Repeat Dose Research Articles

Effects of Sargassum horneri and Ulva australis Extracts on Body Weight and Serum Glucose Levels of Sprague-Dawley Rats.

This study investigated the safety and functionality of a functional additive for humans and animals from Sargassum horneri (SH) and Ulva australis (UA) waste for recycling marine refuse generated in large quantities in Jeju. Sprague-Dawley rats were orally administered functional additives at 2,000 mg/kg to assess 14-day repeated dose toxicity of the two extracts. For female rats, weight gain after administration of SH was 66.2±18.8% vs. controls. Male rats administered UA showed weight gain of 92.3±8.0% vs. controls. SH and UA significantly decreased serum glucose levels in male rats compared with controls (79.8±11.10% and 76.1±9.67%, respectively). Similarly, significant decrease in serum glucose levels were shown for female rats after administration of SH and UA (79.2±1.58% and 82.8±3.21%, respectively). Furthermore, rats showed significant differences vs. controls in several serological parameters after receiving extracts, however results remained within the normal range. Thus, the SH and UA extracts were considered safe substances that may be used as functional additives to help reduce body weight and serum glucose.

Acute and Repeated Dose Toxicity Study of Clevira Syrup – A Polyherbal Formulation

Aim and Objectives: This study evaluated the acute and repeated dose toxicity effects of Clevira Syrup Polyherbal formulation (CSPHF), which was prepared from ten different herbs, well known and widely used in traditional medicine for the management of viral infections and other inflammatory disease conditions. Individually these herbs (Carica papaya, Melia azedarach, Andrographis paniculata, Vetiveria zizanioides, Trichosanthes dioica, Cyperus rotundus, Zingiber officinale, Piper nigrum, Mollugo Cerviana and Tinospora cordifolia )were completely safe, but the polyherbal formulation effects were not known. Thus, this study was done for the investigation of toxicological profile of CSPHF in Wistar Albino rats. Methods: As per OECD(Organisation for Economic Co-operation and Development) guidelines 423 and 407, Acute and Repeated dose toxicity study were proceeded. In the acute toxicity study a single dose of CSPHF (2000mg/kg) was administered orally to female Wistar rats and in repeated dose toxicity study, CSPHF was administered orally in Control group and three different doses (1000, 500 and 250mg/kg body weight) to both male and female wistar albino rats for 28 days. At the end of the study, the animals were euthanized, observed the external and internal morphology (Acute Toxicity) and assessed the effect of CSPHF on histopathological and biochemical parameters (Repeated Dose toxicity study). Results: In acute toxicity study, there were no visual signs of toxicity of CSPHF (2000mg/kg) observed, whereas in Repeated dose toxicity study Ischaemia, inflammation and hematoma of the internal organs were observed at 1000mg/kg dose, but no such toxic features were seen at 500 and 250mg/kg dose of CSPHF. Conclusion: The results of the Acute and Repeated Dose toxicity study could be authenticated in future studies, which will be more useful and evidence based for the management of Viral infectionsduring pandemics.

Booster Dose of Bacille Calmette-Guérin Vaccine for Tuberculosis in Low and Middle-Income Countries: A Systematic Review

Background: The Bacille Calmette-Guérin (BCG) vaccine, given as a single dose, offers variable protection against Tuberculosis (TB). It is plausible that repeat doses could improve the effectiveness of the BCG vaccine in settings where the population remain at risk of the disease.
 Objective: To assess the effectiveness of BCG revaccination as a booster dose in preventing TB in Low- and Middle- Income Countries (LMICs).
 Methods: We searched the electronic databases without language or publication restrictions and followed the procedures for preparing systematic reviews, including assessing the risk of bias as outlined in the Cochrane handbook. We included randomised controlled trials (RCTs) conducted in LMICs involving children and adults receiving one or more BCG vaccine doses after the primary BCG vaccination. The incidence of severe forms of TB, active TB and adverse events were the primary outcomes.
 Results: Five RCTs were included in this systematic review. Revaccination with BCG probably makes little or no difference to the risk of active TB measured after five years (Relative risk (RR) 1.16, 95% CI 0.88 to 1.51; 348,083 participants; one study, moderate certainty evidence) or nine years post-revaccination (RR 0.96, 95% CI 0.82 to 1.12; 348,083 participants; one study, moderate certainty evidence). In populations with HIV co-infection, revaccination probably increases the risk of pulmonary tuberculosis compared to placebo (RR 1.74, 95% CI 1.00 to 3.01; 46,764 participants; one study, moderate certainty evidence).
 Conclusion: The available evidence suggests that BCG revaccination probably makes little or no difference in preventing tuberculosis disease in LMICs.

JNJ-67569762, A 2-Aminotetrahydropyridine-Based Selective BACE1 Inhibitor Targeting the S3 Pocket: From Discovery to Clinical Candidate.

