The thrombopoietin mimetic JNJ-26366821 reduces the late injury and accelerates the onset of liver recovery after acetaminophen-induced liver injury in mice.

Acetaminophen (APAP)-induced hepatotoxicity is comprised of an injury and recovery phase. While pharmacological interventions, such as N-acetylcysteine (NAC) and 4-methylpyrazole (4-MP), prevent injury there are no therapeutics that promote recovery. JNJ-26366821 (TPOm) is a novel thrombopoietin mimetic peptide with no sequence homology to endogenous thrombopoietin (TPO). Endogenous thrombopoietin is produced by hepatocytes and the TPO receptor is present on liver sinusoidal endothelial cells in addition to megakaryocytes and platelets, and we hypothesize that TPOm activity at the TPO receptor in the liver provides a beneficial effect following liver injury. Therefore, we evaluated the extent to which TPOm, NAC or 4-MP can provide a protective and regenerative effect in the liver when administered 2 h after an APAP overdose of 300 mg/kg in fasted male C57BL/6J mice. TPOm did not affect protein adducts, oxidant stress, DNA fragmentation and hepatic necrosis up to 12 h after APAP. In contrast, TPOm treatment was beneficial at 24 h, i.e., all injury parameters were reduced by 42-48%. Importantly, TPOm enhanced proliferation by 100% as indicated by PCNA-positive hepatocytes around the area of necrosis. When TPOm treatment was delayed by 6 h, there was no effect on the injury, but a proliferative effect was still evident. In contrast, 4MP and NAC treated at 2 h after APAP significantly attenuated all injury parameters at 24 h but failed to enhance hepatocyte proliferation. Thus, TPOm arrests the progression of liver injury by 24 h after APAP and accelerates the onset of the proliferative response which is essential for liver recovery.

Development of a clinically relevant ex vivo model of cardiac ablation for testing of ablation catheters.

INTRODUCTION: Reliable ex vivo cardiac ablation models have the potential to increase catheter testing throughput while minimizing animal usage. The goal of this work was to develop a physiologically relevant ex vivo swine model of cardiac ablation displaying minimal variability and high repeatability and identify and optimize key parameters involved in ablation outcomes. METHODS AND RESULTS: A root cause analysis was conducted to identify variables affecting ablation outcomes. Parameters associated with the tissue, bath media, and impedance were identified. Variables were defined experimentally and/or from literature sources to best mimic the clinical cardiac ablation setting. The model was validated by performing three independent replicates of ex vivo myocardial ablation and a direct comparison of lesion outcomes of the ex vivo swine myocardial and in vivo canine thigh preparation (TP) models. Replicate experiments on the ex vivo model demonstrated low variance in ablation depth (6.5 ± 0.6, 6.3 ± 0.6, 6.2 ± 0.4 mm) and width (10.4 ± 1.1, 9.7 ± 1.0, 9.9 ± 0.9 mm) and no significant differences between replicates. In a direct comparison of the two models, the ex vivo model demonstrated ablation depths similar to the canine TP model at 35 W (6.9 ± 1.0, and 7.0 ± 0.9 mm) and 50 W (8.0 ± 0.7, and 8.4 ± 0.7 mm), as well as similar power to depth ratios (15% and 19% for the ex vivo cardiac and in vivo TP models, respectively). CONCLUSION: The ex vivo model exhibited strong lesion reproducibility and power-to-depth ratios comparable to the in vivo TP model. The optimized ex vivo model minimizes animal usage with increased throughput, lesion characteristics similar to the in vivo TP model, and ability to discriminate minor variations between different catheter designs.

JNJ-10450232 (NTM-006), A novel non-opioid with structural similarities to acetaminophen, produces relatively long-lasting analgesia after a single dose in patients undergoing 3rd molar extraction.

