Full Dose-response Curves Research Articles

Spread-out Bragg peak FLASH: quantifying normal tissue toxicity in a murine model.

A favorable effect of ultra-high dose rate (FLASH) radiation on normal tissue-sparing has been indicated in several preclinical studies. In these studies, the adverse effects of radiation damage were reduced without compromising tumor control. Most studies of proton FLASH investigate these effects within the entrance of a proton beam. However, the real advantage of proton therapy lies in the Spread-out Bragg Peak (SOBP), which allows for giving a high dose to a target with a limited dose to healthy tissue at the entrance of the beam. Therefore, a clinically relevant investigation of the FLASH effect would be of healthy tissues within a SOBP. Our study quantified the tissue-sparing effect of FLASH radiation on acute and late toxicity within an SOBP in a murine model. Radiation-induced damage was assessed for acute and late toxicity in the same mice following irradiation with FLASH (Field dose rate of 60 Gy/s) or conventional (CONV, 0.34 Gy/s) dose rates. The right hindleg of unanesthetized female CDF1 mice was irradiated with single-fraction doses between 19.9-49.7 Gy for CONV and 30.4-65.9 Gy for FLASH with 5-8 mice per dose. The leg was placed in the middle of a 5 cm SOBP generated from a mono-energetic beam using a 2D range modulator. Acute skin toxicity quantified by hair loss, moist desquamation and toe separation was monitored daily within 29 days post-treatment. Late toxicity of fibrotic development measured by leg extendibility was monitored biweekly until 30 weeks post-treatment. Comparison of acute skin toxicity following radiation indicated a tissue-sparing effect of FLASH compared to conventional single-fraction radiation with a mean protection ratio of 1.40 (1.35-1.46). Fibrotic development similarly indicated normal tissue sparing with a 1.18 (1.17-1.18) protection ratio. The acute skin toxicity tissue sparing was similar to data from entrance-beam irradiations of Sørensen etal. (4). Full dose-response curves for acute and late toxicity after CONV and FLASH radiation were obtained. Radiation within the SOBP retains the normal-tissue-sparing effect of FLASH with a dose-modifying factor of 40% for acute skin damage and 18% for fibrotic development.

Abstract PO5-23-10: Development of a long-term in vitro assay able to identify compounds that can overcome resistance to CDK4/6 inhibitor plus endocrine therapy in ER+ HER2- breast cancer

Abstract CDK4/6 inhibitor plus endocrine therapy (ET) has emerged as the standard of care first line therapy in ER+ HER2- breast cancer. While this combination has shown the ability to extend overall survival, nearly all patients acquire resistance and progress after 2-3 years. Subsequently, interest has turned to identifying a third partner compound that can be integrated into the CDK4/6i plus ET backbone regimen with the goal of overcoming resistance and driving a deeper response. However, a comprehensive assay-based workflow proficient in identifying compounds with the most promising potential is lacking. To fill this need, we set out to develop a high-throughput in vitro assay with relevant multi-faceted readouts to assess the efficacy of potential triplet combinations using 12 human ER+ breast cancer lines. For this assay, cells were engineered to express nuclear RFP to enable accurate cell counting using basic imaging techniques in 384 well plates. Each third partner compound was tested at two different doses and, in addition to the triplet combination (CDK4/6i + ET + third partner), single agent as well as double agent combinations were investigated. The assay was imaged and re-dosed every 5 days until day 15. On day 15, the compounds were washed out and the cells underwent a 25-day drug holiday to monitor relapse. Duplicate plates of all conditions were monitored and imaged every two hours for 24 hours for live cell staining of apoptosis markers. Additional duplicate plates were fixed and stained on days 5 and 15 for markers of senescence and cell cycle. Images were subsequently fed into a python-based algorithm to determine the percentage of cell population expressing the various markers and phenotypic correlates were extracted. We are looking for triplet combinations that induce a deeper cell cycle arrest, senescence, and/or cell death compared with the CDK4/6 inhibitor plus ET only treatment. Our drug holiday results will help elucidate the treatments able to continue to induce suppression once washed out, indicating treatment options that may induce long-lasting, possibly irreversible effects on the cells. Finally, by extracting phenotypic correlates from brightfield images we hope to cluster the various compounds in their effects across cell lines of diverse backgrounds to help understand the best way to benefit multiple patient populations. Overall, the assay will be able to identify and differentiate various therapeutic combinations based on cell count, senescence, and cell death. The format of this assay enables depth of analysis not accessible using other techniques such as FACS, including full dose-response curves, combination matrices, and longitudinal analyses. Additionally, the assay can be modified to evaluate any number of cellular markers and can be useful across a number of applications. The approach developed here will be foundational in discovering next-step strategies for patients with therapy resistance. Citation Format: Stephanie Kronstadt, Neil Umbreit, Matt Niederst, Rens Janssens. Development of a long-term in vitro assay able to identify compounds that can overcome resistance to CDK4/6 inhibitor plus endocrine therapy in ER+ HER2- breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-23-10.