The discovery of a novel 2-aminotetrahydropyridine class of BACE1 inhibitors is described. Their pKa and lipophilicity were modulated by a pending sulfonyl group, while good permeability and brain penetration were achieved via intramolecular hydrogen bonding. BACE1 selectivity over BACE2 was achieved in the S3 pocket by a novel bicyclic ring system. An optimization addressing reactive metabolite formation, cardiovascular safety, and CNS toxicity is described, leading to the clinical candidate JNJ-67569762 (12), which gave robust dose-dependent BACE1-mediated amyloid β lowering without showing BACE2-dependent hair depigmentation in preclinical models. We show that 12 has a favorable projected human dose and PK and hence presented us with an opportunity to test a highly selective BACE1 inhibitor in humans. However, 12 was found to have a QT effect upon repeat dosing in dogs and its development was halted in favor of other selective leads, which will be reported in the future.

DMD – ANIMAL MODELS: EP.81 Enhanced exon skipping and Dystrophin production in a mouse model of Duchenne muscular dystrophy with EEV-PMO treatment

Antisense oligonucleotide-induced (ASOs) exon skipping is a promising treatment strategy for Duchenne muscular dystrophy (DMD), an X-linked recessive and fatal neuromuscular disease. DMD is caused by mutations in the dystrophin gene and affects approximately 1 in 3,500-5,000 male births. By modulating the splicing of dystrophin pre-mRNA, ASOs can restore the mRNA reading frame and generate a truncated, functional dystrophin protein. This exon skipping approach can address ∼80% of the mutations that cause DMD. However, the poor exposure and endosomal escape in target tissue after systemic administration of ASOs remains the biggest hurdle for the treatment of patients with DMD and results in insufficient dystrophin protein restoration, especially in non-skeletal muscle tissues such as the heart. We have developed a novel ASO delivery system by conjugation of Entrada's proprietary Endosomal Escape Vehicle (EEVTM) to a phosphorodiamidate morpholino oligomer (PMO), an effective ASO for DMD treatment. The EEV is composed of a cyclic cell-penetrating peptide coupled with a nuclear localization sequence, which is rationally designed to facilitate intracellular delivery, endosomal escape and finally localization in the nucleus. The EEV-PMO significantly enhances the systematic delivery of PMOs and results in robust exon skipping and restoration of dystrophin protein in the mdx mouse model skeletal as well as cardiac muscle. A single dose (20 mg/kg) by intravenous administration of EEV-PMO led to >50% exon skipping and >15% correction of dystrophin protein in the heart seven days post injection, and dystrophin protein levels were sustained for up to four weeks. Repeat dosing (10 mg/kg, 4 QW) resulted in >30% of wild-type dystrophin protein in the heart and normalized serum creatine kinase level seven days post last injection. These results have confirmed the significant therapeutic potential of the EEV-PMO delivery system for DMD. Furthermore, these results demonstrate that EEVs can serve as a general and highly efficient delivery platform that possesses great potential for the delivery of other therapeutic ASOs beyond DMD.

Notoedric Mange in a Domestic Cat: Application of Ivermectin and Supportive Therapy

Feline scabies is a disease that is caused by Notoedres cati, a sarcoptic mite that injures the skin tissue. The present clinical case relates a notoedric scabies in a domestic cat, clinical observations, diagnostic test, and treatment. A 4-year-old male neutered domestic short haired cat was brought at a veterinary clinic. Alopecia, erythema, scales, crusts and hyperkeratosis were observed on the head, and pinnae and malodorous from lesions were observed in dermatological examination. Deep skin scrapings were collected. Microscopic examination revealed adult live mites presence. Based on the morphology, the mites isolated were identified as Notoedres cati. Ivermectin oral capsules (300µg/kg), repeat dose at 14 days. Bath with chlorhexidine and Benzoyl peroxide every 5 days for two months were applied. Moreover, multivitamin complexes were supplied daily, orally. An immunostimulant 1mL/10kg. IM, again applied two days. A clinical improvement in the cat was observed after administered this treatment. The present study reports notoedric mange in a domestic short haired cat in Colombia. The application of ivermectin with supportive therapy was useful in the treatment of notoedric scabies in this cat. The application of products other than ivermectin showed positive clinical observations in the recovery of this patient with feline scabies.

Exposure to an Environmental Mixture of Polycyclic Aromatic Hydrocarbons Induces Hepatic Cytochrome P450 Enzymes in Mice.

