, and assess synergistic interactions with immune-based therapies.

Feasibility study utilizing a combinatorial functional precision medicine platform, Optim.AI, to help guide treatment for pancreatic cancer.

e13614 Background: Functional screening methods are increasingly applied to guide patient treatment and could be beneficial in indications with poor survival and response rates, such as pancreatic cancer. However, challenges faced when implementing ex vivo testing include complexities in obtaining sufficient tumor cells for different cancer types and maximizing the number of treatments tested with limited sample material. The latter is especially significant in informing clinical decision making as combination therapies are common with cancer, exponentially increasing the number of regimens to select from. To address this hurdle, we developed Optim.AI, a combinatorial functional precision medicine platform, which utilizes efficiently designed experiments and small data AI to expand to over 530,000 phenotypic response data points and predictively rank all actionable treatments within a 12-drug panel. In this feasibility study, we explored the application of Optim.AI on patient-derived tumor cells in pancreatic cancer. Methods: We established a procedure to run Optim.AI with isolated tumor cells from pancreatic cancer patients. After dissociation, these cells were then incubated to allow for 3D organoid formation within the following week. Upon organoid formation, the cells were treated with an FDA-approved panel of chemotherapy and targeted drugs. Cell viability was quantified post-drug treatment for analysis with Optim.AI software to rank all possible single to 4-drug combinatorial therapies for report generation. Results: The viability of the cells post-processing was high (mean = 97.4% live cells), passing the quality check of having at least 60% live cells before proceeding with Optim.AI testing. The success rate of organoid formation was 100% within 1 week of sample processing. The turnaround time from the receipt of samples to dissemination of the reports to clinicians was approximately a week (mean = 7.67 days). Z’ factor, a measurement of statistical effect size for high throughput screening, was demonstrated to be more than 0.5 for the reports generated, indicative of very good assays. The top ranked drug combinations were sensitive, with their normalized cell viability reported to be under 0.2, while the prior line of treatment was shown to be resistant, 100% concordant with previously observed clinical outcomes. Notably, Gemcitabine with either a HDAC or PI3K inhibitor was ranked high amongst the reports generated. Conclusions: Optim.AI had a high success rate in generating reports within a very short timeframe for pancreatic cancer. Comparing the top ranked therapies against the prior lines of treatment allows the clinicians to better evaluate the alternative treatments. Based on this initial feasibility study, Optim.AI could potentially be viable in guiding clinicians in managing treatment options for pancreatic cancer patients.

Abstract 4433: Secondary electrospray ionization mass spectrometry for volatile analysis of lung cancer

Abstract Lung cancer remains a leading cause of cancer-related mortality globally, necessitating innovative approaches for early detection and improved understanding of its metabolic landscape and consequences. Secondary Electrospray Ionization Mass Spectrometry (SESI-MS), an innovative analytical technique, has emerged as a powerful tool for the analysis of volatile organic compounds (VOCs) associated with various diseases, from metabolic disorders to bacterial infections. In this proof-of-concept study, we first utilized SESI-MS analyses of four representative lung cancer cells of both non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) to demonstrate its capability to profile VOCs released by lung cancer cells. The technique allows for real-time, non-invasive analysis of the headspace above cultured cells, enabling the detection of unique VOC signatures associated with different lung cancer subtypes. Meanwhile, SESI-MS also facilitated the discrimination specific cells based on distinct VOC patterns. Furthermore, we also performed cisplatin treatment experiments and observed the metabolic signature shift post drug treatment. These results collectively offered potential applications in early diagnosis and personalized treatment strategies. Next, we continued with in vivo investigations to further extend the utility of SESI-MS in lung cancer research. Animal models, mimicking human lung cancer pathophysiology, have been subjected to SESI-MS analysis to identify volatile biomarkers indicative of tumor development and progression. Several representative cancer drug treatments were performed to the lung cancer mice model, and the drug-induced responses were also monitored via SESI-MS breath analyses. The in vivo SESI-MS approach detected many interesting breath VOC features that could provide valuable insights into the dynamic changes in VOC profiles during various stages of lung cancer, and therefore has the potential to offer a deeper understanding of the disease's metabolic alterations and potential avenues for targeted therapeutic interventions. In conclusion, SESI-MS-based volatile analysis presents a robust and versatile approach for investigating lung cancer, and our ongoing work has the potential to bridge the gap between in vitro and in vivo models for better translational research outcomes. The detection of unique VOC signatures associated with lung cancer cells and tumors holds significant promise for early detection, subtype classification, and monitoring of treatment responses. As technology continues to advance, SESI-MS stands poised to play a pivotal role in transforming the landscape of lung cancer diagnostics and research. Citation Format: Jiangjiang (Chris) Zhu, Fouad Choueiry. Secondary electrospray ionization mass spectrometry for volatile analysis of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4433.

