Sigmoidal Dose-response Curve Research Articles

In vitro antileishmanial activities of hydro-methanolic crude extracts and solvent fractions of Clematis simensis fresen leaf, and Euphorbia abyssinica latex.

As a result of increasing drug resistance, crossover resistance development, prolonged therapy, and the absence of different agents with innovative methods for implementation, the efficacy of recent antileishmanial medications is severely declining. So, it is vital to look for other medications from botanical remedies that have antileishmanial activity. The latex of Euphorbia abyssinica (E abyssinica) and the leaves of Clematis simensis fresen (C simensis) were macerated in methanol (80%). In vitro antileishmanial activity of the preparation was tried on promastigotes of Leishmania aethiopica (L aethiopica) and Leishmania donovani (L donovani) using resazurin assay, and fluorescence intensity was measured. One percent of dimethyl sulfoxide (DMSO) and media as negative control and amphotericin B as positive control were used. Additionally, hemolytic & phytochemical tests of the preparation were done. The mean and standard errors of each extract were evaluated and interpreted for statistical significance using one-way analysis of variance. From sigmoidal dose-response curves of % inhibition, half maximal inhibitory concentration (IC50) values were determined by GraphPad Prism and Microsoft Excel; outcomes were presented as mean ± standard error of mean of triplicate trials. P < .05 was statistical significance. The phytochemical screening of C simensis and E abyssinica confirmed the existence of steroids, phenols, tannins, saponins, alkaloids, terpenoids, flavonoids and glycosides. C simensis possesses antileishmanial activity with IC50 outcomes of 46.12 ± 0.03 and 8.18 ± 0.10 µg/mL on the promastigotes of L aethiopica and L donovani, respectively. However, E abyssinica showed stronger activity with IC50 outcomes of 16.07 ± 0.05 µg/mL and 4.82 ± 0.07 µg/mL on L aethiopica and L donovani, respectively. C simensis and E abyssinica have a less hemolytic effect on human red blood cells at low concentrations. The outcomes from this investigation demonstrated that the preparation of C simensis and E abyssinica indicated significant antileishmanial activity. Therefore, further in vivo assessment of antileishmanial, cytotoxicity activity and quantitative identification of secondary metabolites are highly recommended.

Dissociation of Centrally and Peripherally Induced Transcranial Magnetic Stimulation Effects in Nonhuman Primates.

Transcranial magnetic stimulation (TMS) is a noninvasive brain stimulation method that is rapidly growing in popularity for studying causal brain-behavior relationships. However, its dose-dependent centrally induced neural mechanisms and peripherally induced sensory costimulation effects remain debated. Understanding how TMS stimulation parameters affect brain responses is vital for the rational design of TMS protocols. Studying these mechanisms in humans is challenging because of the limited spatiotemporal resolution of available noninvasive neuroimaging methods. Here, we leverage invasive recordings of local field potentials in a male and a female nonhuman primate (rhesus macaque) to study TMS mesoscale responses. We demonstrate that early TMS-evoked potentials show a sigmoidal dose-response curve with stimulation intensity. We further show that stimulation responses are spatially specific. We use several control conditions to dissociate centrally induced neural responses from auditory and somatosensory coactivation. These results provide crucial evidence regarding TMS neural effects at the brain circuit level. Our findings are highly relevant for interpreting human TMS studies and biomarker developments for TMS target engagement in clinical applications.SIGNIFICANCE STATEMENT Transcranial magnetic stimulation (TMS) is a widely used noninvasive brain stimulation method to stimulate the human brain. To advance its utility for clinical applications, a clear understanding of its underlying physiological mechanisms is crucial. Here, we perform invasive electrophysiological recordings in the nonhuman primate brain during TMS, achieving a spatiotemporal precision not available in human EEG experiments. We find that evoked potentials are dose dependent and spatially specific, and can be separated from peripheral stimulation effects. This means that TMS-evoked responses can indicate a direct physiological stimulation response. Our work has important implications for the interpretation of human TMS-EEG recordings and biomarker development.

Functional design of experiment for potency assay optimization and in-silico simulation

For biotherapeutic analytics, robust and reliable potency assays are required. Design of experiment (DoE) approaches are used to investigate the impact of multiple assay parameters. Currently, specific assay features (e.g., half effective concentration) are modelled independently from each other. A joint interpretation of several assay features is thus difficult. In our functional DoE approach, we use the functional relationship of the assay features to describe the sigmoidal dose-response curve. With the composed functional form, the direct impact of assay parameters on the dose-response curve shape was modelled. Moreover, a multivariate desirability was defined and used for assay optimization. We believe that functional modelling contributes to understanding the joint influence of assay parameters and helps to design robust biotherapeutic analytics.