Cytochrome P450 enzymes (CYPs) play an important role in bioactivating or detoxifying polycyclic aromatic hydrocarbons (PAHs), common environmental contaminants. While it is widely accepted that exposure to PAHs induces CYPs, effectively increasing rates of xenobiotic metabolism, dose- and time-response patterns of CYP induction are not well-known. In order to better understand dose- and time-response relationships of individual CYPs following induction, we exposed B6129SF1/J mice to single or repeated doses (2-180 μmol/kg/d) of benzo[a]pyrene (BaP) or Supermix-10, a mixture of the top 10 most abundant PAHs found at the Portland Harbor Superfund Site. In hepatic microsomes from exposed mice, we measured amounts of active CYPs using activity-based protein profiling and total CYP expression using global proteomics. We observed rapid Cyp1a1 induction after 6 h at the lowest PAH exposures and broad induction of many CYPs after 3 daily PAH doses at 72 h following the first dose. Using samples displaying Cyp1a1 induction, we observed significantly higher metabolic affinity for BaP metabolism (Km reduced 3-fold), 3-fold higher intrinsic clearance, but no changes to the Vmax. Mice dosed with the highest PAH exposures exhibited 1.7-5-fold higher intrinsic clearance rates for BaP compared to controls and higher Vmax values indicating greater amounts of enzymes capable of metabolizing BaP. This study demonstrates exposure to PAHs found at superfund sites induces enzymes in dose- and time-dependent patterns in mice. Accounting for specific changes in enzyme profiles, relative rates of PAH bioactivation and detoxification, and resulting risk will help translate internal dosimetry of animal models to humans and improve risk assessments of PAHs at superfund sites.

Safety and toxicology assessment of sodium nitrite administered by intramuscular injection

Intramuscular (IM) injection of nitrite (1-10 mg/kg) confers survival benefit and protects against lung injury after exposure to chlorine gas in preclinical models. Herein, we evaluated safety/toxicity parameters after single, and repeated (once daily for 7 days) IM injection of nitrite in male and female Sprague Dawley rats and Beagle dogs. The repeat dose studies were performed in compliance with the Federal Drug Administration's (FDA) Good Laboratory Practices Code of Federal Regulations (21 CFR Part 58). Parameters evaluated consisted of survival, clinical observations, body weights, clinical pathology, plasma drug levels, methemoglobin and macroscopic and microscopic pathology. In rats and dogs, single doses of ≥100 mg/kg and 60 mg/kg resulted in death and moribundity, while repeated administration of ≤30 or ≤ 10 mg/kg/day, respectively, was well tolerated. Therefore, the maximum tolerated dose following repeated administration in rats and dogs were determined to be 30 mg/kg/day and 10 mg/kg/day, respectively. Effects at doses below the maximum tolerated dose (MTD) were limited to emesis (in dogs only) and methemoglobinemia (in both species) with clinical signs (e.g. blue discoloration of lips) being dose-dependent, transient and reversible. These signs were not considered adverse, therefore the No Observed Adverse Effect Level (NOAEL) for both rats and dogs was 10 mg/kg/day in males (highest dose tested for dogs), and 3 mg/kg/day in females. Toxicokinetic assessment of plasma nitrite showed no difference between male and females, with Cmax occurring between 5 mins and 0.5 h (rats) or 0.25 h (dogs). In summary, IM nitrite was well tolerated in rats and dogs at doses previously shown to confer protection against chlorine gas toxicity.

Predicting the in vivo developmental toxicity of benzo[a]pyrene (BaP) in rats by an in vitro\u2013in silico approach

Developmental toxicity testing is an animal-intensive endpoints in toxicity testing and calls for animal-free alternatives. Previous studies showed the applicability of an in vitro–in silico approach for predicting developmental toxicity of a range of compounds, based on data from the mouse embryonic stem cell test (EST) combined with physiologically based kinetic (PBK) modelling facilitated reverse dosimetry. In the current study, the use of this approach for predicting developmental toxicity of polycyclic aromatic hydrocarbons (PAHs) was evaluated, using benzo[a]pyrene (BaP) as a model compound. A rat PBK model of BaP was developed to simulate the kinetics of its main metabolite 3-hydroxybenzo[a]pyrene (3-OHBaP), shown previously to be responsible for the developmental toxicity of BaP. Comparison to in vivo kinetic data showed that the model adequately predicted BaP and 3-OHBaP blood concentrations in the rat. Using this PBK model and reverse dosimetry, a concentration–response curve for 3-OHBaP obtained in the EST was translated into an in vivo dose–response curve for developmental toxicity of BaP in rats upon single or repeated dose exposure. The predicted half maximal effect doses (ED50) amounted to 67 and 45 mg/kg bw being comparable to the ED50 derived from the in vivo dose–response data reported for BaP in the literature, of 29 mg/kg bw. The present study provides a proof of principle of applying this in vitro–in silico approach for evaluating developmental toxicity of BaP and may provide a promising strategy for predicting the developmental toxicity of related PAHs, without the need for extensive animal testing.