Abstract 6492: KRAS inhibitor-based combinations in pancreatic cancer identified through Optim.AI

Abstract PURPOSE: KRAS is the most frequently mutated oncogene in almost all cancers, including pancreatic ductal carcinoma (PDAC) and colorectal cancer. Increased development efforts targeting KRAS mutations have yielded recent FDA approvals of two KRAS G12C inhibitors. However, this is beneficial for only a small proportion of patients especially with indications like PDAC, where G12C mutations are rare (1%) while G12D mutations account for approximately 45%. Hence, there is a need to develop methods to target a broader range of KRAS mutations. Beyond direct gene mutation, activation of oncogenic KRAS and downstream or parallel signaling through other genomic alterations may benefit from KRAS-specific therapeutic strategies, especially combination therapy. In this study, Optim.AI™, a hybrid computational-experimental platform is harnessed to identify mutation-specific optimal therapies and to stratify efficacious drug combinations containing KRAS inhibitors in pancreatic cancer. METHODS: Optim.AI™ utilizes small datasets to rationally converge upon optimal drug combinations within a defined drug search space. By mapping experimental data points to a second-order quadratic function, Optim.AI™ predicts cell killing efficacies for all possible combinations, independent of the background of the models utilized. In this study, pancreatic cancer cell lines, with varying KRAS mutations, were tested with an array of 155 different combinations consisting of 12 drugs at varying concentrations. The drugs include KRAS G12C inhibitors, Sotorasib and Adagrasib, KRAS inhibitors in development, and approved therapies used clinically for PDAC. Post-drug treatment viability was measured and used for Optim.AI™ analysis, to rank and compare the top therapies across the different cell lines. RESULTS: Through Optim.AI™ analysis, we observed a range of sensitivities towards KRAS inhibition, even for both Sotorasib and Adagrasib. Compared against HT-29 cell line, harboring wild type KRAS, we have identified distinct combinations of KRAS inhibitors with standard of care drugs on cell lines with G12D, G12V and G12C KRAS mutants. Subsequent validation of the top and bottom ranked combinations for the respective cell lines correspond to the predicted Optim.AI™ results. CONCLUSIONS: This study has demonstrated the differential sensitivities of the KRAS inhibitors across several pancreatic cancer cell lines, with different KRAS mutational profiles. Notably, we have identified KRAS inhibitor-based combinations that are distinctly different from wild type KRAS cell line. These preliminary findings could be used to drive precision medicine by identifying suitable biomarkers to potentially stratify pancreatic cancer patients and improve treatment outcomes. Future work includes evaluating the clinical significance of these combinations by validating them on patient-derived organoids. Citation Format: Jhin Jieh Lim, Edward Kai-Hua Chow, Masturah Rashid. KRAS inhibitor-based combinations in pancreatic cancer identified through Optim.AI [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6492.

Timeframe Analysis of Novel Synthetic Cannabinoids Effects: A Study on Behavioral Response and Endogenous Cannabinoids Disruption.

This study investigates the impact of SCs consumption by assessing the effects of three novel synthetic cannabinoids (SCs); MDMB-CHMINACA, 5F-ADB-PINACA, and APICA post-drug treatment. SCs are known for their rapid onset (<1 min) and prolonged duration (≥5 h). Therefore, this research aimed to assess behavioral responses and their correlation with endocannabinoids (ECs) accumulation in the hippocampus, and EC's metabolic enzymes alteration at different timeframes (1-3-5-h) following drug administration. Different extents of locomotive disruption and sustained anxiety-like symptoms were observed throughout all-encompassing timeframes of drug administration. Notably, MDMB-CHMINACA induced significant memory impairment at 1 and 3 h. Elevated levels of anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) were detected 1 h post-MDMB-CHMINACA and 5F-ADB-PINACA administration. Reduced mRNA expression levels of fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL) (AEA and 2-AG degrading enzymes, respectively), and brain-derived neurotrophic factor (BDNF) occurred at 1 h, with FAAH levels remaining reduced at 3 h. These findings suggest a connection between increased EC content and decreased BDNF expression following SC exposure. Cognitive disruption, particularly motor coordination decline and progressive loss manifested in a time-dependent manner across all the analyzed SCs. Our study highlights the importance of adopting a temporal framework when assessing the effects of SCs.