Temporal effect of imatinib adherence on time to remission in chronic myeloid leukemia patients.

Adherence to imatinib in chronic myeloid leukemia (CML) patients is estimated to be as low as 70% despite its clinical benefit, and our understanding of the impact of nonadherence in this population is limited. This study presents a novel application of the Alternating Conditional Estimation (ACE) algorithm in newly diagnosed CML patients to map the full dose-response curve (DRC) and determine how the strength of this curve varies over time. We applied the ACE algorithm alongside a backward elimination procedure to detect the presence of time dependence and nonlinearity in the relationship between imatinib adherence and time-to-remission. An extended Cox model allowing for the flexible modeling of identified effects via unpenalized B-splines was subsequently fit and assessed. The substantial improvement in model fit associated with the extended Cox approach suggests that traditional Cox proportional hazards model assumptions do not hold in this setting. Results indicate that the DRC for imatinib is non-linearly increasing, with an attenuated effect above a 74% adherence rate. The strength of this effect on remission varied over time and was strongest in the initial months of treatment, reaching a peak around 90 days post-initiation (log hazard ratio: 2.12, 95% confidence interval: 1.47 to 2.66). Most patients that achieved remission did so by 4 months (120 days) with consistently high adherence, suggesting that this could be a critical time and duration for realizing treatment benefit and patient monitoring. Findings regarding the relationship between adherence and remission can additionally help guide the design of future studies.

Relative reinforcing effects of dibutylone, ethylone, and N-ethylpentylone: self-administration and behavioral economics analysis in rats.

Following the emergence of methylone as one of the most popular synthetic cathinones, this group of novel psychoactive substance with names ending in "-lone," such as dibutylone, ethylone, and N-ethylpentylone, appeared on the recreational drug market. The pharmacological mechanisms of dibutylone, ethylone, and N-ethylpentylone are well understood; however, to date, the reinforcing effects of dibutylone, ethylone, and N-ethylpentylone are still unclear. This study aimed to examine the self-administration of dibutylone, ethylone, and N-ethylpentylone relative to methamphetamine (METH) and to quantify their relative reinforcing effectiveness using behavioral economic analysis. Male Sprague-Dawley rats were trained to self-administer METH (0.05mg/kg) under a fixed-ratio 1 (FR1) schedule. Following the training, dose substitution was used to generate full dose-response curves for METH and the three synthetic cathinones. According to the first doses on the descending limb of the dose-response curves, rats were trained to self-administer METH (0.05mg/kg), dibutylone (0.1mg·kg-1·infusion-1), ethylone (0.4mg·kg-1·infusion-1), or N-ethylpentylone (0.1mg·kg-1·infusion-1) under an FR1 schedule, and a behavioral economic evaluation of their reinforcing effectiveness was then performed. Dibutylone, ethylone, and N-ethylpentylone functioned as reinforcers, and the inverted U-shaped dose-response curves were obtained. The rank order of reinforcing potency in this procedure was METH > N-ethylpentylone ≈ dibutylone > ethylone. In the economic analysis, the comparisons of the essential value (EV) transformed from demand elasticity (α) indicated that the rank order of efficacy as reinforcers was METH (EV = 7.93) ≈ dibutylone (EV = 7.81) > N-ethylpentylone (EV = 5.21) ≈ ethylone (EV = 4.19). These findings demonstrated that dibutylone, ethylone, and N-ethylpentylone function as reinforcers and have addictive potential, suggesting that the modification of α-alkyl and N-alkyl side chains may affect their reinforcing efficacy.

Development of a Quasi-Quantitative Structure-Activity Relationship Model for Prediction of the Immobilization Response of Daphnia magna Exposed to Metal-Based Nanomaterials.