L-citrulline: A preclinical safety biomarker for the small intestine in rats and dogs in repeat dose toxicity studies

IntroductionGastrointestinal (GI) toxicity is still an issue within drug development, especially for novel oncology drugs. The identification of GI mucosal damage at an early stage with high sensitivity and specificity across preclinical species and humans remains difficult. To date, in preclinical studies, no qualified mechanistic, diagnostic or prognostic biomarkers exist for GI mucosal toxicity. L-citrulline is one of the most promising biomarker candidates used in clinical settings to quantify enterocyte integrity in various small intestinal diseases. L-citrulline is an intermediate metabolic amino acid produced mainly by functional enterocytes of the small intestine, whereby enterocyte loss will cause a drop in circulating L-citrulline. MethodsIn several repeat-dose toxicity studies, plasma L-citrulline has been evaluated as a potential safety biomarker for intestinal toxicity in beagle dogs and Wistar (Han) rats treated with different oncological drug candidates in drug development. Clinical observations and body weight determinations were performed during the pretreatment, treatment and treatment-free recovery period as well as toxicokinetic, gross and histopathology examinations. The quantitative determination of plasma L-citrulline levels during the pretreatment (only dogs), treatment and treatment-free recovery period were performed using an HPLC MS/MS assay. In cynomolgus monkeys, the first investigations on baseline L-citrulline levels were performed. ResultsIn dogs, a dose- and exposure-dependent decrease of up to 50% in plasma L-citrulline was seen without histopathological alterations. However, a decrease of more than 50% in comparison to the individual animal pretreatment value of L-citrulline correlated very well with histopathological findings (intestinal crypt necrosis, villus atrophy, enterocyte loss) and clinical signs (bloody faeces and diarrhoea). During a treatment-free recovery period, a trend of increasing levels was observed in dogs. In rats, absolute L-citrulline plasma levels of treated animals decreased compared to the values of the concurrent control group. This decrease also correlated with the histopathological findings in the small intestine (single cell necrosis and mucosa atrophy). Because of a large physiological variation in L-citrulline plasma levels in dogs and rats, a clear cut-off value for absolute L-citrulline levels predictive of intestinal mucosal toxicity was difficult to establish. However, a > 50% decrease in L-citrulline plasma levels during the treatment period strongly correlated with histopathological findings. DiscussionBased on the performed analysis, a longitudinal investigation of L-citrulline plasma levels for individual animals in the control and treatment groups is essential and pretreatment values of L-citrulline levels in rodents would be highly informative. Overall, further cross-species comparison (Cynomolgus monkey, mouse) and implementation in clinical trials as exploratory biomarker is essential to foster the hypothesis and to understand completely the clinical relevance of L-citrulline as a small intestine biomarker.

Antihyperglycemic and hypoglycemic activities of the aqueous leaf extract of Rubus Erlangeri Engl (Rosacea) in mice

BackgroundThe prevalence of diabetes mellitus is on the inexorable rise despite the promises of a wide range of conventional medications. Thus, there is a need to scientifically investigate plants for antidiabetic effect. MethodsAfter the Rubus Erlanrige Engl (Rosaceae) leaf has been decocted, the plant extract's antidiabetic activity was first investigated in vitro and then in vivo. The in vitro activity was assessed using 3, 5-Dinitrosalicylic acid, and 2,2-diphenyl-1-picrylhydrazine method for α-amylase inhibition and antioxidant effect respectively. On the other hand, the in vivo antidiabetic activity was carried out in normoglycemic, glucose loaded (2.5 g/kg) and single dose streptozotocin (200 mg/kg) induced diabetic mice. ResultsAcute toxicity study showed the extract is safe with ≥2 g/kg. The in vitro results demonstrated the extract has an IC50 of 7.34 ± 0.02 and 10.38 ± 0.0.62 μg/ml for antioxidant and α-amylase inhibition activity respectively. On the other hand, the in vivo study revealed that the extract significantly reduced blood glucose level following glucose loading. The extract did not, however, produce a significant reduction of glucose level in normal mice indicating low risk of hypoglycemia. The extract also significantly decreased blood glucose levels in streptozotocin-induced diabetic mice. In the single dose study, the extract lowered blood glucose level all except by lower dose at the 3rd and 4th h (p < 0.05). In repeated dose studies, the reduction in fasting blood glucose was significant with all doses of the extract from the 2nd week onwards. In addition, the extract produced less reduction in body weight after diabetic induction. ConclusionThe findings collectively indicate that the extract has an antidiabetic activity, with low risk of hypoglycemia, probably mediated by various secondary metabolites that act in synergy.

A genome guided evaluation of the Lab4 probiotic consortium

Draft genome sequences of the Lab4 probiotic consortium were deposited in Genbank: Bifidobacterium animalis subsp lactis CUL34 (PRJNA482550), Bifidobacterium bifidum CUL20 (PRJNA559984), Lactobacillus acidophilus CUL60 (PRJNA482335), Lactobacillus acidophilus CUL21 (PRJNA482434). Probiogenomic analyses confirmed existing taxonomies and identified putative gene sequences that were functionally related to the performance of each organism during in vitro assessments of bile and acid tolerability, adherence to enterocytes and susceptibility to antibiotics. Genomic stability predictions identified no significant risk of gene acquisition of both antibiotic resistance and virulence genes. These observations were supported by acute phase and repeat dose tolerability studies in Wistar rats. High doses of Lab4 did not result in mortalities, clinical/histopathological abnormalities nor systemic toxicity. Increased faecal numbers of Lab4 in supplemented rats implied survival through the gastrointestinal tract and/or impact the intestinal microbiota composition. In summary, this study provides multifaceted support for probiotic functionality and the safety of the Lab4 consortium.