JNJ-10450232 (NTM-006) is a non-opioid, non-NSAID analgesic and antipyretic compound with structural similarity to acetaminophen. Preclinical models show comparable analgesia relative to acetaminophen and no evidence of hepatotoxicity associated with overdose. Moreover, it was safe and generally well tolerated in a First-in-Human Study. This single-dose, single-center, inpatient, randomized, double-blind study in moderate-to-severe acute pain following third molar extraction compared efficacy and safety of 250 mg and 1000 mg JNJ-10450232 (NTM-006), 1000 mg acetaminophen, and placebo during the 24 h following administration. While onset of action of 1000 mg JNJ-10450232 (NTM-006) was relatively slower compared with acetaminophen, its duration of action was sustained up to 24 h being superior beginning 7 h after administration. No clinically important differences among treatment groups in nature or severity of adverse events were observed and no serious adverse events were reported. Increased bilirubin, potentially due to UGT1A1 inhibition and ingestion of blood from oral surgery, was the most commonly reported adverse event and the only event reported by ≥ 5% of subjects across treatment groups. These data support further evaluation of JNJ-10450232 (NTM-006) for the treatment of moderate-to-severe pain. CLINICALTRIALS.GOV ID: NCT02209181.

Preclinical safety assessment of JNJ-10450232 (NTM-006), a structural analog of acetaminophen, that does not cause hepatotoxicity at supratherapeutic doses.

JNJ-10450232 (NTM-006) is a new molecular entity that is structurally related to acetaminophen. A comprehensive non-clinical safety program was conducted to support first-in-human and clinical efficacy studies based on preclinical data suggesting that the compound has comparable or enhanced antinociceptive and antipyretic efficacy without causing hepatotoxicity at supratherapeutic doses. No hepatic toxicity was noted in a mouse model sensitive to acetaminophen hepatotoxicity or in rats, dogs, and non-human primates in 28-day repeat dose toxicity studies at and above doses/exposures at which acetaminophen is known to cause hepatotoxicity. In the 28-day toxicity studies, all treatment-related findings were monitorable and reversible. Methemoglobinemia, which was observed in dogs and to a lesser extent in rats, is also observed with acetaminophen. This finding is considered not relevant to humans due to species differences in metabolism. Thyroid hypertrophy and hyperplasia were also observed in dogs and were shown to be a consequence of a species-specific UGT induction also demonstrated with increased thyroid hormone metabolism. Indirect bilirubin elevation was observed in rats as a result of UGT1A1 Inhibition. JNJ-10450232 (NTM-006) had no toxicologically relevant findings in safety pharmacology or genotoxicity studies. Together, these data supported progressing into safety and efficacy studies in humans.

Metabolism and disposition of JNJ-10450232 (NTM-006) in rats, dogs, monkeys and humans.

JNJ-10450232 (NTM-006), a novel non-opioid, non-nonsteroidal anti-inflammatory drug with structural similarities to acetaminophen, demonstrated anti-pyretic and/or analgesic activities in preclinical models and humans and reduced potential to cause hepatotoxicity in preclinical species. Metabolism and disposition of JNJ-10450232 (NTM-006) following oral administration to rats, dogs, monkeys and humans are reported. Urinary excretion was the major route of elimination based on recovery of 88.6% (rats) and 73.7% (dogs) of oral dose. The compound was extensively metabolized based on low recovery of unchanged drug in excreta from rats (11.3%) and dogs (18.4%). Clearance is driven by O-glucuronidation, amide hydrolysis, O-sulfation and methyl oxidation pathways. The combination of metabolic pathways driving clearance in human is covered in at least one preclinical species despite a few species-dependent pathways. O-Glucuronidation was the major primary metabolic pathway of JNJ-10450232 (NTM-006) in dogs, monkeys and humans, although amide hydrolysis was another major primary metabolic pathway in rats and dogs. A minor bioactivation pathway to quinone-imine is observed only in monkeys and humans. Unchanged drug was the major circulatory component in all species investigated. Except for metabolic pathways unique to the 5-methyl-1H-pyrazole-3-carboxamide moiety, metabolism and disposition of JNJ-10450232 (NTM-006) are similar to acetaminophen across species.

First-in-human study to evaluate the safety, tolerability and pharmacokinetics of a novel analgesic and antipyretic drug with structural similarity to acetaminophen.

JNJ-10450232 (NTM-006) is a new molecular entity that comprises structural similarities to acetaminophen and provides comparable analgesia in animals and humans without causing the hepatotoxicity associated with acetaminophen overdose in preclinical models. This double-blind, placebo-controlled, first-in-human study evaluated the safety, tolerability, and pharmacokinetics of JNJ-10450232 (NTM-006) following single (50-6000 mg) and multiple (250-2500 mg twice daily for 8 days) doses in healthy male volunteers. JNJ-10450232 (NTM-006) was absorbed within 1-3 h, except at high doses at which Cmax was delayed and bimodal, while increases in AUC were more than dose proportional. CL/F and Vd/F decreased approximately 3-fold with increasing single doses up to 6000 mg and multiple doses up to 1000 mg, resulting in similar t½ values that ranged from 8 to 10 h across doses. JNJ-10450232 (NTM-006) was generally safe and well tolerated, and no dose-limiting toxicities were observed. Transient increases in indirect bilirubin were noted at post-baseline timepoints due to UGT1A1 inhibition, without any evidence of adverse hepatic effects. Macular rash and generalized erythema were the most common drug-related adverse events after multiple doses.