Abstract 5322: High throughput chemotherapeutic drug screening system for gastric cancer: cure-GA

Abstract To discover clinically applicable anticancer drugs and predict therapeutic response for advanced gastric cancer, we developed a high-throughput drug (HTD) screening system that could rapidly evaluate drug reactivity using 3D cultured primary cells derived from gastric cancer (GC) patients. Primary cancer cells were isolated from fresh surgical specimens that resected from 143 GC patients using Gentle Max tissue dissociation system. Primary cells were mixed with Matrigel, and placed on a micropillar for three-dimensional (3D) culture. After the primary cells were stabilized in the complete culture medium (CCM) than added various chemotherapeutic drugs containing 5-FU, Oxaliplatin, and Paclitaxel in CCM and incubated for 7 days. Cell viability was determined through calcein staining and quantified scanned images. The IC50 for each drug was calculated by a sigmoidal dose-response curve, using the GraphPad Prism 9 program. The average weight of gastric cancer tissue used in the experiment was 300 mg (75 mg ~ 1930 mg), and the average number of dissociated viable cells for each tissue was 3.9 × 10^6 cells/case. About 2.4 × 10^5 live cell was required per drug, we were able to obtain an average of 6.4 (Min.2, Max 14) drug reactivity data per tissue using the HTD screening system. GC tissues obtained from the operating room were dissociated within 16 hours and then loaded into the HTS system within 3 hours. Cells were stabilized for 1 day in 3D culture plate and exposed to the drug for 7 days, and then data reports were made within 3 days. As a result, it was possible to obtain within 14 days from fresh surgical GC tissue to drug response data. Additionally, we confirmed that 3D cultured primary cells derived from GC tissues consistently preserved primary characters using IHC. Similar to their parental cancer tissue, GC 3D cultured primary cells derived from adenocarcinoma large glandular patterns and retain the expression of some marker proteins. In this study, we evaluated the drug response data for 101 cases (success rate 71%; 101/143) to 5-FU, Oxaliplatin, and Palitaxel, etc. using the HTD screening system and it was confirmed that individual patient had a difference response to each drug. Here we established the HTD screening system using 3D cultured GC patient derived primary cells. The advantages of this system were that it is the first model system that directly used patient-derived primary cells for drug screening, and it can rapidly evaluate drug reactivity to various anticancer drugs within 10 days. The HTD screening system based on patient-derived primary cells can provide that information to predict drug response and allow for finding more appropriate therapy for each patient. Citation Format: Jieun Lee, Jung Eun Kim, Sanjun Lee, Tae-Kyeong Lee, In Hee Kim, So Hee Yoon, Mira Yoo, Eunju Lee, Doo-Young Hwang, So Hyun Kang, Bo Sung Ku, Dong Woo Lee, Young Suk Park, Ji-Won K, Jin Won Kim, Sang-Hoon Ahn, Keun-Wook Lee, Hyung-Ho Kim, Hyun Jung Oh, Yun-Suhk Suh. High throughput chemotherapeutic drug screening system for gastric cancer: cure-GA. [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 5322.

Radiobiological modeling of radiation-induced acute rectal mucositis: A single-institutional study of cervical carcinoma.

This study aimed to estimate the fitting parameters of sigmoidal dose-response (SDR) curve of radiation-induced acute rectal mucositis in pelvic cancer patients treated with Intensity Modulated Radiation Therapy (IMRT) for the calculation of normal tissue complication probability (NTCP). Thirty cervical cancer patients were enrolled to model the SDR curve for rectal mucositis. The patients were evaluated weekly for acute radiation-induced (ARI) rectal mucositis toxicity and their scoring was performed as per Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. The radiobiological parameters, namely n, m, TD50, and γ50 were calculated from the fitted SDR curve obtained from the clinical data of cervical cancer patients. ARI toxicity for rectal mucosa in carcinoma of cervical cancer patients was calculated for the endpoint rectal mucositis. The n, m, TD50, and γ50 parameters from the SDR curve of Grade 1 and Grade 2 rectal mucositis were found to be 0.328, 0.047, 25.44 ± 1.21 (confidence interval [CI]: 95%), and 8.36 and 0.13, 0.07, 38.06 ± 2.94 (CI: 95%), and 5.15, respectively. This study presents the fitting parameters for NTCP calculation of Grade 1 and Grade 2 ARI rectal toxicity for the endpoint of rectal mucositis. The provided nomograms of volume versus complication and dose versus complication for different grades of rectal mucositis help radiation oncologists to decide the limiting dose to reduce the acute toxicities.

Radiobiological modeling of radiation-induced acute mucosal toxicity (oral mucositis and pharyngeal mucositis): A single-institutional study of head-and-neck carcinoma.