An evaluation of carcinogenicity predictors from short-term and sub chronic repeat-dose studies of agrochemicals in rats: Opportunities to refine and reduce animal use

The aim of this study was to examine whether short term, repeat dose, rat studies provide sufficient information about potential carcinogenicity to enable predictions about the carcinogenic potential of agrochemicals to be made earlier in compound development. This study aimed to identify any correlations between toxicity findings obtained for short term rat studies (28 day and 90 day) and neoplastic findings obtained from 24 month rat carcinogenicity studies for agrochemical compounds (18 compounds) tested in Han Wistar and Sprague Dawley rats. The macroscopic pathology, microscopic pathology, hematology, biochemistry, organ weights, estrogen receptor activation and genotoxicity results were examined. Seven out of 18 non genotoxic compounds developed tumors in treated rats in the carcinogenicity study and of these, two compounds showed no preneoplastic findings in the affected tissues (false negatives). Of the remaining five true positives, correlations were noted between corneal opacity and keratitis (90 day study) as early indicators of squamous cell carcinoma and papilloma of the cornea of the eye (compound 1, a hydroxyphenylpyruvate dioxygenase inhibitor) and inflammation of the stomach and kidney (90 day study) and gastric squamous cell papilloma and squamous cell carcinoma and renal tubular adenoma and carcinoma, respectively (compound 12, a fungicide with multisite activity). Minor decreases in uterine weight and increases in estradiol hydroxylation activity at 28 days were associated with endometrial adenocarcinoma (compound 18, a mitochondrial complex II electron transport inhibitor). Early liver weight increases and hepatocellular centrilobular hypertrophy (28 day study) were associated with thyroid follicular adenomas (compound 11, a succinate dehydrogenase inhibitor) in female animals only. Hepatic centrilobular hypertrophy (28 day studies) correlated with thyroid adenomas in males in carcinogenicity studies (compound 2, a hydroxyphenylpyruvate dioxygenase inhibitor). In contrast, treatment related, nasopharynx tumors (compound 3, an elongase inhibitor) and uterine adenocarcinoma (compound 9, a succinate dehydrogenase inhibitor) could not be correlated with findings from the short term studies examined. Eleven compounds displayed preneoplastic findings with no tumors (false positives) and there were no compounds with no preneoplastic findings and no tumors (true negatives). This work indicates the value of examining historical, short term studies for specific, nonneoplastic findings which correlate with tumors in carcinogenicity studies, which may obviate the need for further animal carcinogenicity studies.

The Antidepressant-Like and Analgesic Effects of Kratom Alkaloids are accompanied by Changes in Low Frequency Oscillations but not ΔFosB Accumulation.

Mitragyna speciosa (“kratom”), employed as a traditional medicine to improve mood and relieve pain, has shown increased use in Europe and North America. Here, the dose-dependent effects of a purified alkaloid kratom extract on neuronal oscillatory systems function, analgesia, and antidepressant-like behaviour were evaluated and kratom-induced changes in ΔFosB expression determined. Male rats were administered a low or high dose of kratom (containing 0.5 or 1 mg/kg of mitragynine, respectively) for seven days. Acute or repeated low dose kratom suppressed ventral tegmental area (VTA) theta oscillatory power whereas acute or repeated high dose kratom increased delta power, and reduced theta power, in the nucleus accumbens (NAc), prefrontal cortex (PFC), cingulate cortex (Cg) and VTA. The repeated administration of low dose kratom additionally elevated delta power in PFC, decreased theta power in NAc and PFC, and suppressed beta and low gamma power in Cg. Suppressed high gamma power in NAc and PFC was seen selectively following repeated high dose kratom. Both doses of kratom elevated NAc-PFC, VTA-NAc, and VTA-Cg coherence. Low dose kratom had antidepressant-like properties whereas both doses produced analgesia. No kratom-induced changes in ΔFosB expression were evident. These results support a role for kratom as having both antidepressant and analgesic properties that are accompanied by specific changes in neuronal circuit function. However, the absence of drug-induced changes in ΔFosB expression suggest that the drug may circumvent this cellular signaling pathway, a pathway known for its significant role in addiction.