Site-specific cancer risk following cobalt exposure via orthopedic implants or in occupational settings: A systematic review and meta-analysis.

In 2020, the European Commission up-classified metal cobalt as Class 1B Carcinogen (presumed to have carcinogenic potential) based primarily on data from rodent inhalation carcinogenicity studies. This up-classification requires an assessment under the Medical Device Regulations of cobalt cancer risk from medical devices. We performed a systematic review and meta-analysis to evaluate site-specific cancer risks with cobalt exposure from either total joint replacement (TJR) or occupational exposure (OC). Results were stratified by exposure type (OC or TJR), exposure level (metal-on-metal (MoM) or non-MoM), follow-up duration (latency period: <5, 5-10 or >10 years), and cancer incidence or mortality (detection bias assessment). From 30 studies (653,104 subjects, average 14.5 years follow-up), the association between TJR/OC and cancer risk was null for 22 of 27 cancer sites, negative for 3 sites, and positive for prostate cancer and myeloma. Significant heterogeneity and large estimate ranges were observed for many cancer sites. No significant increase in estimates was observed by exposure level or follow-up duration. The current evidence, including weak associations, heterogeneity across studies and no increased association with exposure level or follow-up duration, is insufficient to conclude that there exists an increased risk for people exposed to cobalt in TJR/OC of developing site-specific cancers.

A comprehensive weight of evidence assessment of published acetaminophen genotoxicity data: Implications for its carcinogenic hazard potential.

In 2019, the California Office of Environmental Health Hazard Assessment initiated a review of the carcinogenic hazard potential of acetaminophen, including an assessment of its genotoxicity. The objective of this analysis was to inform this review process with a weight-of-evidence assessment of more than 65 acetaminophen genetic toxicology studies that are of widely varying quality and conformance to accepted standards and relevance to humans. In these studies, acetaminophen showed no evidence of induction of point or gene mutations in bacterial and mammalian cell systems or in in vivo studies. In reliable, well-controlled test systems, clastogenic effects were only observed in unstable, p53-deficient cell systems or at toxic and/or excessively high concentrations that adversely affect cellular processes (e.g., mitochondrial respiration) and cause cytotoxicity. Across the studies, there was no clear evidence that acetaminophen causes DNA damage in the absence of toxicity. In well-controlled clinical studies, there was no meaningful evidence of chromosomal damage. Based on this weight-of-evidence assessment, acetaminophen overwhelmingly produces negative results (i.e., is not a genotoxic hazard) in reliable, robust high-weight studies. Its mode of action produces cytotoxic effects before it can induce the stable, genetic damage that would be indicative of a genotoxic or carcinogenic hazard.

A systematic assessment of the epidemiologic literature regarding an association between acetaminophen exposure and cancer.

Introduced in the 1950s, acetaminophen is one of the most widely used antipyretics and analgesics worldwide. In 1999, the International Agency for Research on Cancer (IARC) reviewed the epidemiologic studies of acetaminophen and the data were judged to be "inadequate" to conclude that it is carcinogenic. In 2019 the California Office of Environmental Health Hazard Assessment initiated a review process on the carcinogenic hazard potential of acetaminophen. To inform this review process, the authors performed a comprehensive literature search and identified 136 epidemiologic studies, which for most cancer types suggest no alteration in risk associated with acetaminophen use. For 3 cancer types, renal cell, liver, and some forms of lymphohematopoietic, some studies suggest an increased risk; however, multiple factors unique to acetaminophen need to be considered to determine if these results are real and clinically meaningful. The objective of this publication is to analyze the results of these epidemiologic studies using a framework that accounts for the inherent challenge of evaluating acetaminophen, including, broad population-wide use in multiple disease states, challenges with exposure measurement, protopathic bias, channeling bias, and recall bias. When evaluated using this framework, the data do not support a causal association between acetaminophen use and cancer.