This study aimed to estimate the fitting parameters of sigmoidal dose-response (SDR) curve of radiation-induced acute oral and pharyngeal mucositis in head-and-neck (H and N) cancer patients treated with Intensity Modulated Radiation Therapy (IMRT) for the calculation of normal tissue complication probability (NTCP). Thirty H-and-N cancer patients were enrolled to model the SDR curve for oral and pharyngeal mucositis. The patients were evaluated weekly for acute radiation-induced (ARI) oral and pharyngeal mucositis toxicity, and their scoring was performed as per the common terminology criteria adverse events version 5.0. The radiobiological parameters, namely n, m, TD50, and γ50 were calculated from the fitted SDR curve obtained from the clinical data of H-and-N cancer patients. ARI toxicity for oral and pharyngeal mucosa in carcinoma of H-and-N cancer patients was calculated for the endpoint oral mucositis and pharyngeal mucositis. The n, m, TD50, and γ50 parameters from the SDR curve of Grade 1 and Grade 2 oral mucositis were found to be [0.10, 0.32, 12.35 ± 3.90 (confidence interval [CI] 95%) and 1.26] and [0.06, 0.33, 20.70 ± 6.95 (CI 95%) and 1.19] respectively. Similarly for pharyngeal mucositis, n, m, TD50, and γ50 parameters for Grade 1 and Grade 2 were found to be [0.07, 0.34, 15.93 ± 5.48 (CI. 95%) and 1.16 ] and [0.04, 0.25, 39.02 ± 9.98(CI. 95%) and 1.56] respectively. This study presents the fitting parameters for NTCP calculation of Grade 1 and Grade 2 ARI toxicity for the endpoint of oral and pharyngeal mucositis. The provided nomograms of volume versus complication and dose versus complication for different grades of oral mucositis and pharyngeal mucositis help radiation oncologists to decide the limiting dose to reduce the acute toxicities.

Radiobiological modelling of radiation-induced acute skin toxicity (dermatitis): A single institutional study of breast carcinoma.

The purpose of the study was to estimate the fitting parameters of the sigmoidal dose response (SDR) curve of radiation-induced acute dermatitis in breast cancer patients treated with intensity-modulated radiation therapy for calculation of normal tissue complication probability (NTCP). Twenty-five breast cancer patients were enrolled to model the SDR curve for acute dermatitis. The acute radiation-induced (ARI) dermatitis toxicity was assessed weekly for all the patients, and their scores were determined using the common terminology criterion adverse events version 5.0. The radiobiological parameters n, m, TD50, and γ50 were derived using the fitted SDR curve obtained from breast cancer Patient's clinical data. ARI dermatitis toxicity in carcinoma of breast patients was calculated for the end point of acute dermatitis. The n, m, TD50, and γ50 parameters from the SDR curve of Grade-1 dermatitis are found to be 0.03, 0.04, 28.65 ± 1.43 (confidence interval [CI] 95%) and 1.02 and for Grade-2 dermatitis are found to be 0.026, 0.028, 38.65 ± 1.93 (CI. 95%) and 1.01 respectively. This research presents the fitting parameters for NTCP calculation of Grade-1 and Grade-2 acute radiation-induced skin toxicity in breast cancer for the dermatitis end point. The presented nomograms of volume versus complication probability and dose versus complication probability assist radiation oncologists in establishing the limiting dose to reduce acute toxicities for different grades of acute dermatitis in breast cancer patients.

An Agent-Based Model of Radiation-Induced Lung Fibrosis.

Early- and late-phase radiation-induced lung injuries, namely pneumonitis and lung fibrosis (RILF), severely constrain the maximum dose and irradiated volume in thoracic radiotherapy. As the most radiosensitive targets, epithelial cells respond to radiation either by undergoing apoptosis or switching to a senescent phenotype that triggers the immune system and damages surrounding healthy cells. Unresolved inflammation stimulates mesenchymal cells' proliferation and extracellular matrix (ECM) secretion, which irreversibly stiffens the alveolar walls and leads to respiratory failure. Although a thorough understanding is lacking, RILF and idiopathic pulmonary fibrosis share multiple pathways and would mutually benefit from further insights into disease progression. Furthermore, current normal tissue complication probability (NTCP) models rely on clinical experience to set tolerance doses for organs at risk and leave aside mechanistic interpretations of the undergoing processes. To these aims, we implemented a 3D agent-based model (ABM) of an alveolar duct that simulates cell dynamics and substance diffusion following radiation injury. Emphasis was placed on cell repopulation, senescent clearance, and intra/inter-alveolar bystander senescence while tracking ECM deposition. Our ABM successfully replicates early and late fibrotic response patterns reported in the literature along with the ECM sigmoidal dose-response curve. Moreover, surrogate measures of RILF severity via a custom indicator show qualitative agreement with published fibrosis indices. Finally, our ABM provides a fully mechanistic alveolar survival curve highlighting the need to include bystander damage in lung NTCP models.

Abstract 6039: Application of triple negative breast cancer patient-derived organoid and xenograft model for drug screening