The conventional Hill equation model is suitable to fit dose–response data obtained from performing (eco)toxicity assays. Models based on quasi–quantitative structure–activity relationships (QSARs) to estimate the Hill coefficient (nH) were developed with the aim of predicting the response of the invertebrate species Daphnia magna to exposure to metal‐based nanomaterials. Descriptors representing the pristine properties of nanoparticles and media conditions were coded to a quasi–simplified molecular input line entry system and correlated to experimentally derived values of nH. Monte Carlo optimization was used to model the set of nH values, and the model was trained on the basis of reported dose–response relationships of 60 data sets (n = 367 individual response observations) of 11 metal‐based nanomaterials as obtained from 20 literature reports. The model simulates the training data well, with only 2.3% deviation between experimental and modeled response data. The technique was employed to predict the dose–response relationships of 15 additional data sets (n = 72 individual observations) not included in model development of seven metal‐based nanomaterials from 10 literature reports, with an average error of 3.5%. Combining the model output with either the median effective concentration value or any other known effect level as obtained from experimental data allows the prediction of full dose–response curves of D. magna immobilization. This model is an accurate screening tool that allows the determination of the shape and slope of dose–response curves, thereby greatly reducing experimental effort in case of novel advanced metal‐based nanomaterials or the prediction of responses in altered exposure media. This screening model is compliant with the 3Rs (replacement, reduction, and refinement) principle, which is embraced by the scientific and regulatory communities dealing with nano‐safety. Environ Toxicol Chem 2022;41:1439–1450. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Interactions between impulsivity and MDPV self-administration in rats.

Synthetic cathinones, such as 3,4-methylenedioxypyrovalerone (MDPV), are recreational drugs of abuse often identified in 'bath salts' preparations. Humans report compulsive patterns of bath salts use, and previous work suggests that a subset of rats develop unusually high levels of MDPV self-administration. This study aims to test the hypothesis that high levels of impulsivity (e.g., inability to withhold responding for a sucrose reward) will predispose rats to high levels of MDPV self-administration relative to rats with lower levels of impulsivity. The 1-choice serial reaction time task (1-CSRTT) was used to assess impulsivity (i.e., premature responding) in 10 female and 10 male Sprague Dawley rats. Rats were then allowed to self-administer 0.032 mg/kg/inf MDPV or 0.32 mg/kg/inf cocaine, after which full dose-response curves for MDPV (0.001-0.1mg/kg/inf) or cocaine (0.01-1mg/kg/inf) were generated under a FR5 schedule of reinforcement. After a history of self-administering MDPV or cocaine, impulsivity was reassessed under the 1-CSRTT, prior to evaluating the acute effects of MDPV (0.032-0.32 mg/kg) or cocaine (0.1-1mg/kg) on impulsivity. Level of impulsivity was not correlated with subsequent levels of either MDPV or cocaine self-administration, and level of drug self-administration was also not correlated with subsequent levels of impulsivity, although acute administration of MDPV and cocaine did increase premature responding. In failing to find direct relationships between either impulsivity and subsequent drug-taking behaviour, or drug-taking behaviour and subsequent assessments of impulsivity, these findings highlight the complexity inherent in the associations between impulsive behaviour and drug-taking behaviour in both animal models and humans.

Bioactivity of 11 keto and hydroxy androgens in yeast and mammalian host cells

Recent studies have highlighted the potential role of 11oxygenated (keto or hydroxy) androgens in human reproductive function with 11keto androgens circulating at concentrations comparable with testosterone in women and children. However, the intrinsic androgenic bioactivities of 11 keto and hydroxy androgens are not fully characterized. We therefore investigated the full androgen dose-response curves using complementary in vitro yeast and mammalian (HEK293) host cell bioassays of 11 keto and hydroxy derivatives of the potent androgens, testosterone (T) and dihydrotestosterone (DHT), compared with their parent non-11 oxygenated steroids together with the pro-androgen precursor (androstenedione (A4)) and metabolites (androstanedione, androsterone). For potent androgens, the mammalian HEK293 host cell bioassay was 22–138 times more sensitive than the yeast host cell bioassay. In both androgen bioassays, 11keto derivatives displayed androgenic bioactivity but significantly lower molar potency than their parent non-keto steroids. By contrast, the 11hydroxy derivatives had minimal or no androgenic bioactivity. In both bioassays 5α-reduction increased androgenic potency. These findings confirm that that 11keto androgens may contribute directly to androgen status in women, children, and other conditions apart from healthy eugonadal men whereas 11hydroxy androgens have negligible androgenic potency although it cannot be excluded that they may be converted to more potent androgens in vivo.