Improved Hand Function in Children With Cerebral Palsy With Repeat Doses of Augmented Pediatric Constraint-Induced Movement Therapy

Abstract Date Presented 04/13/21 Children with unilateral cerebral palsy participating in repeat doses of pediatric constraint-induced movement therapy camp augmented with the Hocoma Armeo®Spring demonstrated significant improvements in hand function and bimanual performance for both doses and showed a generalized upward trend in hand function over time. Primary Author and Speaker: Heather Roberts Additional Authors and Speakers: Sara P. Johnston, David Plutschack

Rituximab for the Treatment of Refractory Cardiac Sarcoidosis: A Single-Center Experience

BackgroundWe sought to examine the effect of anti–B-cell therapy (rituximab) on cardiac inflammation and function in corticosteroid-refractory cardiac sarcoidosis. Cardiac sarcoidosis (CS) is a rare cause of cardiomyopathy characterized by granulomatous inflammation involving the myocardium. Although typically responsive to corticosteroid treatment, there is a critical need for identifying effective steroid-sparing agents for disease control. Despite increasing evidence on the role of B cells in the pathogenesis of sarcoidosis, there is limited data on the efficacy of anti–B-cell therapy, specifically rituximab, for controlling CS. Methods and ResultsWe reviewed the clinical experience at a tertiary care referral center of all patients with CS who received rituximab after failing to improve with initial immunosuppression therapy, which included corticosteroids. Fluorodeoxyglucose positron emission tomography (FDG PET/CT) images before and after rituximab treatment were evaluated. All images were interpreted by 2 experienced nuclear medicine trained physicians. We identified 7 patients (5 men, 2 women; mean age at diagnosis, 49.0 ± 7.9 years) with active CS who were treated with rituximab. The median length of follow-up was 5.1 years. All individuals, but 1, had received prior steroid-sparing agents in addition to corticosteroids. Rituximab was administered either as 1000 mg intravenously ×1 or ×2 doses, separated by 2 weeks. Repeat dosing, if appropriate, was considered after 6 months. All tolerated the infusions well. Inflammation as assessed by maximum standardized uptake value on cardiac FDG PET/CT uptake significantly decreased in 6 of 7 patients (median 6.0–4.5, Wilcoxon signed rank z –1.8593, W 3), whereas the left ventricular ejection fraction improved or stabilized in 4 patients but decreased in 3. The mean left ventricular ejection fraction was 40.1% and 43.3% before and after treatment, respectively (P = .28). Three patients reported improved physical capacity, and 5 patients showed improved arrhythmic burden on Holter monitoring or implantable cardioverter-defibrillator interrogation. One patient subsequently developed a fungal catheter-associated infection and sepsis requiring discontinuation. ConclusionsRituximab was well-tolerated and seemed to decrease inflammation, as assessed by cardiac FDG PET/CT in all but 1 patient with active CS. These data suggest that rituximab may be a promising therapeutic option for CS, which deserves merits further study.

ImmTOR nanoparticles enhance AAV transgene expression after initial and repeat dosing in a mouse model of methylmalonic acidemia

A major barrier to adeno-associated virus (AAV) gene therapy is the inability to re-dose patients due to formation of vector-induced neutralizing antibodies (Nabs). Tolerogenic nanoparticles encapsulating rapamycin (ImmTOR) provide long-term and specific suppression of adaptive immune responses, allowing for vector re-dosing. Moreover, co-administration of hepatotropic AAV vectors and ImmTOR leads to an increase of transgene expression even after the first dose. ImmTOR and AAV Anc80 encoding the methylmalonyl-coenzyme A (CoA) mutase (MMUT) combination was tested in a mouse model of methylmalonic acidemia, a disease caused by mutations in the MMUT gene. Repeated co-administration of Anc80 and ImmTOR was well tolerated and led to nearly complete inhibition of immunoglobulin (Ig)G antibodies to the Anc80 capsid. A more profound decrease of plasma levels of the key toxic metabolite, plasma methylmalonic acid (pMMA), and disease biomarker, fibroblast growth factor 21 (FGF21), was observed after treatment with the ImmTOR and Anc80-MMUT combination. In addition, there were higher numbers of viral genomes per cell (vg/cell) and increased transgene expression when ImmTOR was co-administered with Anc80-MMUT. These effects were dose-dependent, with the higher doses of ImmTOR providing higher vg/cell and mRNA levels, and an improved biomarker response. Combining of ImmTOR and AAV can not only block the IgG response against capsid, but it also appears to potentiate transduction and enhance therapeutic transgene expression in the mouse model.