Retention of Histidine-Rich Protein II Rapid Diagnostic Tests in Pregnant Women after Antimalarial Treatment

Malaria is a preventable and curable disease and malaria diagnosis is critical to reduce malaria burden. This study is aimed at investigating the persistent positivity of Histidine-Rich Protein II (HRP2) rapid diagnostic test in pregnant women after antimalarial treatment. The study was carried out in Turai Yar’adua Maternity and Children’s Hospital Katsina and Federal Medical Centre Katsina. Blood samples were collected with the help of trained physicians. The result from this study shows that HRP2 persist more in the blood of pregnant women after treatment with fensider. It was observed that in all the age groups, day 3, day 7 and day 14 post drug treatment, were still positive for malaria using HRP2 malaria rapid diagnostic tool. The HRP2 was positive for all the different age group after 21 and 28 days post treatment except the age group 30-34 (97.5%). The percentage in the positivity result of pregnant women testing positive after treatment with malaria drug changed at 35 and 42 days post treatment in almost all the age groups. There was no statistical significant difference between the different age groups (p&lt;0.05) in HRP2. The persistent positivity decreases in all the trimester at 35 and 42 days post treatment in HRP2. There was no statistical significant difference between the age of pregnancy p&gt;0.05. The highest negative result was observed in the second trimester in 42 days post treatment. This therefore provides a useful data for improved malaria diagnosis, treatment and guide for pregnant women who are susceptible to malaria parasite.

Abstract A128: Analytical and clinical evaluation of a functional combinatorial precision medicine platform

Abstract Non-Hodgkin’s lymphoma (NHL) is a heterogenous malignancy with many different subtypes. Although patients initially respond to treatments, approximately 40% of this cohort will subsequently relapse. After exhausting several lines of treatment, determining the next option is largely empirical. There is a need to develop approaches that are able to identify personalized therapies. There are existing ex vivo approaches that use single-drug sensitivity assays but lack the ability to truly evaluate drug combinations. In this clinical study, we aim to evaluate the potential of an ex vivo combinatorial drug sensitivity platform, Optim.AI™, to identify therapeutic options for relapsed/refractory (RR) NHL. Repeatability, between-run and intermediate precision parameters were used to evaluate the analytical precision of the approach. Optim.AI™ was carried out using K422 and SU-DHL-4 cell lines, either on same or different days by two operators. The cell viability was quantified post-drug treatment and used to calculate the standard deviation (SD) and coefficient of variation (CV) between runs. 98 patients with RR-NHL of both B-NHL and NK/T-NHL origin were analyzed. Single cell suspensions from tumor biopsies or blood aspirates were treated with a panel of drugs with known efficacy. Post-drug treatment cell viability was used as phenotypic input for Optim.AI™ analysis, mapping experimental data points to a second-order quadratic function to predict all other cell killing efficacies. Optim.AI™ results were shared with treating physicians and Optim.AI™-guided therapy was offered in the absence of standard options. For the analytical precision validation, the percentage of replicates with CV ≤ 30% followed the same trend for both cell lines, where 92.2%, 96.1% and 92.6% for repeatability, between-run and intermediate precision parameters respectively were observed for K422 cell line. Out of the 98 successfully generated reports from the processed samples, 35 patient results were evaluable upon analysis where 19 patients were given Optim.AI™ top ranked therapies while the rest were given non-top ranked options, as per physicians’ discretion. Approximately 67% of those with guided therapies achieved clinical benefit, with 7 patients observing complete response while 6 patients attained partial response. For the 35 patients analyzed, the sensitivity of the study was found to be 85% (95% CI, 64-94.8%) while the specificity was at 86.7% (95% CI, 62.1-96.3%). This prospective study demonstrated the precision utility of Optim.AI™, evident from the high percentage of replicates falling within the defined CV. More importantly, it highlights the clinical utility of Optim.AI™ in identifying patient-specific therapies that improved clinical outcomes. The high sensitivity and specificity scores provide confidence in the reliability of the approach to accurately predict efficacious options. The results of this study show the potential of functional precision medicine in identifying patient-specific therapy, while improving precision and personalized cancer treatment. Citation Format: Masturah Mohd Abdul Rashid, Jhin Jieh Lim, Lisa Chow, Edward Kai-Hua Chow. Analytical and clinical evaluation of a functional combinatorial precision medicine platform [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A128.

