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.

Neural stem cell delivery of an oncolytic adenovirus in newly diagnosed malignant glioma: a first-in-human, phase 1, dose-escalation trial.

BACKGROUND: Malignant glioma is the most common and lethal primary brain tumour, with dismal survival rates and no effective treatment. We examined the safety and activity of NSC-CRAd-S-pk7, an engineered oncolytic adenovirus delivered by neural stem cells (NSCs), in patients with newly diagnosed high-grade glioma. METHODS: This was a first-in-human, open-label, phase 1, dose-escalation trial done to determine the maximal tolerated dose of NSC-CRAd-S-pk7, following a 3 + 3 design. Patients with newly diagnosed, histologically confirmed, high-grade gliomas (WHO grade III or IV) were recruited. After neurosurgical resection, NSC-CRAd-S-pk7 was injected into the walls of the resection cavity. The first patient cohort received a dose starting at 6·25 × 1010 viral particles administered by 5·00 × 107 NSCs, the second cohort a dose of 1·25 × 1011 viral particles administered by 1·00 × 108 NSCs, and the third cohort a dose of 1·875 × 1011 viral particles administered by 1·50 × 108 NSCs. No further dose escalation was planned. Within 10-14 days, treatment with temozolomide and radiotherapy was initiated. Primary endpoints were safety and toxicity profile and the maximum tolerated dose for a future phase 2 trial. All analyses were done in all patients who were included in the trial and received the study treatment and were not excluded from the study. Recruitment is complete and the trial is finished. The trial is registered with ClinicalTrials.gov, NCT03072134. FINDINGS: Between April 24, 2017, and Nov 13, 2019, 12 patients with newly diagnosed, malignant gliomas were recruited and included in the safety analysis. Histopathological evaluation identified 11 (92%) of 12 patients with glioblastoma and one (8%) of 12 patients with anaplastic astrocytoma. The median follow-up was 18 months (IQR 14-22). One patient receiving 1·50 × 108 NSCs loading 1·875 × 1011 viral particles developed viral meningitis (grade 3) due to the inadvertent injection of NSC-CRAd-S-pk7 into the lateral ventricle. Otherwise, treatment was safe as no formal dose-limiting toxicity was reached, so 1·50 × 108 NSCs loading 1·875 × 1011 viral particles was recommended as a phase 2 trial dose. There were no treatment-related deaths. The median progression-free survival was 9·1 months (95% CI 8·5-not reached) and median overall survival was 18·4 months (15·7-not reached). INTERPRETATION: NSC-CRAd-S-pk7 treatment was feasible and safe. Our immunological and histopathological findings support continued investigation of NSC-CRAd-S-pk7 in a phase 2/3 clinical trial. FUNDING: US National Institutes of Health.

Single dose GLP toxicity and biodistribution study of a conditionally replicative adenovirus vector, CRAd-S-pk7, administered by intracerebral injection to Syrian hamsters.

BACKGROUND: CRAd-S-pk7 is a conditionally replicative oncolytic adenoviral vector that contains a survivin promoter and a pk7 fiber modification that confer tumor-specific transcriptional targeting and preferential replication in glioma while sparing the surrounding normal brain parenchyma. METHODS: This IND-enabling study performed under GLP conditions evaluated the toxicity and biodistribution of CRAd-S-pk7 administered as a single intracerebral dose to Syrian hamsters, a permissive model of adenoviral replication. Two hundred and forty animals were stereotactically administered either vehicle (n = 60) or CRAd-S-pk7 at 2.5 × 10(7), 2.5 × 10(8), or 2.5 × 10(9) viral particles (vp)/animal (each n = 60) on day 1. The animals were closely monitored for toxicology evaluation, assessment of viral distribution, and immunogenicity of CRAd-S-pk7. RESULTS: Changes in hematology, clinical chemistry, and coagulation parameters were minor and transient, and consistent with the inflammatory changes observed microscopically. These changes were considered to be of little toxicological significance. The vector remained localized primarily in the brain and to some degree in the tissues at the incision site. Low levels of vector DNA were detected in other tissues in a few animals suggesting systemic circulation of the virus. Viral DNA was detected in brains of hamsters for up to 62 days. However, microscopic changes and virus-related toxicity to the central nervous system were considered minor and decreased in incidence and severity over time. Such changes are not uncommon in studies using adenoviral vectors. CONCLUSION: This study provides safety and toxicology data justifying a clinical trial of CRAd-S-pk7 loaded in FDA-approved HB1.F3.CD neural stem cell carriers administered at the tumor resection bed in humans with recurrent malignant glioma.

Species and gender differences in the carcinogenic activity of trimethylolpropane triacrylate in rats and mice.

