REL-1017 (Esmethadone), A Novel NMDAR Blocker for the Treatment of MDD is Not Neurotoxic in Sprague-Dawley Rats.

REL-1017 (esmethadone; dextromethadone; (S)-methadone) is the opioid-inactive dextro-isomer of the racemic mixture, (R, S)-methadone. REL-1017 acts as a low affinity, low potency N-methyl-D-aspartate receptor (NMDAR) channel blocker with rapid, robust, and sustained therapeutic effects in patients with major depressive disorder (MDD). Systemic administration of NMDAR blockers may cause transient and reversible pathomorphological alterations in brain cortical neurons characterized by cytoplasmic vacuolization, which are called Olney's lesions, and may also lead to irreversible neuronal necrosis. We determined whether REL-1017 administration via oral gavage for 1-4 days to Sprague-Dawley rats could produce Olney's lesions and cortical neuronal death and microgliosis as compared with MK-801, a known neurotoxic potent NMDAR blocker. As previously reported, MK-801 produced Olney's lesions, neuronal necrosis and cortical microgliosis, and impaired behavior and activity. In contrast, administration of REL-1017 at low (20-31.25 mg/kg in females and males), medium (40-62.5 mg/kg) or high (80-110 mg/kg) doses did not cause pathomorphological changes in brain neurons and did not cause impaired behavior and activity. In conclusion, REL-1017 did not produce initial or cumulative neurotoxic effects or other evidence of damage to cortical neurons, further encouraging the development of REL-1017 as a potentially safe novel candidate for rapid treatment of MDD.

Hemangiosarcoma in mice administered pregabalin: analysis of genotoxicity, tumor incidence, and tumor genetics.

Pregabalin, (S)-3-(aminomethyl)-5-methylhexanoic acid, binds with high affinity to the α(2)δ subunit of voltage-gated calcium channels and exerts analgesic, anxiolytic, and antiseizure activities. Two-year carcinogenicity studies were completed in B6C3F1 and CD-1 mice and two separate studies in Wistar rats. Doses in mice were 200, 1000, and 5000 mg/kg/day, with systemic exposures (AUC(0-24 h)) up to 31 times the mean exposure in humans, given the maximum recommended clinical dose. In rats, doses were 50, 150, and 450 mg/kg/day in males and 100, 300, and 900 mg/kg/day in females; systemic exposures up to 24 times were achieved in clinical trials. In both strains of mice, pregabalin treatment was associated with an increased incidence of hemangiosarcoma primarily in liver, spleen, and bone marrow. The incidence of hemangiosarcoma was higher in B6C3F1 mice than in CD-1 mice, consistent with its spontaneous incidence. Pregabalin did not increase the incidence of any other tumor type in rats and was not genotoxic, based on an extensive battery of in vivo and in vitro tests in bacterial and mammalian systems. Thus, pregabalin is a single-species, single tumor-type, nongenotoxic mouse carcinogen. Hemangiosarcomas occurring in mice treated with pregabalin were genotypically distinct from hemangiosarcomas induced by genotoxic carcinogens in humans with respect to ras and p53 mutation patterns and were similar to spontaneous tumors. Furthermore, there was a strong association between pregabalin treatment and bone marrow changes in these studies in mice, suggesting a possible link between the effects observed in bone marrow and the increase in tumor incidence in pregabalin-treated mice.

Evaluation of an ex vivo murine local lymph node assay: multiple endpoint comparison.