Abstract A127: Identification of therapeutic drug combinations targeting KRAS

Abstract KRAS is the most frequently mutated oncogene and increased development efforts targeting KRAS mutations have yielded recent FDA approvals of two KRAS G12C inhibitors. However, for some indications, there are other KRAS mutations more common than G12C. For Pancreatic ductal adenocarcinoma (comprising more than 90% of pancreatic cancer cases), 94% harbor KRAS mutations, where G12D mutations account for around 45% while G12C mutations are rare (1%). Hence, there is a need to develop methods to target a broader range of KRAS mutations. Beyond direct gene mutation, activation of oncogenic KRAS and downstream MAPK signaling through other genomic alterations may benefit from KRAS-specific therapeutic strategies, especially combination therapy. We have developed a platform, Optim.AI™, that rationally converges upon optimal drug combinations within a defined drug search space, without any prior knowledge of the background of the models. In this study, we utilized the platform to identify efficacious drug combinations that contain a histone deacetylase 4/6 inhibitor, KYAN-001 in the context of KRAS mutations. 7 cell lines (6 prostate and pancreatic cancer with normal, epithelial THLE-2 cell line) were evaluated with Optim.AI. The cancer cells and THLE-2 were seeded into 384-well plates and treated with 155 different combinations consisting of indication-specific drugs the following day. Post-drug treatment cell viability was used for Optim.AI™ analysis, which maps experimental data points to a second-order quadratic function to predict and rank cancer cell killing efficacies for all other permutations of drug combinations. In androgen resistant prostate cancer, Optim.AI identified synergistic interaction between KYAN-001 and a MEK inhibitor. With MEK being downstream of the KRAS pathway, we wanted to evaluate the efficacy of this combination on pancreatic cancer. Interestingly, this combination was demonstrated to be top ranked against PANC-1 and AsPC-1 cell lines, both harboring G12D KRAS mutation, as compared to a cell line with wild-type KRAS, BxPC-3. This was further validated via half-maximal inhibitory curves (IC50), where synergism was also observed for the combination on AsPC-1, but not BxPC-3. KYAN-001 was also shown to exhibit monotherapy sensitivity against pancreatic cancer patient-derived organoids (PDOs), with a mean of 4 µM. This study has successfully demonstrated the ability of Optim.AI™ to identify novel, optimal drug combinations for both prostate and pancreatic cancers, without any prior background knowledge on these cell lines. With MEK being downstream of the KRAS pathway and 94% of those diagnosed with pancreatic cancer have KRAS mutations, the differential sensitivities of this combination across the cell lines presents the utility of the platform in precision medicine, especially for undruggable targets like KRAS. With promising and demonstrated monotherapy efficacy of KYAN-001 on PDOs, further experiments could be done to evaluate the clinical relevance of both KYAN-001 and MEK inhibition on other relevant ex vivo models. Citation Format: Masturah Mohd Abdul Rashid, Lisa Chow, Edward Kai-Hua Chow. Identification of therapeutic drug combinations targeting KRAS [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A127.

Biomarker-Guided Drug Repurposing and Molecular Validation of Angiotensin-2 Receptor Type-1 in Brain Tumor

Glioma, the most frequent and malignant brain tumor, is one of the most lethal forms of cancer. Among a wide range of aberrantly expressed genes, Angiotensin-2 Receptor Type-1 (AGTR1) is reported to be up-regulated in glioma and associated with aggressive tumor traits and progression. The current study evaluates the anticancer potential of commonly used angiotensin receptor blocker (ARB) telmisartan. A total of eleven, Food and Drug Administration (FDA) approved ARBs were selected, and eight drugs were docked against the AGTR1 receptor. The antihypertensive drug with the highest docking score was selected for in vitro experimentation. Half maximal inhibitory concentration was determined and subsequently applied to patient-derived glioma cell lines. Quantitative gene expression post-drug treatment was determined through real-time polymerase chain reaction (PCR). In vitro growth inhibitory assays revealed that telmisartan at a dose of 45±0.06 μM was able to inhibit 50% of the cell population in malignant glioma U87 cell lines. The PCR results show that AGTR1 expression in the untreated sample was high, as evidenced by 2-ΔΔCt values (342.4, 138.5, 1467.3) for sample IK148-Glioblastma multiform (GBM), IK163-low grade glioma (LGG), and IK231-Medulloblastoma (MDB), respectively. The comparison was made with positive control U-87 cell lines. Following the drug treatment, an increase in the cycle threshold (Ct) mean value for AGTR1 was observed, coinciding with a decrease in mean fold changes of 0.06±0.8, 0.5±2.0, and 0.02±0.02 for GBM, LGG, and MDB samples, respectively. Based on the findings of this study, it can be concluded that telmisartan exhibits successful inhibitory effects against AGTR1 expression in glioma cell lines.

Abstract 5485: Clinical evaluation of a functional combinatorial precision medicine platform reveals combination-specific sub-populations in t-cell lymphoma