Trimethylolpropane triacrylate (TMPTA) is a multifunctional monomer with industrial applications. To determine the carcinogenic potential, male and female F344/N rats and B6C3F1/N mice were administered TMPTA (0, 0.3, 1.0, or 3.0mg/kg) in acetone dermally for 2 years. There were no differences in the body weights and survival in the treated animals compared to controls. Nonneoplastic skin lesions at the site of application included epidermal hyperplasia and hyperkeratosis in both rats and mice. There were no incidences of tumors at the site of application in rats and mice. Rare malignant liver neoplasms were observed in female mice that included hepatoblastoma in the 0.3 and 3.0mg/kg groups, and hepatocholangiocarcinoma in the 1.0 and 3.0mg/kg groups. The incidences of uterine stromal polyp and stromal polyp or stromal sarcoma (combined) in female mice occurred with positive trends and the incidences were significantly increased in the 3.0mg/kg group. A marginal increase in the incidences of malignant mesothelioma in male rats may have been related to TMPTA treatment. In conclusion, our studies show that TMPTA is a dermal irritant in both rats and mice of either sex. Increased incidences of tumor formation were observed in female mice and male rats.

Comparison of the effects of hexavalent chromium in the alimentary canal of F344 rats and B6C3F1 mice following exposure in drinking water: implications for carcinogenic modes of action.

Exposure to high concentrations of hexavalent chromium (Cr[VI]) in drinking water is reported to induce oral mucosa tumors in F344 rats and intestinal tumors in B6C3F1 mice. To investigate the modes of action underlying these tumors, 90-day drinking water studies (with interim necropsy at day 8) were conducted with concentrations of 0.1-182 mg/l Cr(VI), administered as 0.3-520 mg/l sodium dichromate dihydrate. Blood and tissue samples were analyzed for chromium content, oxidative stress, iron levels, and gross and microscopic lesions. Results for the F344 rats are described herein and compared with results from B6C3F1 mice published previously. After 90 days of exposure, total chromium concentrations in the rat and mouse oral mucosae were comparable, yet significant dose-dependent decreases in the reduced-to-oxidized glutathione ratio (GSH/GSSG) were observed only in rats. In the duodenum, changes in GSH/GSSG were only observed in mice. Levels of 8-hydroxydeoxyguanosine were not increased in the oral or duodenal mucosae of either species. Glutathione levels were increased in the duodenum but decreased in the jejunum of both species, indicating potential differential responses in the intestinal segments. Histiocytic infiltration was observed in the duodenum of both species, yet duodenal cytokines were repressed in mice but increased in rats. Serum and bone marrow iron levels were more decreased in rats than mice. Collectively, these data suggest that Cr(VI)-induced carcinogenesis in the rodent alimentary canal involves oxidative stress; however, differences in histopathology, cytokines, and iron status suggest potential contributions from other factors as well.

Investigation of the mode of action underlying the tumorigenic response induced in B6C3F1 mice exposed orally to hexavalent chromium.

Chronic ingestion of high concentrations of hexavalent chromium [Cr(VI)] in drinking water induces intestinal tumors in mice. To investigate the mode of action (MOA) underlying these tumors, a 90-day drinking water study was conducted using similar exposure conditions as in a previous cancer bioassay, as well as lower (heretofore unexamined) drinking water concentrations. Tissue samples were collected in mice exposed for 7 or 90 days and subjected to histopathological, biochemical, toxicogenomic, and toxicokinetic analyses. Described herein are the results of toxicokinetic, biochemical, and pathological findings. Following 90 days of exposure to 0.3-520 mg/l of sodium dichromate dihydrate (SDD), total chromium concentrations in the duodenum were significantly elevated at ≥ 14 mg/l. At these concentrations, significant decreases in the reduced-to-oxidized glutathione ratio (GSH/GSSG) were observed. Beginning at 60 mg/l, intestinal lesions were observed including villous cytoplasmic vacuolization. Atrophy, apoptosis, and crypt hyperplasia were evident at ≥ 170 mg/l. Protein carbonyls were elevated at concentrations ≥ 4 mg/l SDD, whereas oxidative DNA damage, as assessed by 8-hydroxydeoxyguanosine, was not increased in any treatment group. Significant decreases in the GSH/GSSG ratio and similar histopathological lesions as observed in the duodenum were also observed in the jejunum following 90 days of exposure. Cytokine levels (e.g., interleukin-1ß) were generally depressed or unaltered at the termination of the study. Overall, the data suggest that Cr(VI) in drinking water can induce oxidative stress, villous cytotoxicity, and crypt hyperplasia in the mouse intestine and may underlie the MOA of intestinal carcinogenesis in mice.

Toxicity and carcinogenicity studies of methylene blue trihydrate in F344N rats and B6C3F1 mice.