In vivo validation and tissue sparing factor for acute damage of pencil beam scanning proton FLASH

Background and purposePreclinical studies indicate a normal tissue sparing effect using ultra-high dose rate (FLASH) radiation with comparable tumor response. Most data so far are based on electron beams with limited utility for human treatments.This study validates the effect of proton FLASH delivered with pencil beam scanning (PBS) in a mouse leg model of acute skin damage and quantifies the normal tissue sparing factor, the FLASH factor, through full dose response curves. Materials and methodsThe right hind limb of CDF1 mice was irradiated with a single fraction of proton PBS in the entrance plateau of either a 244 MeV conventional dose rate field or a 250 MeV FLASH field. In total, 301 mice were irradiated in four separate experiments, with 7–21 mice per dose point. The endpoints were the level of acute moist desquamation to the skin of the foot within 25 days post irradiation. ResultsThe field duration and field dose rate were 61–107 s and 0.35–0.40 Gy/s for conventional dose rate and 0.35–0.73 s and 65–92 Gy/s for FLASH. Full dose response curves for five levels of acute skin damage for both conventional and FLASH dose rate revealed a distinct normal tissue sparing effect with FLASH: across all scoring levels, a 44–58% higher dose was required to give the same biological response with FLASH as compared to the conventional dose rate. ConclusionsThe normal tissue sparing effect of PBS proton FLASH was validated. The FLASH factor was quantified through full dose response curves.

Inductively Coupled Electrical Stimulation - Part 4: Effect of PEMF on seed germination; evidence of triphasic inverse hormesis

Our objectives were to study the effects of PEMF on a non-animal living system with a simple and unambiguous biomarker: seed germination rate. We selected seeds representing three groups: high- intermediate- and low-germination-rate seeds (lettuce, bell pepper, and strawberry, respectively). PEMF was applied at 15 pulses per second (pps or Hz) to strawberry and lettuce seeds, and 3 or 15 pps to bell pepper seeds, using only one well-defined PEMF pulse waveform shape and intensity. The only PEMF variables between groups were therefore PEMF pulse rate and total number of pulses, which was taken to be the PEMF dose, equivalent to the duration of exposure at a known pulse rate. In the case of lettuce, we studied the effects of germination using only one PEMF dose compared to no-dose (PEMF -) controls, and no interactions with other pre-planting procedures. In the case of strawberry seeds, we studied possible interactions between PEMF, pre-freezing and pre-soaking of seeds immediately before planting. For bell peppers, extensive dose-response curves are reported. Total dose was calculated as D = R * t, where R = pulse rate (pulses per second) and t = treatment duration (seconds), the product yielding D (dose, i.e. total number of pulses in the PEMF treatment). Bell pepper seeds have an intermediate germination rate that allows the possibility for large and commercially important swings in germination rate both up and down, so we attempted to construct a full spectrum dose-response curve, beginning with PEMF dosing thought to be below threshold then extending into what was thought to potentially be the excessive and toxic range. We also attempted to identify which PEMF parameters are most relevant for dosing: total number of pulses versus “frequency” (pulse rate) and duration of exposure. Results: Lettuce seeds showed evidence of a positive effect of PEMF on germination rate (79% unstimulated, 92% stimulated), with no effect on average time to germination. Strawberry seeds showed little effect of PEMF on germination, if any, and no apparent interaction with pre-freezing or pre-soaking of seeds. Bell Pepper seeds showed a clear inverse hormesis response within the dose range studied, and suggested a tri-phasic response to PEMF exposure for doses in excess of those applied. Specifically, extremely low PEMF doses had a negative effect on bell pepper seed germination rate, whereas intermediate doses had significant positive effects on germination rate, and germination rate began trending down again for the highest PEMF doses, suggesting a third phase of inhibited germination for extreme PEMF dosages. This latter trend was not fully elucidated by the present data set, but is only suggested by data points at the most extreme upper dosages included in this study. Overall, these results were entirely unexpected and may be of importance, therefore independent replication of these results is advised. To explain the unexpected results, a detailed discussion of various multi-phasic dose-response curves is also included. All negative findings were included in this report, specifically to counteract the dominant practice of publication bias in the life sciences.

MDPV "high-responder" rats also self-administer more oxycodone than their "low-responder" counterparts under a fixed ratio schedule of reinforcement.