Abstract T-cell lymphoma (TCL) is a heterogenous subgroup of non-Hodgkin’s lymphoma with poor prognosis. Approved targeted monotherapies have seen limited success due to lack of effective combinations or identification of sensitive patient populations. Hence, there is a need to develop alternative strategies to identify effective combinations for individual patients and specific TCL sub-populations. We interrogated the potential for an ex vivo combinatorial drug sensitivity platform, Quadratic Phenotypic Optimization Platform (QPOP), in a clinical study for relapsed/refractory Non-Hodgkin Lymphoma (RR-NHL) to identify effective subtype-specific combinations through a functional combinatorial precision medicine approach. We analyzed 37 primary samples from patients with RR-NHL of both T- and NK-cell origin in Singapore, with disease amenable to biopsy or blood/marrow aspiration. Single cell suspensions from tumor samples were treated with a panel of 12 drugs with known preclinical or clinical efficacy against TCL. Post-drug treatment cell viability was used as a phenotypic input for QPOP analysis, which maps experimental data points to a second-order quadratic function to predict cell killing efficacies for all possible permutations of drug combinations. Romidepsin-based combinations appeared among the top ranked QPOP-derived 2-drug combinations with predicted output cutoff &amp;lt; 0.55 in 91.8% of samples treated with similar QPOP drug panel. Among these combinations, combinations with copanlisib, ifosfamide and venetoclax were the most frequently occurring doublet, appearing in 24, 14 and 8 samples, respectively. A total of 10 patients received QPOP-derived romidepsin-based combinations at physicians’ discretion. We noted outcomes of stable disease or better in 7/10 (70%) patients, with clinical benefit seen in 2 complete responses and 2 partial responses (40%). Thus, romidepsin-based combinations showed improved outcomes for TCL patients compared to the prior reported overall response rate of 25% that led to romidepsin’s approval as monotherapy. This study showed the clinical utility of QPOP in identifying combination therapy-specific sub-populations in TCL. Targeted agents, such as romidepsin, copanlisib and venetoclax, that have previously been explored as single agents in TCL were predicted by QPOP to be more effective in the proper combination for the proper patient. This study demonstrates that functional combinatorial precision medicine platforms, such as QPOP, may improve clinical outcomes through identification of more appropriate combination therapies. This work, as well as additional studies in other cancer types, provide evidence towards the need for optimal combination therapy design and the potential of functional precision medicine to improve precision and personalized cancer treatment. Citation Format: Edward Kai-Hua Chow, Masturah Rashid, Sanjay Prasad De Mel, Jasmine Goh, Anand Jeyasekharan. Clinical evaluation of a functional combinatorial precision medicine platform reveals combination-specific sub-populations in t-cell lymphoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5485.

A Metabolomic and Transcriptomic Study Revealed the Mechanisms of Lumefantrine Inhibition of Toxoplasma gondii.

Toxoplasma gondii is an obligate protozoon that can infect all warm-blooded animals including humans. T. gondii afflicts one-third of the human population and is a detriment to the health of livestock and wildlife. Thus far, traditional drugs such as pyrimethamine and sulfadiazine used to treat T. gondii infection are inadequate as therapeutics due to relapse, long treatment period, and low efficacy in parasite clearance. Novel, efficacious drugs have not been available. Lumefantrine, as an antimalarial, is effective in killing T. gondii but has no known mechanism of action. We combined metabolomics with transcriptomics to investigate how lumefantrine inhibits T. gondii growth. We identified significant alternations in transcripts and metabolites and their associated functional pathways that are attributed to lumefantrine treatment. RH tachyzoites were used to infect Vero cells for three hours and subsequently treated with 900 ng/mL lumefantrine. Twenty-four hours post-drug treatment, we observed significant changes in transcripts associated with five DNA replication and repair pathways. Metabolomic data acquired through liquid chromatography-tandem mass spectrometry (LC-MS) showed that lumefantrine mainly affected sugar and amino acid metabolism, especially galactose and arginine. To investigate whether lumefantrine damages T. gondii DNA, we conducted a terminal transferase assay (TUNEL). TUNEL results showed that lumefantrine significantly induced apoptosis in a dose-dependent manner. Taken together, lumefantrine effectively inhibited T. gondii growth by damaging DNA, interfering with DNA replication and repair, and altering energy and amino acid metabolisms.

Early Reduction of Glucose Consumption Is a Biomarker of Kinase Inhibitor Efficacy Which Can Be Reversed with GLUT1 Overexpression in Lung Cancer Cells.