Abstract Background: Breast cancer Patient Derived Organoids (PDOs) and patient-derived xenografts (PDXs) which recapitulate the tumor heterogeneity and molecular signatures of original tumor tissue, are useful tools to enable greater precision of both approved and experimental medicines through personalized therapeutic approaches. We focus on use of ex vivo PDOs, PDX-derived organoids (PDXOs), and vivo PDXs models for drug testing and personalized therapy. In this study, we investigated the oncogenic role and therapeutic potency of targeting WEE1 in breast cancer. Methods: Fresh surgical specimens of primary TNBC breast tumor (N=3) were used for PDOs. PDOs and PDX-derived organoids (PDXOs) were cultured in previously reported media compositions based on ECM hydrogel. PODs and PDXOs were subjected to drug screening for 22 anticancer drugs including chemoreagents and targeted drugs in the 3D HTS system. Drug sensitivity was tested in triplicate in different concentration ranges for 7 days. The IC50 for each drug was calculated by a sigmoidal dose-response curve, using the GraphPad Prism 9 program. For drug screening in PDXs models, PDOs were injected into the fourth mammary fat pads of NOD.Cg-Prkdcscid Il2rgtm1wjl/SzJ mice. WEE1 inhibitor (AZD1775) was administered by oral gavage (AZD1775, 30 mg/kg) for 3 weeks. Immunostaining of ER, PR, Her2, CK5 and Ki67 was performed in PODs, PODXs and PDX tumor tissues. Results: All PDOs, PDXs, and PDXOs were ER-, PR- and HER2- negative. PDOs and PDXOs (172T, 185T, 207T) showed differential response to 22 anticancer drugs. In PDOs and PDXOs models, 172T and 207T PDOs and PDXOs are highly sensitive to AZD1775, but 185T PDOs and PDXOs less sensitive or never respond to treatment with AZD1775 (10 µM). In PDX models, treatment with AZ1775 decreased tumor growth (P&amp;lt;0.0079) as well as distance metastasis in 207T-PDX mice, whereas in 172T- and 185T-PDX mice, the tumor growth and metastasis were not reduced compared with the vehicle group. Conclusion: Consequently, PDOs, PDXOs and PDXs recapitulated immunohistological signatures of original tumor tissues. The results of WEE1 inhibitor response between PDOs, PDXOs and PDXs were consistent. This study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified drugs. Citation Format: Seungyeon Ryu, Hoe Suk Kim, Jung Eun KIM, So-Hyun Yoon, Sangeun Lee, Moonjou Baek, Han-Byoel Lee, Dong Woo Lee, Bosung Ku, Wonshik Han. Application of triple negative breast cancer patient-derived organoid and xenograft model for drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6039.

Abstract B025: Antifragile therapy

Abstract We present a novel paradigm of evolutionary cancer therapy based on the “antifragility” of the drug dose-response function. Antifragility is the word originally coined to describe the opposite of fragility. Systems or organisms can be described as antifragile if they derive a benefit from systemic variability, volatility, randomness, or disorder. In this work, we quantify the evolutionary and ecological benefit (or harm) derived from increasing dose volatility in treatment scheduling. Nonlinear sigmoidal dose-response curves are ubiquitous in medicine and have both convex and concave regions. Despite the ubiquity of dose response assays in biological sciences, these curves are typically used to measure differential response in first-order effects (mean value of drug dose delivered), while second-order effects (variance of drug dose) are generally ignored. Analysis of the convexity of dose response curves provides a direct prediction of response to continuous treatment (“even” schedules with zero volatility) in comparison to high-dose/low-dose treatment (“uneven” schedules with high volatility). For example, if the dose response function is antifragile (concave) near a dose of ‘x’, continuous administration of x may have a less efficacious response compared to a regimen that switches equally between 120% of x and 80% of x, even though both regimens use the same total drug. Mathematical analysis of dose response curves in vitro for a H3122 ALK-positive non-small cell lung cancer (NSCLC) cell line predicts that evolved-resistance cell lines can be more effectively treated using volatile treatment scheduling regimens, while treatment-naïve cell lines are most effectively treated by continuous treatment. However, selection pressure due to treatment selects for resistant phenotypes over time. We construct a mathematical model of gradual resistance, parameterized to data, and predict time-dependent antifragility in continuous (8 weeks), volatile (8 weeks) ALK inhibition in vivo. The key insight is that dose-response concavity (“anti-fragility”) increases in proportion to the amount of resistance in the tumor population. Antifragility provides a time-dependent metric which 1) predicts the emergence of resistance and 2) determines the optimal subsequent dosing strategy. Previous work indicates that resistance to ALK inhibitors occurs gradually, through the acquisition of multiple cooperating genetic and epigenetic adaptive changes. This observation led us to hypothesize that there is a critical point in the evolution of ALK-positive tumors where it is optimal to switch from continuous treatment to volatile dosing to optimally control the onset of gradual resistance. This hypothesis is also tested in vivo, comparing continuous and volatile treatment schedules of ALK inhibitors to a switching schedule of continuous-volatile (4 weeks each). We end by discussing the implications for adaptive therapy. Citation Format: Jeffrey West, Bina Desai, Maximilian Strobl, Luke Pierik, Richard Miles, Cole Armagost, Mark Robertson-Tessi, Andriy Marusyk, Alexander R. A. Anderson. Antifragile therapy [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B025.

Radiobiological modeling of radiation-induced acute proctitis: A single-institutional study of prostate carcinoma.