Assessment of the biochemical pathways for acetaminophen toxicity: Implications for its carcinogenic hazard potential.

In 2019 California's Office of Environmental Health Hazard Assessment (OEHHA) initiated a review of the carcinogenic hazard potential of acetaminophen. In parallel with this review, herein we evaluated the mechanistic data related to the steps and timing of cellular events following therapeutic recommended (≤4 g/day) and higher doses of acetaminophen that may cause hepatotoxicity to evaluate whether these changes indicate that acetaminophen is a carcinogenic hazard. At therapeutic recommended doses, acetaminophen forms limited amounts of N-acetyl-p-benzoquinone-imine (NAPQI) without adverse cellular effects. Following overdoses of acetaminophen, there is potential for more extensive formation of NAPQI and depletion of glutathione, which may result in mitochondrial dysfunction and DNA damage, but only at doses that result in cell death - thus making it implausible for acetaminophen to induce the kind of stable, genetic damage in the nucleus indicative of a genotoxic or carcinogenic hazard in humans. The collective data demonstrate a lack of a plausible mechanism related to carcinogenicity and are consistent with rodent cancer bioassays, epidemiological results reviewed in companion manuscripts in this issue, as well as conclusions of multiple international health authorities.

Carcinogenic assessment of cobalt-containing alloys in medical devices or cobalt in occupational settings: A systematic review and meta-analysis of overall cancer risk from published epidemiologic studies.

In 2020, the European Commission up-classified pure cobalt metal to a Category 1B hazard, based primarily on data from rodent inhalation carcinogenicity studies of metallic cobalt. The European Commission review did not evaluate cobalt-containing alloys in medical devices, which have very different properties vs. pure cobalt metal and did not include a systematic epidemiologic review. We performed a systematic review and meta-analysis of published, peer-reviewed epidemiologic studies evaluating the association between overall cancer risk and exposure to orthopedic implants containing cobalt alloys or cobalt particulates in occupational settings. Study-specific estimates were pooled using random-effects models. Analyses included 20 papers on orthopedic implants and 10 occupational cohort papers (~1 million individuals). The meta-analysis summary estimates (95% confidence intervals) for overall cancer risk were 1.00 (0.96-1.04) overall and 0.97 (0.94-1.00) among high-quality studies. Results were also similar in analyses stratified by type of exposure/data sources (occupational cohort, implant registry or database), comparators (general or implant population), cancer incidence or mortality, follow-up duration (latency period), and study precision. In conclusion, meta-analysis found no association between exposure to orthopedic implants containing cobalt alloys or cobalt particulates in occupational settings and overall cancer risk, including an analysis of studies directly comparing metal-on-metal vs. non-metal-on-metal implants.

A hazard evaluation of the reproductive/developmental toxicity of cobalt in medical devices.

Cobalt (Co) is an essential element with human exposure occurring from the diet, supplement ingestion, occupational sources, and medical devices. The European Chemical Agency (ECHA) recently voted to classify Co metal as a Reproductive Hazard Category 1B; presumed human reproductive toxicant due to adverse testicular effects in male rodents. A weight of evidence evaluation of the preclinical reproductive and developmental toxicity studies and available clinical data was performed to critically evaluate the relevance of this proposed classification for Co in medical devices. Reproductive responses to Co are limited to the male testes and sperm function following high systemic exposure in rodents, only at Co concentrations/doses that result in overt toxicity (i.e., above the maximum tolerable dose (MTD)). The potential mechanisms of Co reproductive/developmental toxicity, including its indirect mode of action in the testes and relevance to humans, are discussed. The available preclinical and clincial evidence suggests that it would be more appropriate to classify Co as a Reproductive Hazard Category 2 compound: suspected human reproductive toxicant and, in the case of Co-containing medical devices, it should not be considered a reproductive hazard.

An integrated benefit-risk assessment of cobalt-containing alloys used in medical devices: Implications for regulatory requirements in the European Union.