Small molecule inhibitors that target oncogenic driver kinases are an important class of therapies for non-small cell lung cancer (NSCLC) and other malignancies. However, these therapies are not without their challenges. Each inhibitor works on only a subset of patients, the pharmacokinetics of these inhibitors is variable, and these inhibitors are associated with significant side effects. Many of these inhibitors lack non-invasive biomarkers to confirm pharmacodynamic efficacy, and our understanding of how these inhibitors block cancer cell growth remains incomplete. Limited clinical studies suggest that early (< 2weeks after start of therapy) changes in tumor glucose consumption, measured by [18F]FDG PET imaging, can predict therapeutic efficacy, but the scope of this strategy and functional relevance of this inhibition of glucose consumption remains understudied. Here we demonstrate that early inhibition of glucose consumption as can be measured clinically with [18F]FDG PET is a consistent phenotype of efficacious targeted kinase inhibitors and is necessary for the subsequent inhibition of growth across models of NSCLC. We tested nine NSCLC cell lines (A549, H1129, H1734, H1993, H2228, H3122, H460, HCC827, and PC9 cells) and ten targeted therapies (afatinib, buparlisib, ceritinib, cabozantinib, crizotinib, dovitinib, erlotinib, ponatinib, trametinib, and vemurafenib) across concentrations ranging from 1.6nM to 5µM to evaluate whether these inhibitors block glucose consumption at 24-h post-drug treatment and cell growth at 72-h post-drug treatment. We overexpressed the facilitative glucose transporter SLC2A1 (GLUT1) to test the functional connection between blocked glucose consumption and cell growth after treatment with a kinase inhibitor. A subset of these inhibitors and cell lines were studied in vivo. Across the nine NSCLC cell lines, ten targeted therapies, and a range of inhibitor concentrations, whether a kinase inhibitor blocked glucose consumption at 24-h post-drug treatment strongly correlated with whether that inhibitor blocked cell growth at 72-h post-drug treatment in cell culture. These results were confirmed in vivo with [18F]FDG PET imaging. GLUT1 overexpression blocked the kinase inhibitors from limiting glucose consumption and cell growth. Our results demonstrate that the early inhibition of lung cancer glucose consumption in response to a kinase inhibitor is a strong biomarker of and is often required for the subsequent inhibition of cell growth. Early inhibition of glucose consumption may provide complementary information to other biomarkers in determining whether a drug will effectively limit tumor growth.

3D Bioprinted Silk-Based In Vitro Osteochondral Model for Osteoarthritis Therapeutics.

3D bioprinting of osteochondral tissue offers unique opportunities for enabling precise pharmacological interventions in osteoarthritis (OA). The current study investigates the screening potential of anti-inflammatory drugs using bioprinted inflamed human osteochondral units. The biomimetic hierarchical geometry is bioprinted using silk-based bioinks encapsulating pre-differentiated stem cells, creating an in vitro model. Inflammation is stimulated in the model, using tumor necrosis factor-alpha and Interleukin-1 beta pro-inflammatory cytokines. The resultant degeneration, akin to OA, is flagged by key markers like sulfated glycosaminoglycan, collagen, alkaline phosphatase, and downregulation of osteochondral transcript levels. In the next step, the screening of anti-inflammatory drugs is validated using celecoxib and rhein. Consequently, in the inflamed constructs, the initial upregulation of the key inflammatory mediators (nitric oxide, Prostaglandin E2), is subsequently downregulated, post-drug treatment. In addition, catabolic markers (matrix metalloproteinases and aggrecanase-1), indicative of hypertrophic and apoptosing chondrocytes, are significantly downregulated in the treatment groups; while the transcript and protein levels required for osteochondral health are attenuated. Therefore, the in vitro model mimicks the inflammation in the early stages of OA, and corroborates a potential high-throughput platform for screening novel anti-inflammatory drugs in OA therapeutics.

Effects of anti-TNF biologic drugs on uveitis severity in Behçet patients: systematic review and Meta-analysis.

To investigate effects of anti-TNF biologic drugs on uveitis severity (comparing visual acuity logMAR levels) in Behçet patients. Three databases PubMed, Scopus, and the Web of Science were searched for qualified papers focusing on the anti-TNF-α factors treatment in Behçet's disease (BD)-associated uveitis. Studies that were designed pre and post anti-TNF drug treatment, were selected. After determining the search strategy for this study, the relevant data were extracted. The initial search was performed in the target databases and a total of about 1458 articles were found. Fifteen articles were selected for systematic review and only 12 of them had inclusion criteria for Meta-analysis (with visual acuity data). The mean dose of prednisolone before and after biological treatments was reported in 5 studies (28.56 and 7.56 mg/kg, respectively). Also, the preliminary results indicate a significant reduction in visual acuity logMAR levels (MD=-1.5 IU/L, 95%CI: -2.1, -0.01). Biological drugs significantly reduce the dose of prednisolone and affect visual acuity values.

Application of Urine and Copro Antigen Assays after Primary Infection and Drug Treatment in an Experimental Opisthorchiasis Animal Model.