To estimate the fitting parameters of the sigmoidal dose response (SDR) curve of radiation-induced acute proctitis in prostate cancer patients treated with intensity modulated radiation therapy (IMRT) for the calculation of normal tissue complication probability (NTCP). Twenty-five prostate cancer patients were enrolled and evaluated weekly for acute radiation-induced (ARI) proctitis toxicity. Their scoring was performed as per common terminology criteria for adverse events version 5.0. The radiobiological parameters namely n, m, TD50, and γ50 were calculated from the fitted SDR curve obtained from the clinical data of prostate cancer patients. ARI toxicity for rectum in carcinoma of prostate patients was calculated for the endpoint of acute proctitis. The n, m, TD50, and γ50 parameters from the SDR curve of Grade 1 and Grade 2 acute proctitis are found to be 0.13, 0.10, 30.48 ± 1.52 (confidence interval [CI] 95%), 3.18 and 0.08, 0.10, 44.37 ± 2.21 (CI 95%), 4.76 respectively. This study presents the fitting parameters for NTCP calculation of Grade-1 and Grade-2 ARI rectum toxicity for the endpoint of acute proctitis. The provided nomograms of volume versus complication and dose versus complication for different grades of acute proctitis in the rectum help radiation oncologists to decide the limiting dose to reduce the acute toxicities.

Abstract P5-02-02: Development of automated 3D high-throughput drug screening platform for patient-derived breast cancer organoids

Abstract Background Patient-derived cancer organoids, which reliably conserve original features of tumors, are emerging as an excellent model for predicting therapy response and drug screening. Developing optimized 3D high-throughput drug screening platform to establish patient-derived cancer organoids and simultaneously perform drug screening is essential for personalized medicine. Methods We established normal breast organoids (n=4) and breast cancer organoids (n=10) from 20 fresh surgical specimen (normal 7, tumor 13 cases). A number (500-2000) of normal and cancer cells were automatically dispensed with the ASFA™ Spotter ST and organoids were generated by hydrogel hanging-drop culture on Cellvitro™ Pillar platform (Medical &amp; Bio Decision, South Korea). Organoids were subjected to drug screening for 17 anticancer drugs including chemoreagents and targeted drugs in the 3D HTS system. Drug sensitivity was tested in triplicate in different concentration ranges for 5 days. Drug cytotoxic effect was assessed by calcein AM staining. Acquisition and analysis of high-content 3D organoid images were peformed using ASFA™ SCANNER (Medical &amp; Bio Decision, South Korea). The IC50 for each drug was calculated by a sigmoidal dose-response curve, using the GraphPad Prism 9 program. We analyzed a drug response index (DRI) using a prediction alogorithm to evalute drug sensitivity (DRI&amp;lt;-0.5) and resistance (DRI&amp;gt;0.5). Results We summarized the DRI value of patient-derived breast organoids of 7 drugs (Table 1). 6 tumor organoids (2T, 6T, 8T, 10T, 13T, 15T) showed high sensitivity to Docetaxel, Doxorubicin, Paclitaxel, and Gemcitabin while 4 tumor organoids (1T, 9T, 14T, 16T) were less sensitive and resistant. 2 tumor organoids (10T, 16T) were sensitive to Tamoxifen and 2 tumor organoids (6T, 8T) show high sensitivity to palbociclib and erlotinib. Normal organoids show less sensitivity and resistance to chemotherapeutic drugs. Drug response index &amp;gt;0.5 : resistancy top 30%, Drug response index &amp;lt;-0.5 : sensitivity top 30%. Conclusions Herein, we developed the hydrogel hanging-drop culture on Cellvitro™ Pillar platform for easily and rapidly high-throughput drug screening in patient-derived organoids using a small number of cells by testing clinically actionable drugs at different concentrations. There were different drug response indeces for each individual organoids to chemoreagents and targeted drugs. We anticipate that 3D high-throughput drug screenings platform based on patient-derived organoids can provide the information to predict drug response and allow for finding more appropriate therapy for individual patients. Table 1.DRI values of patient-derived breast organoids of 7 drugs.Patient No.Tumor/NormalDocetaxelPaclitaxelDoxorubicinTamoxifenGemcitabinePalbociclibErlotinib1TTumor1.051.261.080.060.590.26-2TTumor-0.64-0.72-0.60-0.08-0.79--6TTumor-0.98-0.99-2.330.00-1.44-2.31-1.608TTumor-0.32-0.50-0.250.64-1.07-1.47-0.669TTumor1.050.980.610.870.430.610.4410TTumor-0.84-0.77-0.78-1.680.790.610.0913TTumor-0.87-0.430.560.70-0.510.611.1714TTumor0.861.261.180.481.81--15TTumor-1.59-1.45-1.41--0.71-0.13-16TTumor1.051.261.18-1.70-1.22-2NNormal1.051.260.650.741.810.610.7810NNormal1.05-0.910.810.870.880.610.084NNormal-0.31-0.25-0.530.25-0.190.61-1.575NNormal1.051.130.380.75-0.630.610.10 Citation Format: Jungeun Kim, Hoe Suk Kim, Ga Yeon Kim, Kyung hyeun Park, Seung yeon Ryu, Sangeun Lee, Dong Woo Lee, Bosung Ku, Han-Byoel Lee, Wonshik Han. Development of automated 3D high-throughput drug screening platform for patient-derived breast cancer organoids [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-02-02.

Oxytocin Receptor Activation Rescues Opioid-Induced Respiratory Depression by Systemic Fentanyl in the Rat.