Safety and Efficacy Of Ferumoxytol For The Episodic Treatment Of Iron Deficiency Anemia In Patients With a History Of Unsatisfactory Oral Iron Therapy: Results Of a Phase III Open-Label, Six Month Extension Study

▪ BackgroundThe efficacy of intravenous iron replacement in treating iron deficiency anemia (IDA) has been studied in patients with various underlying conditions. In some conditions like abnormal uterine and gastrointestinal bleeding, there may be ongoing blood loss leading to a continuing need for treatment that may require episodic iron replacement. MethodsA 6 month Phase III, open-label extension study was designed to evaluate the safety and efficacy of ferumoxytol (FER) for the episodic treatment of IDA patients with a history of unsatisfactory oral iron therapy or in whom oral iron could not be used. The study enrolled patients from a double-blind, placebo (P)-controlled 5-week Phase III study, who had been randomized 3:1 to treatment with FER or P and completed that study. Enrolled patients were evaluated for IDA monthly throughout the 6-month Observation Period. Those with persistent or recurrent IDA (defined as Hgb <11.0 g/dL and TSAT <20%) at any evaluation visit, began a 5-week Treatment Period (TP), receiving a course of FER (2 x 510 mg) with response assessed at TP Week 5. ResultsThis extension study enrolled 78.5% of patients from the previous Phase III study (163/200 P, 471/608 FER), of whom 93% of the P- and 39% of the FER-treated patients met treatment criteria during the 6-month extension and received at least 1 course of FER. In the remaining (12 P, 285 FER) patients, mean monthly Hgb remained ≥12.1 g/dL without further treatment. Mean Hgb increased significantly (p<0.0001) from TP Baseline to TP Week 5 following Treatment Course 1 (2.6 g/dL; N=151) and repeat courses (Course 2: 1.5 g/dL, N=244 [38.5%]; Course 3: 1.1 g/dL, N=69 [10.9%]); mean Week 5 Hgb was comparable following Courses 1, 2 and 3. The few patients receiving Treatment Courses 4 (N=18) or 5 (N=4) limited further analyses. Overall, 61% of FER treated patients never required a 2nd course of treatment. Statistically significant (p<0.0001) increases in mean TSAT from TP Baseline to TP Week 5 followed Course 1 and subsequent courses.FER was also well-tolerated and no new safety signals were observed among patients who received repeat dosing. The incidence of treatment emergent adverse events (TEAEs) was 47.7%, 38.1% and 37.7% in patients who received Course 1, 2 and 3 of FER, respectively. The pattern of related TEAEs decreased in subsequent courses, and none were reported in patients who received more than 3 courses. The incidence of serious adverse events (SAEs) was comparable in patients who received Course 1, 2 and 3, and most were attributable to comorbidities and/or procedural complications; no related SAEs or deaths were reported. For all other TEAE categories, no clear pattern of incidence was observed between Treatment Courses. [Display omitted] ConclusionFER (2x 510 mg) delivered over multiple treatment courses was generally well tolerated in IDA patients with a history of unsatisfactory oral iron therapy or in whom oral iron could not be used with persistent or recurrent IDA; Week 5 post-treatment Hgb was comparable following Courses 1, 2 and 3. No trend of increasing incidence of TEAEs was identified with repeat dosing, and a consistent, positive and robust treatment effect was observed. Durability of the treatment effect following FER was demonstrated by the majority of patients not requiring further iron treatment following the initial course during this 6 month extension study. Disclosures:Vadhan-Raj:AMAG Pharmaceuticals, Inc: Research Funding. Off Label Use: Feraheme (ferumoxytol) injection. For treatment of iron deficiency anemia in non-CKD patients. Dahl:AMAG Pharmaceuticals, Inc: Employment. Lau:AMAG Pharmaceuticals, Inc: Employment. Bernard:AMAG Pharmaceuticals, Inc: Employment. Li:AMAG Pharmaceuticals, Inc: Employment. De:AMAG Pharmaceuticals, Inc: Employment. Allen:AMAG Pharmaceuticals, Inc: Consultancy. Strauss:AMAG Pharmaceuticals, Inc: Employment.FER Received in Extension Study (N=337) Treatment Course‡Course 1 (N=151)Course 2 (N=244)Course 3 (N=69)Efficacy Results (ITT Population)Mean Change in Hgbµ2.6 (1.6)*1.5 (1.3)*1.1 (1.3)*% Patients with Increase in Hgb ≥ 2.0 g/dL¥78.843.937.7Mean Change in TSAT (%)µ12.8 (10.2)*11.7 (12.5)*7.5 (9.1)*Safety Results (Safety Population)All AEs47.7%38.1%37.7%Related AEs12.6%5.3%2.9%SAEs4.6%4.9%4.3%Related SAEs000AEs of Special Interest- protocol-defined€00.4%0Cardiovascular AE Composite EndpointΩ1.3%0.8%1.4%AEs Resulting in Study Discontinuation2.6%04.3%Death000* p-value <0.0001 based on paired t-test‡ Course numbering based on treatment received in placebo-controlled studyµ from Baseline to Week 5¥ at any time from Baseline to Week 5€ includes protocol defined signs and symptoms of hypotension and hypersensitivityΩ includes myocardial infarction, heart failure, moderate to severe hypertension and hospitalization due to any cardiovascular cause

Repeat and single dose administration of gadodiamide to rats to investigate concentration and location of gadolinium and the cell ultrastructure