Infection by Opisthorchis viverrini causes significant health problems, including cholangiocarcinoma (CCA); thus control and elimination of this trematode is an important strategy for the reduction of CCA. Currently, urine and copro antigen detection is more sensitive than parasitological examination of the feces for the diagnosis of opisthorchiasis. Given limitations in human studies, we used an animal model to quantify the parasite antigen profiles in urine and feces in O. viverrine-infected hamsters, and postdrug treatment. The positive detections of O. viverrini antigen began from week 1 in urine and week 2 in feces after infection until week 28 of the study. The recoveries of O. viverrini worms were detected starting from week 1 and eggs of O. viverrini were detected in feces from week 3 after infection and remained detectable throughout the study period. There was a significant positive correlation of urine and copro antigen levels with the number of fecal egg counts (P < 0.01) and worm recovery (P < 0.01). In the drug-treatment experiment, treatment of infected hamsters with praziquantel significantly reduced worm burden, fecal egg output, and antigen in urine and feces compared with the untreated controls (P < 0.001). At 4 weeks posttreatment, the egg and worm reduction rates were 100% and 95.5%, respectively. The positive antigen detections in urine and feces corresponded with partial worm clearance from praziquantel treatment. This study demonstrated a direct link of urine and copro antigen tests with worms infecting the liver thereby reaffirming the reliability of urine and copro antigen assay in opisthorchiasis diagnosis.

Pramipexole treatment attenuates mechanical hypersensitivity in male rats experiencing chronic inflammatory pain

Opioids are commonly prescribed for pain despite growing evidence of their low efficacy in the treatment of chronic inflammatory pain and the high potential for misuse. There is a clear need to investigate non-opioid alternatives for the treatment of pain. In the present study, we tested the hypothesis that acute and repeated dopamine agonist treatment would attenuate mechanical hypersensitivity in male Long-Evans rats experiencing chronic inflammatory pain. We used two clinically available therapeutics, l-DOPA (precursor of dopamine biosynthesis) and pramipexole (dopamine D2/3 receptor agonist), to examine the functional role of dopamine signaling on mechanical hypersensitivity using an animal model of chronic inflammatory pain (complete Freund's adjuvant, CFA). We found that both acute and repeated pramipexole treatment attenuated hyperalgesia-like behavior in CFA-treated animals but exhibited no analgesic effects in control animals. In contrast, there was no effect of acute or repeated l-DOPA treatment on mechanical hypersensitivity in either CFA- or saline-treated animals. Notably, we discovered some extended effects of l-DOPA and pramipexole on decreasing pain-like behavior at three days and one week post-drug treatment. We also examined the effects of pramipexole treatment on glutamatergic and presynaptic signaling in pain- and reward-related brain regions including the nucleus accumbens (NAc), dorsal striatum (DS), ventral tegmental area (VTA), cingulate cortex (CC), central amygdala (CeA), and periaqueductal gray (PAG). We found that pramipexole treatment decreased AMPA receptor phosphorylation (pGluR1845) in the NAc and DS but increased pGluR1845 in the CC and CeA. A marker of presynaptic vesicle release, pSynapsin, was also increased in the DS, VTA, CC, CeA, and PAG following pramipexole treatment. Interestingly, pramipexole increased pSynapsin in the NAc of saline-treated animals, but not CFA-treated animals, suggesting blunted presynaptic vesicle release in the NAc of CFA-treated animals following pramipexole treatment. To examine the functional implications of impaired presynaptic signaling in the NAc of CFA animals, we used ex vivo electrophysiology to examine the effects of pramipexole treatment on the intrinsic excitability of NAc neurons in CFA- and saline-treated animals. We found that pramipexole treatment reduced NAc intrinsic excitability in saline-treated animals but produced no change in NAc intrinsic excitability in CFA-treated animals. These findings indicate alterations in dopamine D2/3 receptor signaling in the NAc of animals with a history of chronic pain in association with the anti-hyperalgesic effects of pramipexole treatment.

A Novel Therapeutic Mechanism of Imipridones ONC201/ONC206 in MYCN-Amplified Neuroblastoma Cells via Differential Expression of Tumorigenic Proteins.

Neuroblastoma is the most common extracranial nervous system tumor in children. It presents with a spectrum of clinical prognostic measures ranging from benign growths that regress spontaneously to highly malignant, treatment evasive tumors affiliated with increased mortality rates. MYCN amplification is commonly seen in high-risk neuroblastoma, rendering it highly malignant and recurrence prone. In our current study, we investigated the therapeutic potential of small molecule inducers of TRAIL, ONC201, and ONC206 in MYCN-amplified IMR-32 and non-MYCN-amplified SK-N-SH human neuroblastoma cell lines. Our results exhibit potent antitumor activity of ONC201 and ONC206 via a novel inhibition of EGF-induced L1CAM and PDGFRβ phosphorylation in both cell lines. Drug treatment significantly reduced cellular proliferation, viability, migration, invasion, tumorsphere formation potential, and increased apoptosis in both cell lines. The protein expression of tumorigenic NMYC, Sox-2, Oct-4, FABP5, and HMGA1 significantly decreased 48 h post-drug treatment, whereas cleaved PARP1/caspase-3 and γH2AX increased 72 h post-drug treatment, compared with vehicle-treated cells in the MYCN-amplified IMR-32 cell line. We are the first to report this novel differential protein expression after ONC201 or ONC206 treatment in human neuroblastoma cells, demonstrating an important multitarget effect which may yield added therapeutic benefits in treating this devastating childhood cancer.