In 2017, the European Union (EU) Committee for Risk Assessment (RAC) recommended the classification of metallic cobalt (Co) as Category 1B with respect to its carcinogenic and reproductive hazard potential and Category 2 for mutagenicity but did not evaluate the relevance of these classifications for patients exposed to Co-containing alloys (CoCA) used in medical devices. CoCA are inherently different materials from Co metal from a toxicological perspective and thus require a separate assessment. CoCA are biocompatible materials with a unique combination of properties including strength, durability, and a long history of safe use that make them uniquely suited for use in a wide-range of medical devices. Assessments were performed on relevant preclinical and clinical carcinogenicity and reproductive toxicity data for Co and CoCA to meet the requirements under the EU Medical Device Regulation triggered by the ECHA re-classification (adopted in October 2019 under the 14th Adaptation to Technical Progress to CLP) and to address their relevance to patient safety. The objective of this review is to present an integrated overview of these assessments, a benefit-risk assessment and an examination of potential alternative materials. The data support the conclusion that the exposure to CoCA in medical devices via clinically relevant routes does not represent a hazard for carcinogenicity or reproductive toxicity. Additionally, the risk for the adverse effects that are known to occur with elevated Co concentrations (e.g., cardiomyopathy) are very low for CoCA implant devices (infrequent reports often reflecting a unique catastrophic failure event out of millions of patients) and negligible for CoCA non-implant devices (not measurable/no case reports). In conclusion, the favorable benefit-risk profile also in relation to possible alternatives presented herein strongly support continued use of CoCA in medical devices.

Carcinogenic hazard assessment of cobalt-containing alloys in medical devices: Review of in vivo studies.

Cobalt (Co) alloys have been used for over seven decades in a wide range of medical devices, including, but not limited to, hip and knee implants, surgical tools, and vascular stents, due to their favorable biocompatibility, durability, and mechanical properties. A recent regulatory hazard classification review by the European Chemicals Agency (ECHA) resulted in the classification of metallic Co as a Class 1B Carcinogen (presumed to have carcinogenic potential for humans), primarily based on inhalation rodent carcinogenicity studies with pure metallic Co. The ECHA review did not specifically consider the carcinogenicity hazard potential of forms or routes of Co that are relevant for medical devices. The purpose of this review is to present a comprehensive assessment of the available in vivo preclinical data on the carcinogenic hazard potential of exposure to Co-containing alloys (CoCA) in medical devices by relevant routes. In vivo data were reviewed from 33 preclinical studies that examined the impact of Co exposure on local and systemic tumor incidence in rats, mice, guinea pigs, and hamsters. Across these studies, there was no significant increase of local or systemic tumors in studies relevant for medical devices. Taken together, the relevant in vivo data led to the conclusion that CoCA in medical devices are not a carcinogenic hazard in available in vivo models. While specific patient and implant factors cannot be fully replicated using in vivo models, the available in vivo preclinical data support that CoCA in medical devices are unlikely a carcinogenic hazard to patients.

Application of the DILIsym® Quantitative Systems Toxicology drug-induced liver injury model to evaluate the carcinogenic hazard potential of acetaminophen.

In 2019, the California Office of Environmental Health Hazard Assessment (OEHHA) initiated a review of the carcinogenic hazard potential of acetaminophen. The objective of the analysis herein was to inform this review by assessing whether variability in patient baseline characteristics (e.g. baseline glutathione (GSH) levels, pharmacokinetics, and capacity of hepatic antioxidants) leads to potential differences in carcinogenic hazard potential at different dosing schemes: maximum labeled doses of 4 g/day, repeated doses above the maximum labeled dose (>4-12 g/day), and acute overdoses of acetaminophen (>15 g). This was achieved by performing simulations of acetaminophen exposure in thousands of diverse virtual patients scenarios using the DILIsym® Quantitative Systems Toxicology (QST) model. Simulations included assessments of the dose and exposure response for toxicity and mode of cell death based on evaluations of the kinetics of changes of: GSH, N-acetyl-p-benzoquinone-imine (NAPQI), protein adducts, mitochondrial dysfunction, and hepatic cell death. Results support that, at therapeutic doses, cellular GSH binds to NAPQI providing sufficient buffering capacity to limit protein adduct formation and subsequent oxidative stress. Simulations evaluating repeated high-level supratherapeutic exposures or acute overdoses indicate that cell death precedes DNA damage that could result in carcinogenicity and thus acetaminophen does not present a carcinogenicity hazard to humans at any dose.

A critical review of the acetaminophen preclinical carcinogenicity and tumor promotion data and their implications for its carcinogenic hazard potential.