Opioid overdose intervention by naloxone, a high affinity receptor antagonist, reverses opioid-induced respiratory depression (OIRD) and analgesia by displacing opioids. Systemic naloxone stimulates release of the hypothalamic neuropeptide oxytocin, which has analgesic properties and participates in cardiorespiratory homeostasis. To test the hypothesis that oxytocin can reverse OIRD, we assessed the rescue potential of graded doses (0, 0.1, 2, 5, 10, 50 nmol/kg, i.v.) of oxytocin to counter fentanyl (60 nmol/kg, i.v.)-induced depression of neural inspiration indexed by recording phrenic nerve activity (PNA) in anesthetized (urethane/α-chloralose), vagotomized, and artificially ventilated rats. Oxytocin dose-dependently rescued fentanyl OIRD by almost immediately reversing PNA burst arrest (P = 0.0057) and restoring baseline burst frequency (P = 0.0016) and amplitude (P = 0.0025) at low but not high doses, resulting in inverted bell-shaped dose-response curves. Oxytocin receptor antagonism (40 nmol/kg, i.v.) prevented oxytocin reversal of OIRD (arrest: P = 0.0066, frequency: P = 0.0207, amplitude: P = 0.0022). Vasopressin 1A receptor (V1aR) antagonism restored high-dose oxytocin efficacy to rescue OIRD (P = 0.0170 to P < 0.0001), resulting in classic sigmoidal dose-response curves, and prevented (P = 0.0135) transient hypertension from V1aR cross-activation (P = 0.0275). Alone, vasopressin (5 nmol/kg, i.v.) failed to reverse fentanyl respiratory arrest (P = 0.6184). The nonpeptide oxytocin receptor agonist WAY-267464 (75 nmol/kg, i.v.), which has V1aR antagonist properties, quickly reversed fentanyl OIRD (P < 0.0001), with rapid recovery of PNA frequency (P = 0.0011) and amplitude (P = 0.0044) without adverse hemodynamic consequences (P = 0.9991). Findings indicate that peptide and nonpeptide agonist activation of oxytocin receptors without V1aR cross-activation rescues fentanyl OIRD. Oxytocin receptor agonists could be lifesaving resuscitation agents that enhance rather than interrupt opioid analgesia. SIGNIFICANCE STATEMENT: Oxytocin receptor activation produces analgesia. Here, we demonstrate that activation by the US Food and Drug Administration-approved agonist oxytocin and the nonpeptide partial agonist WAY-267464 can each reverse fentanyl cardiorespiratory depression. Selective targeting of oxytocin receptors for resuscitation from opioid overdose, alone or in combination with an opioid antagonist, could eliminate or attenuate negative side effects associated with traditional opioid receptor antagonism.

Erythrocyte Aggregation in Hyperthermic Hypoxic Conditions Significantly Related to Self‐identified Race

Our purpose was to identify the kinetics of erythrocyte (RBC) reversible aggregation (Agg) during stop flow (SF) experiments as a function of temperature and O2 for RBC from healthy subjects self-identified as Black (B, n=8), Hispanic (H, 4) or Caucasian (C, 4) (48+/- 9 y). Whole blood (EDTA, BioIVT, Westbury, NY), vendor verified to be viral free, and used within 1 week of donation, was centrifuged and the platelet poor plasma (PPP) was recovered. All RBCs were resuspended in autologous PPP, and incubated at specified temperatures (37, 41, 45, 49 C) and O2 (0, 5, 10%, saturating). Flow was initiated in a microchannel system at 0.1-0.2 Re, then stopped while recording the entire microchannel width. The greyscale range of the images showed a significant Fahraeus effect with flow (cell free layer at wall). With SF, as Agg proceeded, the aggregates accumulated within the centerline of the microchannel, increasing the variability of the greyscale range from white at the microchannel wall to black in the centerline. The change in variability indicated kinetics of Agg that occurred over 3 min of recording. The primary data of greyscale vs time (custom software) was fit to a sigmoid (dose response) curve, where the fitted EC50 was the T1/2 for Agg and with the fitted slope (p) indicated the kinetics of Agg. At 37 C the T1/2 was 93 s in 0% O2, 75 s with venous (5%) or arterial (10%) and was not different by group. As temperature increased, the T1/2 became significantly faster in blood from B or H (to 65 s), but not C. Correlation between temperature and the fitted T1/2 were significant in B (slope, R2; -2.9, 0.92), H (-2.7, 0.97) and C (-1.1, 0.6), but the F-test on the slope was significant for B (p=0.02) and H (0.04) not C (0.6). This coincided with a significantly steeper p for B and H. At venous, arterial or saturating O2, the effect of increasing temperature to decrease the T1/2 was much less pronounced, and not different between groups. This data shows that in only hypoxic conditions, with temperatures of 41 C (105.8 F) or higher, RBC from self-identified B or H subjects aggregates more readily than C. These results pertain to our focus of microvascular compromise in thermal burn injury progression, but also clinical conditions of localized cautery to control bleeding, high fevers or malignant hyperthermia.