Oxycodone is one of the most commonly prescribed and most frequently abused opioid analgesics, yet little is known regarding individual vulnerabilities to oxycodone abuse. The synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV) has been shown to produce a "high-responder" phenotype characterized by increased drug intake and responding during periods of signaled drug unavailability (e.g., during post-infusion timeouts) in ~ 40% of male Sprague-Dawley rats. This phenotype also transfers to other psychostimulants (e.g., cocaine and methamphetamine), but it is unknown whether this phenotype transfers to other (non-stimulant) drugs of abuse. The present study aimed to (1) reestablish the "high-responder" phenotype in male Sprague-Dawley rats (n = 11) that acquired self-administration of MDPV (0.032 mg/kg/inf) on a fixed ratio 1 (FR1) schedule of reinforcement and (2) compare full dose-response curves for MDPV and oxycodone self-administration under an FR5 schedule of reinforcement. MDPV was ~ 3-fold more potent at maintaining peak levels of behavior and resulted in greater overall drug intake than oxycodone. High levels of timeout responding were noted in a subset of rats that acquired MDPV self-administration ("high-responders", n = 5), and the FR5 dose-response curve for MDPV was shifted upward for these rats relative to their "low-responder" (n = 6) counterparts. "High-responders" also self-administered more infusions of oxycodone under an FR5 schedule of reinforcement than "low-responders"; however, this was not coupled with increased levels of timeout responding. The present data suggest thata subset of individuals with a history of using synthetic cathinones may be particularly vulnerable to the abuse of oxycodone.

Does the combination of hyperthermia with low LET (linear energy transfer) radiation induce anti-tumor effects equivalent to those seen with high LET radiation alone?

Introduction The combination of hyperthermia with low LET (linear energy transfer) radiation may have similar anti-tumor effects as high LET radiation alone. This pre-clinical study determined the optimal heating temperature and time interval between radiation and heat to achieve this equivalent effect. Methods C3H mammary carcinomas (200 mm3 in size) growing in the right rear foot of CDF1 mice was used in all experiments. Tumors were locally irradiated with graded doses of either 240 kV ortho- or 6 MV mega-voltage X-rays to produce full dose-response curves. Heating (41.0–43.5 °C; 60 min) was achieved by immersing the tumor bearing foot in a water-bath applied at the same time, or up to 4-hours after, irradiating. The endpoint was the percentage of mice showing local tumor control at 90 days, with enhancements calculated from the ratios of the radiation doses causing 50% tumor control (± 95% confidence intervals). Results Previous published results in this tumor model reported that carbon ions were 1.3–1.7 times more effective than low LET radiation at inducing tumor control. Similar enhancements occurred with a temperature of only 41.0 °C with a simultaneous heat and radiation treatment. However, higher temperatures were needed with the introduction of any interval; at 42.5 °C, the enhancement was 2.5 with a simultaneous treatment, decreasing to a value within the carbon ion range with a 4-hour interval. Conclusions Combining hyperthermia with low LET radiation can be as effective as high LET at inducing tumor control, but the temperature needed depended on the time interval between the two modalities.

Predicting Early Life Stage Mortality in Birds and Fishes from Exposure to Low-Potency Agonists of the Aryl Hydrocarbon Receptor: A Cross-Species Quantitative Adverse Outcome Pathway Approach.

Dioxin-like compounds (DLCs) cause early life stage mortality of vertebrates through activation of the aryl hydrocarbon receptor (AhR). A prior study developed a cross-species quantitative adverse outcome pathway (qAOP) which can predict full dose-response curves of early life stage mortality for any species of bird or fish exposed to DLCs using the species- and chemical-specific 50% effect concentration (EC50) from an in vitro AhR transactivation assay with COS-7 cells. However, calculating a reliable EC50 for input into this qAOP requires the maximal response of the concentration-response curve to be known, which is not always possible for low-potency agonists, such as some polychlorinated biphenyls (PCBs). To enable predictions for these low-potency agonists, the present study revised this qAOP to use the effect concentration threshold (ECThreshold ) from the in vitro AhR transactivation assay as input. Significant linear relationships were demonstrated between ECThreshold and the dose to cause 0, 10, 50, or 100% mortality among early life stages of 3 species of birds and 7 species of fish for 4 DLCs: 2,3,7,8-tetrachlorodibenzo-p-dioxin, PCB 126, PCB 77, and PCB 105. These 4 linear relationships were combined to form the revised qAOP. This qAOP using the ECThreshold enables prediction of experimental dose-response curves for lower-potency agonists to within an order of magnitude on average, but the prior qAOP using EC50 predicts experimental dose-response curves for higher-potency agonists with greater accuracy. Environ Toxicol Chem 2020;39:2055-2064. © 2020 SETAC.

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays.