In 2019 the California Office of Environmental Health Hazard Assessment (OEHHA) initiated a review of the carcinogenic hazard potential of acetaminophen, including an assessment of the long-term rodent carcinogenicity and tumor initiation/promotion studies. The objective of the analysis herein was to inform this review process with a weight-of-evidence assessment of these studies and an assessment of the relevance of these models to humans. In most of the 14 studies, there were no increases in the incidences of tumors in any organ system. In the few studies in which an increase in tumor incidence was observed, there were factors such as absence of a dose response and a rodent-specific tumor supporting that these findings are not relevant to human hazard identification. In addition, we performed qualitative analysis and quantitative simulations of the exposures to acetaminophen and its metabolites and its toxicity profile; the data support that the rodent models are toxicologically relevant to humans. The preclinical carcinogenicity results are consistent with the broader weight of evidence assessment and evaluations of multiple international health authorities supporting that acetaminophen is not a carcinogenic hazard.

Effect of Opioids on Testosterone Levels: Cross-Sectional Study using NHANES.

OBJECTIVE: Opioids can suppress gonadal hormone production, which may result in low testosterone levels. To date, there have been no large-scale population-based studies examining the extent to which opioid use may contribute to changes in testosterone levels. DESIGN: Cross-sectional study. SETTING: 2011-2012 National Health and Nutrition Examination Survey. SUBJECTS: Participants 17 years and older who had data on prescription medication usage and serum testosterone levels available. Participants were divided in two groups, opioid exposed and unexposed. METHODS: Testosterone levels of participants who responded that they had been exposed (n = 320) to prescription opioids in the past 30 days were compared with those who were unexposed (n = 4909). The number of participants with low testosterone levels was calculated and unadjusted and adjusted analyses were performed. RESULTS: Participants on opioids had higher odds of having low testosterone levels than those unexposed, odd ratio (OR) = 1.40, 95% confidence interval (CI) (1.07-1.84). After controlling for opioid exposure, as the age and the number of comorbidities increased, the odds of having low testosterone levels significantly increased in all categories. Compared with participants between 17 and 45 years of age, participants >70 years had OR = 1.70, 95% CI (1.16-2.50). Compared with participants with no comorbidities, participants with >2 comorbidities had OR = 1.69 95% CI (1.24-2.30). CONCLUSION: When assessing the impact of opioids on testosterone, the effects of age and medical conditions should be considered.

Thrombopoietin mimetic stimulates bone marrow vascular and stromal niches to mitigate acute radiation syndrome.

BACKGROUND: Acute radiation syndrome (ARS) manifests after exposure to high doses of radiation in the instances of radiologic accidents or incidents. Facilitating regeneration of the bone marrow (BM), namely the hematopoietic stem and progenitor cells (HSPCs), is key in mitigating ARS and multi-organ failure. JNJ-26366821, a PEGylated thrombopoietin mimetic (TPOm) peptide, has been shown as an effective medical countermeasure (MCM) to treat hematopoietic-ARS (H-ARS) in mice. However, the activity of TPOm on regulating BM vascular and stromal niches to support HSPC regeneration has yet to be elucidated. METHODS: C57BL/6J mice (9-14 weeks old) received sublethal or lethal total body irradiation (TBI), a model for H-ARS, by 137Cs or X-rays. At 24 h post-irradiation, mice were subcutaneously injected with a single dose of TPOm (0.3 mg/kg or 1.0 mg/kg) or PBS (vehicle). At homeostasis and on days 4, 7, 10, 14, 18, and 21 post-TBI with and without TPOm treatment, BM was harvested for histology, BM flow cytometry of HSPCs, endothelial (EC) and mesenchymal stromal cells (MSC), and whole-mount confocal microscopy. For survival, irradiated mice were monitored and weighed for 30 days. Lastly, BM triple negative cells (TNC; CD45-, TER-119-, CD31-) were sorted for single-cell RNA-sequencing to examine transcriptomics after TBI with or without TPOm treatment. RESULTS: At homeostasis, TPOm expanded the number of circulating platelets and HSPCs, ECs, and MSCs in the BM. Following sublethal TBI, TPOm improved BM architecture and promoted recovery of HSPCs, ECs, and MSCs. Furthermore, TPOm elevated VEGF-C levels in normal and irradiated mice. Following lethal irradiation, mice improved body weight recovery and 30-day survival when treated with TPOm after 137Cs and X-ray exposure. Additionally, TPOm reduced vascular dilation and permeability. Finally, single-cell RNA-seq analysis indicated that TPOm increased the expression of collagens in MSCs to enhance their interaction with other progenitors in BM and upregulated the regeneration pathway in MSCs. CONCLUSIONS: TPOm interacts with BM vascular and stromal niches to locally support hematopoietic reconstitution and systemically improve survival in mice after TBI. Therefore, this work warrants the development of TPOm as a potent radiation MCM for the treatment of ARS.