Methylene blue trihydrate has a variety of biomedical and biologically therapeutic applications. Groups of 50 male and 50 female rats and mice were administered methylene blue trihydrate in 0.5% aqueous methylcellulose solution by gavage at doses of 0, 5, 25, or 50mg/kg bw/day (rats) or 0, 2.5, 12.5, and 25mg/kg bw/day (mice), 5 days per week for 2 years. In rats survival of all dosed groups was similar to that of the vehicle controls, whereas mice exhibited a dose-dependent increase in survival. Rats receiving 25 and 50mg/kg bw/day and mice receiving 25mg/kg bw/day developed mild anemia. The incidences of pancreatic islet cell adenoma and adenoma or carcinoma (combined) were increased in all dosed groups of male rats, but increases were statistically significant in 25mg/kg bw/day males only and the dose-response was non-linear. There was a corresponding increase in the incidence of pancreatic islet cell hyperplasia but statistically significant only in the 50mg/kg bw/day male rats. There were no significant increases in neoplastic transformation observed in the mice; however, positive trends were noted for adenoma or carcinoma (combined) of the small intestine and malignant lymphoma.

In-vitro and in-vivo metabolic studies of the candidate chemopreventative pentamethylchromanol using liquid chromatography/tandem mass spectrometry.

OBJECTIVES: This study focuses on the in-vitro metabolic profiles of pentamethylchromanol in human, rat, dog and non-human primates, and characterizes the associated metabolic kinetics and specific human isozymes responsible for metabolism. Additional investigations compare in-vitro data with in-vivo metabolic data from rats and dogs. METHODS: In-vitro metabolites were generated from commercially available microsomes, S9 fractions and cytochrome P450 isozymes. Reaction mixtures were analysed using liquid chromatography/tandem mass spectrometry for metabolite identification, stability, phenotyping and kinetic profiles. Plasma samples were collected from 28-day toxicology studies in rats and dogs, and analysed using the same methodology as for the identification of in-vitro metabolites. KEY FINDINGS: Samples from in-vitro experiments produced a total of eight identified metabolites while five were observed in the in-vivo samples. Kinetic analysis of metabolites in human microsomes generated Michaelis constants (K(M)) ranging from 10.9 to 104.9 mum. Pentamethylchromanol metabolic stability varied by species and multiple isozymes were identified for the observed biotransformation pathways. Pentamethylchromanol is susceptible to multiple metabolic pathways and differential metabolic stability, which is species dependent. CONCLUSIONS: In-vitro metabolism was not a strong predictor of in-vivo metabolism for the samples assays but showed glucuronidation and sulfation as common biotransformation pathways.

Indole-3-carbinol, but not its major digestive product 3,3'-diindolylmethane, induces reversible hepatocyte hypertrophy and cytochromes P450.

Indole-3-carbinol (I-3-C) and 3,3'-diindolylmethane (DIM) have been shown to reduce the incidence and multiplicity of cancers in laboratory animal models. Based on the observation that I-3-C induced hepatocyte hypertrophy when administered orally for 13 weeks to rats, a treatment and recovery study was undertaken to test the hypothesis that the induction of hepatocyte hypertrophy and cytochrome P450 (CYP) activity by I-3-C are adaptive, reversible responses. Additionally, we directly compared the effects of I-3-C to those of its principle metabolite DIM. Rats were treated orally for 28 days with 2 doses of I-3-C (5 and 50 mg I-3-C/kg body weight/day) and DIM (7.5 and 75 mg DIM/kg body weight/day) and then one-half of the animals were not treated for an additional 28 days. Organ weights, histopathology, and the CYP enzyme activities of 1A1/2, 2B1/2, 2C9, 2D6, 2E1, 3A4, and 19 A were measured both after treatment and after recovery. Oral administration of 50 mg I-3-C/kg body weight/day to rats for 28 days significantly increased liver weights and CYP enzyme activities. The effects in males were more pronounced and persistent after recovery than the effects in females. The increased organ weights returned to control values after treatment. Conversely, DIM did not alter liver weights and had no effect on CYP activities after the 28-day treatment. Some changes in CYP activities were measured after the DIM recovery period but the magnitudes of the changes were considered biologically insignificant. The results show that I-3-C, but not DIM, induces reversible adaptive responses in the liver.

Sensory nerve conduction of the plantar nerve compared with other nerve conduction tests in rats.

OBJECTIVE: In rats the available techniques for evaluation of sensory nerve conduction are limited. We report a new method of sensory nerve conduction of the plantar nerve using needle electrodes as the recording electrodes behind the medial malleolus and ring electrodes as the stimulating electrodes around the three middle toes. METHODS: We performed this sensory nerve conduction test in 25 rats during their growth over a 6 weeks' period and compared this method with the motor nerve conduction and H-reflex sensory nerve conduction of the tibial nerve in 10 rats, and with the motor and mixed nerve conductions of the tail nerve in 15 rats. RESULTS: There was a highly or moderately significant correlation between the body weight and sensory nerve conduction velocity (NCV) of the plantar nerve, mixed NCV and motor NCV of the tail nerve, indicating a growth-related increase in the NCV. The growth-related increase in the NCV was not observed in the motor and H-reflex sensory nerve conductions of the tibial nerves. CONCLUSIONS: This test is simple and reliable and can be used for the sensory nerve conduction test in rats.