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample's impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

A mixture of persistent organic pollutants relevant for human exposure inhibits the transactivation activity of the aryl hydrocarbon receptor in vitro

While humans are exposed to mixtures of persistent organic pollutants (POPs), their risk assessment is usually based on a chemical-by-chemical approach. To assess the health effects associated with mixed exposures, knowledge on mixture toxicity is required. Several POPs are potential ligands of the Aryl hydrocarbon receptor (AhR), which involves in xenobiotic metabolism and controls many biological pathways. This study assesses AhR agonistic and antagonistic activities of 29 POPs individually and in mixtures by using Chemical-Activated LUciferase gene eXpression bioassays with 3 transgenic cell lines (rat hepatoma DR-H4IIE, human hepatoma DR-Hep G2 and human mammary gland carcinoma DR-T47-D). Among the 29 POPs, which were selected based on their abundance in Scandinavian human blood, only 4 exerted AhR agonistic activities, while 16 were AhR antagonists in DR-H4IIE, 5 in DR-Hep G2 and 7 in DR-T47-D when tested individually. The total POP mixture revealed to be AhR antagonistic. It antagonized EC50 TCDD inducing AhR transactivation at a concentration of 125 and 250 and 500 fold blood levels in DR-H4IIE, DR-T47-D and DR-Hep G2, respectively, although each compound was present at these concentrations lower than their LOEC values. Such values could occur in real-life in food contamination incidents or in exposed populations. In DR-H4IIE, the antagonism of the total POP mixture was due to chlorinated compounds and, in particular, to PCB-118 and PCB-138 which caused 90% of the antagonistic activity in the POP mixture. The 16 active AhR antagonists acted additively. Their mixed effect was predicted successfully by concentration addition or generalized concentration addition models, rather than independent action, with only two-fold IC50 underestimation. We also attained good predictions for the full dose-response curve of the antagonistic activity of the total POP mixture.

Sex Differences in Anti‐allodynic Tolerance to Δ9‐THC in Desensitization‐resistant S426A/S430A and Wild‐type Mice

Tolerance to the anti‐allodynic effects of cannabinoids has the potential to limit their therapeutic potential. A growing body of clinical and preclinical evidence suggests that sex influences a variety of cannabinoid‐related outcomes, including cannabinoid‐induced anti‐allodynia. Male and female mice expressing a desensitization‐resistant form of the cannabinoid type‐1 (CB1) receptor (S426A/S430A) show delayed tolerance to Δ9‐tetrahydrocannabinol (Δ9‐THC) in the tail‐flick assay. Likewise, using the tail‐flick test, male wild‐type mice are more sensitive than females to the anti‐nociceptive effects of Δ9‐THC across the full dose response curve. While the tail‐flick is an acute model of thermal pain, it remains unknown whether these sex‐ and/or genotype differences extend to a chronic model of neuropathic pain. The purpose of the current study was to determine whether the S426A/S430A mutation also alters tolerance to anti‐allodynic effects of Δ9‐THC in a sex‐specific manner. Male and female wild‐type and S426A/S430A mice were assessed for tolerance to the anti‐allodynic effects of 6 and 10 mg/kg of Δ9‐THC in a cisplatin‐evoked model of neuropathic pain. Female mice were less sensitive to the anti‐allodynic effects of 6 mg/kg Δ9‐THC than males. Treatment with 6 mg/kg Δ9‐THC was able to completely reverse allodynia in male, but not female, mice. Female mice showed a complete reversal of allodynia following treatment with 10 mg/kg Δ9‐THC. Interestingly, the S426A/S430A mutation did not alter sensitivity or tolerance development to the anti‐allodynic effects of Δ9‐THC compared to wild‐type mice of either sex, suggesting that the action of Δ9‐ THC at the mutant form of CB1 may be masked by its substantial anti‐allodynic effects through CB2. This study highlights the therapeutic potential of Δ9‐THC in a model of neuropathic pain and suggests that these effects might be mediated in a sex‐specific manner that may have clinical implications for the use of cannabinoids in chronic pain management.Support or Funding InformationNational Institute on Drug Abuse grant DA037355 (DJM); Penn State University College of Medicine Department of Anesthesiology & Perioperative Medicine; Pennsylvania Department of Health using CURE Tobacco Settlement Funds (DJM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Response‐Contingent and Non‐Contingent Drug History on the Development of High Levels of MDPV Self‐Administration