Oxytocin Receptor Activation: An Alternative to Naloxone for Rescuing Opioid‐Induced Respiratory Depression by Systemic Fentanyl

Opioid overdose intervention by naloxone, a high affinity receptor antagonist, reverses opioid-induced respiratory depression (OIRD) and analgesia by displacing opioids. Systemic naloxone stimulates release of the hypothalamic neuropeptide oxytocin (OXTN), which has analgesic properties and participates in cardiorespiratory homeostasis. To test the hypothesis that OXTN can reverse OIRD, we assessed the rescue potential of graded doses (0, 0.1, 2, 5, 10, 50 nmol/kg, i.v., n=5-6/dose) of OXTN to counteract fentanyl (60 nmol/kg, i.v.)-induced depression of neural inspiration indexed by recording phrenic nerve activity (PNA) in anesthetized (urethane/α-chloralose), vagotomized, and artificially ventilated rats. OXTN rescued fentanyl OIRD by reducing fentanyl-induced burst arrest (P=0.0057) and recovery time of burst frequency (P=0.0016) and amplitude (P=0.0025), resulting in parabolic inhibitory dose-response curves.OXTN receptor (OXTNR) antagonism (40 nmol/kg, i.v., n=5) impaired OXTN restoration of PNA post-fentanyl (P=0.0207) and prolonged recovery time of PNA frequency (P=0.0066) and amplitude (P=0.0022). Vasopressin (AVP) receptor 1A (V1aR) antagonism restored the efficacy of high dose OXTN to rescue fentanyl OIRD (P<0.0001, n=4-6/dose), resulting in classic sigmoidal inhibitory dose-response curves, and prevented (P>0.9999) transient hypertension from V1aR cross-activation (P=0.0173). Consistent with this finding, post-fentanyl AVP (5 nmol/kg, i.v., n=5) produced acute hypertension (P=0.0004) and failed to reverse OIRD (PNA arrest P=0.6184, PNA freq. P=0.2369, PNA ampl. P=0.9571). The non-peptide OXTNR agonist WAY-267464 (75 nmol/kg, i.v., n=5), which also has V1aR antagonist properties, quickly reversed fentanyl OIRD (P<0.0001), with rapid recovery to baseline PNA frequency (P=0.0011) and amplitude (P=0.0044) without adverse hemodynamic consequences (P=0.4022). Findings indicate that peptide and non-peptide agonist activation of OXTNR without cross-activation of V1aR rescues fentanyl OIRD.OXTNR agonists could be lifesaving resuscitation agents that enhance rather than interrupt opioid analgesia.

Effects of Oxytocin Analogs on Neurite Outgrowth and Cellular Signaling in Human SH‐SY5Y Neuroblastoma Cell Line

Introduction In the central nervous system, arginine vasopressin (AVP) and oxytocin (OT) stimulate neural networks that regulate social behaviors, including social attachment, aggression, and complex social cognition. AVP and OT are neuropeptides that are expressed in the social behavioral network and bind to G-protein coupled receptors, stimulating cellular signaling patterns that ultimately affect social behaviors at the organismal level. Consensus mammalian AVP and OT sequences are highly conserved. In addition to sequence homology between AVP and OT, there is ~85% structural homology between the OT receptor (OTR) and vasopressin 1a receptor (V1aR) resulting in significant cross-reactivity between the ligands and receptors. Perturbations in OT and/or OT receptor expression results in social behavioral deficits, and is associated with a number of psychopathologies including autism spectrum disorder, schizophrenia, anxiety, and depression. Methods The human neuroblastoma derived SH-SY5Y cell line demonstrates morphological characteristics of neurons including neurites, endogenously expresses OTR, and is often used as an in vitro model for human neurons. Functional assays were performed using Fluo8-AM to measure ligand-induced Ca2+mobilization. Sigmoidal dose-response curves for OT analogs were generated using three-parameter nonlinear regression analysis in GraphPad Prism 8.3 software. To assess effects on neurite outgrowth, cells were treated with logarithmic doses of the consensus mammalian OT sequence (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly; Leu8-OT) and a new world monkey analog (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Pro-Gly; Pro8-OT) for 24 hours, fixed and stained with phalloidin and DAPI. The Nikon A1R confocal microscope was used to collect 60X images of GFP-expressing neurons assessed for effects on neurite outgrowth using the Image J package FIJI with the NeuronJ plugin. Results In SH-SY5Y cells, both OT analogs were more potent than AVP at inducing Ca2+ mobilization, which is consistent with OT being more potent and efficacious at the human OTR. Specificity of the signaling at the OTR were confirmed using the vasopressin 1a receptor antagonist SR49059 and the oxytocin receptor antagonist L-371,257. Additionally, both OT analogs show a dose-dependent increase in neurite outgrowth. Conclusions Together, these data suggest that OTR mediated signaling pathways lead to neurite outgrowth in SH-SY5Y cells. Integrative studies of behavior, genetics and ligand-receptor interaction are crucial for translating signaling activation at the cellular level to effects of AVP and OT ligands on social behavior. Knowledge of how OT alters neuronal structure and function has the potential to both identify mechanisms that produce social dysfunction and to inform the development of therapeutic agents.