Inhibition of XPO1 By KPT-330 (Selinexor) Enhances Cell Death Induced By the Bcl-2 Selective Inhibitor ABT-199 (Venetoclax) through Downregulation of Mcl-1 in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a serious disease and despite an improved understanding of the biology of AML, little has changed in the treatment of the disease over the last four decades. The 5 year survival rates of AML remain frustratingly low. Resistance to frontline chemotherapy remains the main cause of treatment failure; therefore a new treatment modality is urgently needed.Bcl-2 is a promising therapeutic target; it is overexpressed in both bulk AML cells and leukemia-initiating cells. The Bcl-2-selective inhibitor ABT-199 (Venetoclax) shows promising anti-leukemic activity in patients with high-risk relapsed/refractory AML or unfit for intensive chemotherapy. However, resistance through another Bcl-2 family protein, Mcl-1, was shown by our lab and others to limit the anti-leukemic activity of ABT-199. Previous studies from our lab have shown that ABT-199 monotherapy decreases the association of Bim with Bcl-2 but is compensated for by its increased association with Mcl-1. We also found that ABT-199 increases cellular Mcl-1 levels by extending the protein's half-life. Based on these previous studies we tested the efficacy of direct inhibition of Bcl-2 by ABT-199 and Mcl-1 by A-1210477. ABT-199 and A-1210477 synergistically induced apoptosis in both ABT-199-resistant and sensitive AML cells. This combination treatment disrupted binding of Bim with Bcl-2 and Mcl-1 concurrently, with no observed binding of Bim to Bcl-xL. This work has demonstrated that targeting both Bcl-2 and Mcl-1 is necessary for AML treatment. However, our work also indicated that direct targeting of Mcl-1 with A-1210477 to enhance the anti-leukemic activity of ABT-199 might cause increased toxicity. Thus, indirect targeting of Mcl-1 may represent a better approach to enhance the anti-leukemic activity of ABT-199 in AML.It was recently reported that targeting exportin 1 (XPO1), a nuclear exporter overexpressed in AML cells, selectively targets leukemia-initiating cells and can decrease Mcl-1 levels in cancer cells, though the underlying mechanism is unknown. KPT-330 (Selinexor) is a XPO1-selective inhibitor which is currently being tested in clinical trials for AML. Preliminary results have shown that KPT-330 induces a response at well-tolerated doses in AML and other hematological malignancies. Thus, XPO1 inhibition may represent a viable strategy for downregulating Mcl-1 to synergize with ABT-199 treatment in AML.To test our hypothesis we tested various concentrations of ABT-199 and KPT-330 alone and in combination in AML cell lines and primary patient samples. At 24 h, synergistic induction of apoptosis was observed between the two drugs in THP-1, CTS, Molm-13, and OCI-AML3 AML cell lines. Synergy was also found in 4 relapsed and 6 newly diagnosed primary AML patient samples. To test the effect of drug treatment on leukemia-initiating cells, we performed colony formation assays on primary AML patient samples post drug treatment and found that combination treatment significantly enhanced inhibition of colony formation when compared to single drug treatment, demonstrating that KPT-330 combined with ABT-199 is effective in targeting the leukemic bulk and the leukemia-initiating cells.To determine the mechanism by which the combination works, we measured the protein levels of various Bcl-2 family members. Decreased protein levels for Mcl-1 and surprisingly Bim were observed post KPT-330 or KPT-330 plus ABT-199 treatment, while protein levels for Bcl-2 and Bcl-xL remained unchanged. A time course experiment revealed that Mcl-1 levels decreased at apoptosis initiation while Bim levels decreased after apoptosis initiation, suggesting that Mcl-1 plays a critical role in apoptosis induced by the drug combination, while Bim may not. Mcl-1 knockdown enhanced combination treatment of ABT-199 and KPT-330. Reciprocal Mcl-1 overexpression partially rescued the cells from drug treatment but was incomplete, likely due to Mcl-1 overexpression not being high enough and/or non-Mcl-1 pathways through which KPT-330 acts. In summary, Mcl-1 is modulated by KPT-330 treatment alone and in combination with ABT-199 and this modulation of Mcl-1 plays an important role in initiation of apoptosis in response to treatment. Additional studies are underway to further elucidate the molecular mechanisms of combination ABT-199 and KPT-330 treatment and to determine the in vivo anti-leukemic efficacy in NSG mouse AML models. DisclosuresNo relevant conflicts of interest to declare.