A subset of rats that self‐administer synthetic cathinones, such as 3,4‐methylenedioxypyrovalerone (MDPV), develop high levels of drug intake and compulsive‐like patterns of responding that may be related to the compulsive, binge‐like patterns of cathinone (e.g., “bath salts”) use reported in humans. Once this “high‐responder” phenotype is established, it transfers to other stimulants; however, a prior history of responding for cocaine, but not food, has been shown to prevent or delay the development of this phenotype. These findings suggested that cocaine produces neuroplastic changes that can prevent or delay the transition to high levels of MDPV intake. The present studies aimed to evaluate whether 1) response contingent and non‐contingent cocaine histories are equally effective at preventing high levels of MDPV intake; and 2) response contingent and non‐contingent MDPV histories are sufficient to establish the high‐responder phenotype in rats self‐administering cocaine. Four groups of male Sprague‐Dawley rats were assigned to initially receive MDPV or cocaine in a response contingent or non‐contingent manner using a “yoked” self‐administration paradigm under a fixed ratio (FR) 1 schedule (i.e., response contingent drug infusions delivered to rat A were paired with non‐contingent infusions of the same drug to rat B). After 20 sessions of this yoked MDPV or cocaine self‐administration procedure (10 sessions at FR1, 10 sessions at FR5), all rats were allowed to self‐administer the alternate drug under an FR5 schedule for an additional 20 sessions (e.g. both cocaine groups now self‐administered MDPV). Subsequently, full FR5 dose response curves were generated by dose substitution. These studies found that, unlike response‐contingent cocaine delivery, non‐contingent cocaine is unable to prevent the development of high levels of MDPV self‐administration. Additionally, non‐contingent MDPV is not sufficient to establish high levels of cocaine intake. These results suggest that the contingencies surrounding drug delivery are important for both establishing and preventing the high‐responder phenotype, indicating that both a behavioral and pharmacological history may be important for the transition from regulated to compulsive patterns of drug intake.Support or Funding InformationThis work was supported by NIH/NIDA grant (R01 DA039146; GTC), NIDA‐ and NIAAA‐IRPs (KCR), and NIH Predoctoral Training Program in the Neurosciences (T32 NS082145; MRD).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of artificial sweeteners on Lemna minor

Artificial sweeteners are common micropollutants in the aquatic environment. They were detected both in surface waters and in groundwater. Human toxicity has also been studied quite intensively but their ecotoxicity has not been studied so far. To assess the impact of four artificial sweeteners (aspartame, sucralose, saccharine, and acesulfame K) and one natural sweetener (stevioside) on freshwater plants, a growth inhibition test was set up in the macrophyte duckweed (Lemna minor). Subsequently full dose-response curves were established by exposing L. minor plants to concentrations of each individual sweetener ranging from 6.25 mg/l up to 100 mg/l for 7 days. Three different endpoints were tested: frond number, frond area and total chlorophyll content. Tests were performed under sterile conditions. Sweeteners had various effects on Lemna plants. Saccharine, acesulfame K and stevioside did not cause any significant negative effects on any of the measured parameters. On the contrary, stevioside and saccharine caused slowly stimulative effects. Aspartame and sucralose inhibited growth parameters (frond number and frond area) but the chlorophyll content was not affected.

Behavioral economic analysis of the reinforcing effects of "bath salts" mixtures: studies with MDPV, methylone, and caffeine in male Sprague-Dawley rats.

"Bath salts" preparations often contain combinations of synthetic cathinones (e.g., 3,4-methylenedioxymethcathinone [methylone], 3,4-methylenedioxypyrovalerone [MDPV]), and caffeine, and evidence suggests that mixtures of synthetic cathinones and caffeine (e.g., MDPV + caffeine or methylone + caffeine) can be more potent and/or effective reinforcers than predicted for an additive interaction. To use demand curve analyses to compare the reinforcing effectiveness of MDPV and methylone to mixtures of MDPV + caffeine and methylone + caffeine. Male Sprague-Dawley rats acquired methylone self-administration (0.32mg/kg/inf) under a fixed ratio (FR) 1 schedule of reinforcement and generated full dose-response curves for methylone (0.01-1mg/kg/inf) under an FR5 schedule of reinforcement. Demand curves were then obtained for methylone, MDPV, caffeine, and methylone + caffeine and MDPV + caffeine mixtures by increasing the FR across sessions according to the following series: 3, 10, 18, 32, 56, 100, 178, etc. RESULTS: Self-administration of methylone was rapidly acquired by 87.5% of rats and was maintained across a range of doses, producing an inverted U-shaped dose-response curve. Rank order demand for the individual constituents was MDPV > methylone > caffeine. Demand for the 3:1 (but not 10:1) methylone + caffeine mixture was greater than that for methylone alone, and demand for MDPV alone was similar to both MDPV + caffeine mixtures evaluated. These studies provide additional evidence that although methylone is an effective reinforcer, combining methylone with caffeine results in an enhanced reinforcing effectiveness compared to methylone alone. Thus, abused "bath salts" preparations containing synthetic cathinones and caffeine may have higher abuse liability than preparations containing only synthetic cathinones.