The local lymph node assay (LLNA) is used to assess the skin sensitization potential of chemicals. In the standard assay, mice are treated topically on the dorsum of both ears with test substance for 3 days. Following 2 days of rest, the initiation of the hypersensitivity response is evaluated by injecting (3)H-thymidine into a tail vein, and then measuring the levels of radioisotope incorporated into the DNA of lymph node cells draining the ears. In the current study, BALB/c mice were treated with the contact sensitizers hexylcinnamic aldehyde (HCA) and oxazolone, and the nonsensitizer methyl salicylate. The proliferative response of lymph node cells was evaluated in an ex vivo assay, in which isolated cells were cultured in vitro with (3)H-thymidine. Treatment of mice with HCA at 5-50% resulted in concentration-related increases in (3)H-thymidine incorporation, with stimulation indices ranging from 3 to 14. Low animal-to-animal variability was seen in three replicate assays testing HCA at 25%. As anticipated, the proliferative response induced by the potent sensitizer oxazolone at 0.25% was greater than HCA at all concentrations tested. Stimulation indices of 1.5 and 3 were seen in two independent experiments with methyl salicylate. These equivocal findings were likely due to the irritancy properties of the compound. Importantly, measuring ex vivo (3)H-thymidine incorporation was more sensitive than evaluating lymph node weight and cellularity, and in vitro bromodeoxyuridine incorporation. Furthermore, the results of the ex vivo LLNA were comparable to the standard assay. This study provided evidence that supports the use of an ex vivo LLNA for hazard assessment of contact hypersensitivity.

Development of an internally controlled antibody microarray.

Antibody microarrays are a high throughput technology used to concurrently screen for protein expression. Most antibody arrays currently used are based on the ELISA sandwich approach that uses two antibodies to screen for the expression of a limited number of proteins. Also because antigen-antibody interactions are concentration-dependent, antibody microarrays need to normalize the amount of antibody that is used. In response to the limitations with the currently existing technology we have developed a single antibody-based microarray where the quantity of antibody spotted is used to standardize the antigen concentration. In addition, this new array utilizes an internally controlled system where one color represents the amount of antibody spotted, and the other color represents the amount of the antigen that is used to quantify the level of protein expression. When compared with median fluorescence intensity alone, normalization for antibody spot intensity decreased variability and lowered the limits of detection. This new antibody array was tested using standard cytokine proteins and also cell lysates obtained from mouse macrophages stimulated in vitro and evaluated for the expression of the cytokine proteins interleukin (IL)-1beta, IL-5, IL-6, and macrophage inflammatory proteins 1alpha and 1beta. The levels of protein expression seen with the antibody microarray was compared with that obtained with Western blot analysis, and the magnitude of protein expression observed was similar with both technologies with the antibody array actually showing a greater degree of sensitivity. In summary, we have developed a new type of antibody microarray to screen for protein expression that utilizes a single antibody and controls for the amount of antibody spotted. This type of array appears at least as sensitive as Western blot analysis, and the technology can be scaled up for high throughput screening for hundreds of proteins in complex biofluids such as blood.

Induction of apoptosis by cationic amphiphilic drugs amiodarone and imipramine.

Phospholipidosis is the excessive accumulation of intracellular phospholipids in cell lysosomes. Drugs that induce this disease often share common physiochemical properties and are collectively classified as cationic amphiphilic drugs (CADs). Although the cause of phospholipidosis and morphologic appearance of affected lysosomes have been studied extensively, less is known about the physiologic effects of the condition. In the current study, U-937 cells were incubated with the CADs amiodarone (2.5-10 microg/mL) and imipramine (2.5-20 microg/mL). Treatment of U-937 cells with these compounds for 96 h resulted in concentration-related increases in phospholipids, as assessed by flow cytometry using the fluorophore nile red. These results were verified by measuring the concentrations of choline-derived phospholipids, which were significantly increased in drug-treated cells. Cell number in amiodarone (10 microg/mL) and imipramine (20 microg/mL) cultures following the 96-h incubation period were markedly reduced compared to control cultures. These observations suggested that accumulation of cellular phospholipids could inhibit cell proliferation. Flow cytometric analysis revealed a decrease in the percentage of cells in the S-phase of the cell cycle following drug treatment, yet DNA replication still occurred in a significant portion of cells. Interestingly, amiodarone and imipramine induced apoptosis in U-937 cells as shown by annexin V-FITC staining and DNA fragmentation. Enzymatic assays demonstrated that amiodarone and imipramine induced the activity of caspases 2 and 3. These results suggest that disruption of cell lysosomes in U-937 cells following accumulation of phospholipids does not cause a cell cycle arrest but instead induces apoptosis by activation of caspase pathways.