Thrombopoietin mimetic stimulates bone marrow vascular and stromal niches to mitigate acute radiation syndrome.

Background: Acute radiation syndrome (ARS) manifests after exposure to high doses of radiation in the instances of radiologic accidents or incidents. Facilitating the regeneration of the bone marrow (BM), namely the hematopoietic stem and progenitor cells (HSPCs), is a key in mitigating ARS and multi-organ failure. JNJ-26366821, a PEGylated thrombopoietin mimetic (TPOm) peptide, has been shown as an effective medical countermeasure (MCM) to treat hematopoietic-ARS (H-ARS) in mice. However, the activity of TPOm on regulating BM vascular and stromal niches to support HSPC regeneration has not yet been elucidated. Methods: C57BL/6J mice (9-14 weeks old) received sublethal or lethal total body irradiation (TBI), a model for H-ARS, by 137Cs or X-rays. At 24 hours post-irradiation, mice were subcutaneously injected with a single dose of TPOm (0.3 mg/kg or 1.0 mg/kg) or PBS (vehicle). At homeostasis and on days 4, 7, 10, 14, 18, and 21 post-TBI with and without TPOm treatment, BM was harvested for histology, BM flow cytometry of HSPCs, endothelial (EC) and mesenchymal stromal cells (MSC), and whole-mount confocal microscopy. For survival, irradiated mice were monitored and weighed for 30 days. Lastly, BM triple negative cells (TNC; CD45-, TER-119-, CD31-) were sorted for single-cell RNA-sequencing to examine transcriptomics after TBI with or without TPOm treatment. Results: At homeostasis, TPOm expanded the number of circulating platelets and HSPCs, ECs, and MSCs in the BM. Following sublethal TBI, TPOm improved BM architecture and promoted recovery of HSPCs, ECs, and MSCs. Furthermore, TPOm elevated VEGF-C levels in normal and irradiated mice. Following lethal irradiation, mice improved body weight recovery and 30-day survival when treated with TPOm after 137Cs and X-ray exposure. Additionally, TPOm reduced vascular dilation and permeability. Finally, single-cell RNA-seq analysis indicated that TPOm increased the expression of collagens in MSCs to enhance their interaction with other progenitors in BM and upregulated the regeneration pathway in MSCs. Conclusions: TPOm interacts with BM vascular and stromal niches to locally support hematopoietic reconstitution and systemically improve survival in mice after TBI. Therefore, this work warrants the development of TPOm as a potent radiation MCM for the treatment of ARS.

PEGylated thrombopoietin mimetic, JNJ­26366821 a novel prophylactic radiation countermeasure for acute radiation injury.

Thrombopoietin (TPO) is the primary regulator of platelet generation and a stimulator of multilineage hematopoietic recovery following exposure to total body irradiation (TBI). JNJ­26366821, a novel PEGylated TPO mimetic peptide, stimulates platelet production without developing neutralizing antibodies or causing any adverse effects. Administration of a single dose of JNJ­26366821 demonstrated its efficacy as a prophylactic countermeasure in various mouse strains (males CD2F1, C3H/HeN, and male and female C57BL/6J) exposed to Co-60 gamma TBI. A dose dependent survival efficacy of JNJ­26366821 (- 24 h) was identified in male CD2F1 mice exposed to a supralethal dose of radiation. A single dose of JNJ­26366821 administered 24, 12, or 2 h pre-radiation resulted in 100% survival from a lethal dose of TBI with a dose reduction factor of 1.36. There was significantly accelerated recovery from radiation-induced peripheral blood neutropenia and thrombocytopenia in animals pre-treated with JNJ­26366821. The drug also increased bone marrow cellularity and megakaryocytes, accelerated multi-lineage hematopoietic recovery, and alleviated radiation-induced soluble markers of bone marrow aplasia and endothelial damage. These results indicate that JNJ­26366821 is a promising prophylactic radiation countermeasure for hematopoietic acute radiation syndrome with a broad window for medical management in a radiological or nuclear event.