Sample-size calculation for preclinical dose–response experiments using heterogeneous tumour models

BackgroundIn preclinical radio-oncological research, local tumour control is considered the most relevant endpoint as it reflects the inactivation of cancer stem cells. Preclinical tumour-control assays may compare dose–response curves between different radiotherapy strategies, e.g., assessing additional targeted drugs and immunotherapeutic interventions, or between different radiation modalities. To mimic the biological heterogeneity of human tumour populations and to accommodate for approaches of personalized oncology, preclinical studies are increasingly performed combining larger panels of tumour models. For designing the study protocols and to obtain reliable results, prospective sample-size planning has to be developed that accounts for such heterogeneous cohorts. MethodsA Monte-Carlo-based method was developed to estimate the sample size of a comparative 1:1 two-arm prospective tumour-control assay. Based on repeated logistic regression analysis, pre-defined dose levels, assumptions on the dose–response curves of the included tumour models and on the dose-modifying factors (DMF), the power is calculated for a given number of animals per dose group. ResultsTwo applications are presented: (i) For a simple tumour-control assay with the head and neck squamous cell carcinoma (HNSCC) model FaDu, 10 animals would be required for each of 7 dose levels per arm to reveal a DMF of 1.25 with a power of 0.82 without drop out (total: 140 animals). (ii) In a more complex experiment combining six different lung tumour models to a heterogeneous population, 21 animals would be required for each of 11 dose levels per arm to reveal a DMF of 1.25 with a power of 0.81 without drop out (total: 462 animals). Analyzing the heterogeneous cohort reduces the required animal number by more than 50% compared to six individual tumour-control assays. ConclusionAn approach for estimating the required animal number for comparative tumour-control assays in a heterogeneous population is presented, allowing also the inclusion of different treatments as a personalized approach in the experimental arm. The software is publicly available and can be applied to plan comparisons of sigmoidal dose–response curves based on logistic regression.

New cytotoxic compounds from the leaves of Caesalpinia benthamiana (Baill.) Herend. & Zarucchi (Fabaceae).

The incidence of multi-drug resistant cancer and the adverse effects associated with available chemotherapy have necessitated the search for new drug candidates. This study investigates the cytotoxic activity of Caesalpinia benthamiana. Column chromatography (CC) and preparative thin layer chromatography (PTLC) were used to isolate compounds. Structural elucidation was done by spectroscopic analysis. MTT assay was used to evaluate cytotoxicity of the compounds against three human adenocarcinoma cells, using methotrexate and dimethyl sulfoxide (DMSO) as positive and negative controls, respectively. CyQuant direct cell proliferation and caspase-3/7 green detection assays were used to investigate the dichloromethane fraction. IC50 values of isolated compounds were determined from sigmoidal dose-response curve. Four new cytotoxic compounds, benthamianoate (2), benthamiacone (3), benthamianin (5) and benthamianol (6), and two known compounds, methyl gallate (1) and 2-methoxyacrylic acid (4) were identified. All the compounds were active with the new monoterpenoid characterized as benthamiacone exhibiting the highest activity (IC50 13.23-21.97 μg/ml) across cancer cell lines investigated. CyQuant direct cell proliferation assay showed significant reduction in the number of live carcinoma cells, while caspase-3/7 green detection assay showed significant increase in the number of dead carcinoma cells. This study revealed potential cytotoxic compounds which are here reported for the first time from C. benthamiana.

Bioinspired Design of Reversible Fluorescent Probes for Tracking Nitric Oxide Dynamics in Live Cells

Bioinspired Design of Reversible Fluorescent Probes for Tracking Nitric Oxide Dynamics in Live Cells

Uncovering the biofumigant capacity of allyl isothiocyanate from several Brassicaceae crops against Fusarium pathogens in maize

Driven by environmental concerns, chemical fumigants are no longer allowed in many countries. Therefore, other strategies for reducing fungal inoculum in soils and on crop debris are being explored. In the present study, several Brassicaceae crops were screened for their potential to control Fusarium gramineaum and Fusarium poae mycelial growth in an in vitro inverted Petri dish experiment. Volatile production was measured using gas chromatography-mass spectrometry headspace analysis. A selection of cultivars from each crop species was further investigated using a pot experiment with maize. Ethiopian mustard (Brassica carinata) and brown mustard (Brassica juncea) released volatile allyl isothiocyanate (AITC) and a higher concentration of AITC was correlated with a better fungal growth reduction in the in vitro screening. Brown mustard cultivar Etamine completely inhibited growth of both Fusarium spp. Pure AITC in a solution with methanol resulted in a sigmoid dose-response curve for both Fusarium spp. tested. Fusarium poae appeared to be more tolerant to AITC than F. graminearum. A pot experiment revealed that the incorporation of brown mustard plant material could alleviate the clear negative effect of F. graminearum infection on maize growth. The present study demonstrated the correlation between the fungistatic effect of biofumigation crops on Fusarium spp. and their production of volatile AITC in vitro, without the addition of exogenous enzymes, and confirmed the biofumigation potential of brown mustard in a pot experiment with maize. These results may help farmers when selecting a green manure crop suitable for biofumigation. © 2020 Society of Chemical Industry.