Gadolinium based contrast agents (GBCA) are used to image patients using magnetic resonance (MR) imaging. In recent years, there has been controversy around gadolinium retention after GBCA administration. We sought to evaluate the potential toxicity of gadolinium in the rat brain up to 1-year after repeated gadodiamide dosing and tissue retention kinetics after a single administration. Histopathological and ultrastructural transmission electron microscopy (TEM) analysis revealed no findings in rats administered a cumulative dose of 12 mmol/kg. TEM-energy dispersive X-ray spectroscopy (TEM-EDS) localization of gadolinium in the deep cerebellar nuclei showed ~ 100 nm electron-dense foci in the basal lamina of the vasculature. Laser ablation-ICP-MS (LA-ICP-MS) showed diffuse gadolinium throughout the brain but concentrated in perivascular foci of the DCN and globus pallidus with no observable tissue injury or ultrastructural changes. A single dose of gadodiamide (0.6 mmol/kg) resulted in rapid cerebrospinal fluid (CSF) and blood clearance. Twenty-weeks post administration gadolinium concentrations in brain regions was reduced by 16–72-fold and in the kidney (210-fold), testes (194-fold) skin (44-fold), liver (42-fold), femur (6-fold) and lung (64-fold). Our findings suggest that gadolinium does not lead to histopathological or ultrastructural changes in the brain and demonstrate in detail the kinetics of a human equivalent dose over time in a pre-clinical model.

Abstract 1038: PMD-026, a first in class oral RSK inhibitor, demonstrates synergy when combined with standard of care in breast cancer tumor models

Abstract PMD-026 is a first in class, reversible, oral small molecule inhibitor of p90 ribosomal S6 kinase (RSK), a kinase family activated by the MAPK and PDK-1 pathways, which regulate substrates involved in cancer cell proliferation and drug resistance. Specifically, RSK2 has been identified as a major driver in breast cancer (BC). In preclinical studies and a Phase I clinical trial in metastatic BC, PMD-026 demonstrated a good safety profile, making it an attractive candidate for combinations with standard of care therapies. Here in we show that RSK2 is activated in 87% of triple negative breast cancer (TNBC), as well as in other subtypes (80% of ER+/PR+ and 81% of HER2+). In a screen of 28 BC cell lines across a broad mutational spectrum, PMD-026 induced apoptosis in most of the models. Next, to determine the role of PMD-026 in the context of conventional BC treatment, we combined it with standard of care chemotherapies fulvestrant, paclitaxel, or doxorubicin, in vitro and in vivo. PMD-026 synergized with fulvestrant, with combination drug index (CDI) values ranging from 0.73 - 0.92 in the HR+ cell line MCF-7. Likewise, PMD-026 synergized with paclitaxel and doxorubicin in TNBC cell lines with CDI values ranging from 0.40 - 0.84 and 0.58 - 0.92 for paclitaxel and doxorubicin, respectively. Consistent with in vitro screening data, PMD-026 synergized with paclitaxel in vivo in the SUM149PT TNBC xenograft model after 38 days of treatment. The combination inhibited tumor growth by 66% (P &amp;lt; 0.0001), whereas paclitaxel and PMD-026 as single agents inhibited tumor growth by 22% (P = 0.3051) and 41% (P = 0.0041), respectively. The synergy of this combination was further supported by a CDI value of 0.75. Treatment among the groups was well tolerated with no changes in body weight observed. Similarly, the combination of PMD-026 and paclitaxel was synergistic in SUM149PT(48 days, CDI: 0.78) and MDA-MB-231 (28 days, CDI: 0.66) xenografts and additive in a PDx model of metastatic TNBC (18 days, CDI: 0.96), resulting in improved survival. To address the safety of combining PMD-026 and paclitaxel, drug-drug interactions (DDI) were assessed. In cytochrome P450-mediated in vitro metabolism assays, PMD-026 showed weak inhibition of Cyp2C8 and Cyp3A4, the main enzymes responsible for paclitaxel metabolism. To understand whether PMD-026 might alter the metabolism of paclitaxel, this potential DDI was assessed in vivo. Pharmacokinetic analysis of PMD-026 (7 days repeat dosing) combined with paclitaxel (8 mg/kg IV dose Day 1 and Day 7) was evaluated in CD-1 mice, however, PMD-026 did not change the absorption, distribution, or blood levels of paclitaxel. Together, these data support adding PMD-026 to standard of care therapies in breast cancer, as they have demonstrated synergy. In particular, PMD-026 is synergistic with paclitaxel in multiple TNBC models, in which it improves efficacy without added toxicity. Citation Format: Aarthi Jayanthan, My-my Huynh, Jangsoon Lee, Gerrit Los, Lambert Yue, Mary Rose Pambid, Naoto T. Ueno, Sandra E. Dunn. PMD-026, a first in class oral RSK inhibitor, demonstrates synergy when combined with standard of care in breast cancer tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1038.