Abstract 3901: Characterizing antibody-drug conjugate cytotoxicity using four different real-time assays

Abstract Recent clinical successes using antibody drug conjugates (ADC) directed to hematological or solid tumor cancers have re-invigorated immunotherapeutic drug development interest. Unfortunately, efficacious ADC are inherently complex entities comprised of a targeting antibody, a stable but cleavable linker, and a toxic payload, for which the best combinations are often identified through iterative trial and error. Therefore, an efficient and systematic method for rank ordering ADC candidates is sought to evaluate their intrinsic pharmacodynamic properties. We chose SKBR3 (Her2+) and T47D (Her2-) cell models to define the on- and off-target cytotoxic potential of trastuzumab emtansine. We employed a suite of four, newly developed real-time assays to measure exposure-dependent changes in viability, caspase activation, phosphatidylserine (PS) exposure and loss of membrane integrity to define potency and magnitude of response, as well as primary mode of cell death. We discovered that trastuzumab emtansine mediated dose-dependent effects on SKBR3, with cytostasis beginning at 17h near 1μg/ml, caspase induction at 20h, which led to PS exposure at about 21h and secondary necrosis at 30h. During this same time course, full dose-response curves were generated for each biomarker as EC50 values evolved and matured to their respective maximal responses. Conversely, the T47D non-target cell population exhibited only minor cytotoxicity at the latest time points, presumably owing to linker degradation and non-specific payload release. Taken together, these data define the in vitro pharmacological disposition of trastuzumab emtansine, including the kinetic component of functionality related to antigen/antibody engagement, internalization, linker mediated payload release and resulting toxicity. This study may provide a tangible roadmap for the critical evaluation of other ADC under development. Citation Format: Andrew L. Niles, Kevin Kupcho, Sarah Duellman, Jolanta Vidugiriene, Dan Lazar, James J. Cali. Characterizing antibody-drug conjugate cytotoxicity using four different real-time assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3901.

Reinforcing Potency and Effectiveness of Synthetic Cathinones: Potency versus Selectivity for DAT

Synthetic cathinones found in “bath salts” preparations interact with monoamine transporters and function as either cocaine‐like monoamine uptake inhibitors or amphetamine‐like substrates. Abuse of these synthetic cathinones has resulted in regulation of some cathinones internationally. Modifications to the chemical structure of regulated cathinones (e.g., MDPV) produce a series of new cathinones (e.g., α‐PVP, α‐PPP, MDPBP, and MDPPP) which may not be subject to existing regulations. Although MDPV is a more potent and effective reinforcer than cocaine, little is known about the reinforcing effects of other synthetic cathinones. Thus, this study examined intravenous self‐administration of cocaine, MDPV, α‐PVP, α‐PPP, MDPBP, and MDPPP to test the hypotheses that 1) stimulants with greater potency to inhibit the dopamine transporter (DAT) are more potent reinforcers, and 2) stimulants with greater selectivity for DAT relative to the serotonin transporter (SERT) are more effective reinforcers. Potencies to inhibit monoamine uptake at DAT and SERT were determined using synaptosomes prepared from rat brains. Separate groups of male Sprague‐Dawley rats (n=12 per group) were used to generate full dose‐response curves under a progressive ratio (PR) schedule of reinforcement for α‐PVP, α‐PPP, MDPBP, and MDPPP; full dose‐response curves for MDPV and cocaine were also generated in each rat (n=48). Rank order potency to inhibit DAT was MDPV > α‐PVP ≈ MDPBP > MDPPP ≈ α‐PPP ≈ cocaine, while rank order DAT/SERT selectivity was α‐PVP > α‐PPP > MDPV > MDPBP > MDPPP > cocaine. The potency to inhibit DAT correlated with the potency of each drug to maintain responding (MDPV > α‐PVP ≈ MDPBP > MDPPP ≈ α‐PPP ≈ cocaine). Relative reinforcing effectiveness as determined by comparing the maximum number of infusions obtained for each drug under a PR schedule of reinforcement was α‐PVP > α‐PPP ≈ MDPV > MDPBP ≈ MDPPP > cocaine, which correlated with DAT/SERT selectivity. These data suggest that (1) potency at DAT is an important determinant of the potency of each drug to function as a reinforcer, (2) each of these synthetic cathinones functions as a more effective reinforcer than cocaine, and (3) DAT/SERT selectivity is related to reinforcing effectiveness and may predict the abuse‐related effects of novel synthetic cathinones in humans.Support or Funding InformationThese experiments were supported by NIH grants R01DA039146 (GTC) and T32DA031115 (BMG) and the Intramural Research Programs of NIDA (MHB, KCR) and NIAAA (KCR).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.