In vitro pharmacological characterization of SPN-810M (molindone).

BACKGROUND: Impulsive aggression (IA) is considered a maladaptive form of aggression that is reactive and overt and occurs outside of the acceptable social context. Many children and adolescents with attention-deficit/hyperactivity disorder (ADHD) display clinically significant aggression, with the predominant subtype being IA. However, there is currently no Food and Drug Administration-approved medication specifically to treat IA. The pathophysiology of IA is not fully understood, although it has been suggested to include the dopamine, norepinephrine, and serotonin systems. METHODS: SPN-810 (extended-release molindone) is being developed for the novel indication of IA and is currently being studied in patients treated for ADHD. Molindone is an indole derivative and a dopamine D2 receptor antagonist. RESULTS: The in vitro pharmacological studies described in the current manuscript demonstrate that the active substance molindone (SPN-810M) is a potent antagonist for the dopamine receptors, D2S and D2L, and the serotonin receptor, 5-HT2B, at therapeutic concentrations. The in vitro studies further demonstrate that the antagonist effect of SPN-810M is due to the parent drug and not the metabolites, and that the antagonism is not affected by the presence of norepinephrine or dopamine neurotransmitters. In addition, studies investigating the potential differential effects of the enantiomers of SPN-810M have demonstrated that the R(-) enantiomer is more potent than S(+), showing greater regulatory effect on D2S and D2L receptors. CONCLUSION: Overall, the results of the in vitro SPN-810M pharmacological studies provide some insight into how SPN-810M modulates the serotonin and dopamine pathways that play a role in IA.

Reproductive toxicology studies supporting the safety of molindone, a dopamine receptor antagonist.

BACKGROUND: An extended-release molindone (a dopamine D2 and serotonin antagonist) is currently being developed as a novel treatment for impulsive aggression (IA) in patients optimally treated for ADHD. Oral Good Laboratory Practice reproductive toxicology studies (fertility and early embryonic [FEE], prenatal/postnatal [PPN], embryo-fetal development [EFD]) were conducted with molindone HCl using International Conference on Harmonisation (ICH) S5(R2)-compliant protocols. METHODS: In the FEE study, 0, 5, 15, or 30 mg kg-1 day-1 was administered to female (2 weeks premating through implantation) and male (4 weeks premating for 57 days) rats, and fertility parameters were evaluated. In the EFD studies, rats received 0, 5, 20, or 40 mg kg-1 day-1 on gestational days (GDs) 6-17; rabbits received 0, 5, 10, or 15 mg kg-1 day-1 on GDs 6-18. Ovarian/uterine and fetal parameters were evaluated at term. In the PPN study, F0 rats received 0, 5, 20, or 40 mg kg-1 day-1 (GD6-LD21); behavior and reproduction were evaluated in F1 offspring. RESULTS: Parental hypoactivity and reduced body weight gain occurred in all studies. In the FEE, prolonged estrous cycles and delayed mating occurred at ≥15 mg kg-1 day-1 , without effects on fertility or embryonic development. No developmental toxicity occurred in F1 fetuses. In F1 pups, reduced preweaning growth was observed at 40 mg kg-1 day-1 , but there were no effects on postweaning growth, behavior, or reproduction. CONCLUSIONS: Molindone was not developmentally toxic in rats or rabbits at 69X and 6X clinical exposures, confirming the reproductive safety of molindone. Changes in estrous cyclicity were related to species-specific pharmacological effects of molindone in rodents and are not considered relevant to human risk.

An Optimal Seed Based Compression Algorithm for DNA Sequences.

This paper proposes a seed based lossless compression algorithm to compress a DNA sequence which uses a substitution method that is similar to the LempelZiv compression scheme. The proposed method exploits the repetition structures that are inherent in DNA sequences by creating an offline dictionary which contains all such repeats along with the details of mismatches. By ensuring that only promising mismatches are allowed, the method achieves a compression ratio that is at par or better than the existing lossless DNA sequence compression algorithms.

Interfacing living cells and spherically supported bilayer lipid membranes.

Spherically supported bilayer lipid membranes (SS-BLMs) exhibiting co-existing membrane microdomains were created on spherical silica substrates. These 5 µm SiO2-core SS-BLMs are shown to interact dynamically when interfaced with living cells in culture, while keeping the membrane structure and lipid domains on the SS-BLM surface intact. Interactions between the SS-BLMs and cellular components are examined via correlating fluorescently labeled co-existing microdomains on the SS-BLMs, their chemical composition and biophysical properties with the consequent organization of cell membrane lipids, proteins, and other cellular components. This approach is demonstrated in a proof-of-concept experiment involving the dynamic organization of cellular cytoskeleton, monitored as a function of the lipid domains of the SS-BLMs. The compositional versatility of SS-BLMs provides a means to address the relationship between the phenomenon of lipid phase separation and the other contributors to cell membrane lateral heterogeneity.

Lipid-Conjugation of Endogenous Neuropeptides: Improved Biotherapy against Human Pancreatic Cancer.

Neuropeptides are small neuronal signaling molecules that act as neuromodulators for a variety of neural functions including analgesia, reproduction, social behavior, learning, and memory. One of the endogenous neuropeptides-Met-Enkephalin (Met-Enk), has been shown to display an inhibitory effect on cell proliferation and differentiation. Here, a novel lipid-modification approach is shown to create a small library of neuropeptides that will allow increased bioavailability and plasma stability after systemic administration. It is demonstrated, on an experimental model of human pancreatic adenocarcinoma, that lipid conjugation of Met-Enk enhances its tumor suppression efficacy compared to its nonlipidated counterparts, both in vitro and in vivo. More strikingly, the in vivo studies show that a combination therapy with a reduced concentration of Gemcitabine has suppressed the tumor growth considerably even three weeks after the last treatment.

A novel sequence and context based method for promoter recognition.

UNLABELLED: Identification of promoters in DNA sequence using computational techniques is a significant research area because of its direct association in transcription regulation. A wide range of algorithms are available for promoter prediction. Most of them are polymerase dependent and cannot handle eukaryotes and prokaryotes alike. This study proposes a polymerase independent algorithm, which can predict whether a given DNA fragment is a promoter or not, based on the sequence features and statistical elements. This algorithm considers all possible pentamers formed from the nucleotides A, C, G, and T along with CpG islands, TATA box, initiator elements, and downstream promoter elements. The highlight of the algorithm is that it is not polymerase specific and can predict for both eukaryotes and prokaryotes in the same computational manner even though the underlying biological mechanisms of promoter recognition differ greatly. The proposed Method, Promoter Prediction System - PPS-CBM achieved a sensitivity, specificity, and accuracy percentages of 75.08, 83.58 and 79.33 on E. coli data set and 86.67, 88.41 and 87.58 on human data set. We have developed a tool based on PPS-CBM, the proposed algorithm, with which multiple sequences of varying lengths can be tested simultaneously and the result is reported in a comprehensive tabular format. The tool also reports the strength of the prediction. AVAILABILITY: The tool and source code of PPS-CBM is available at http://keralabs.org.

Lipidome and proteome map of myelin membranes.

To understand the molecular anatomy of myelin membranes, we performed a large-scale, liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS)-based lipidome and proteome screen on freshly purified human and murine myelin fractions. We identified more than 700 lipid moieties and above 1,000 proteins in the two species, including 284 common lipids and 257 common proteins. This study establishes the first comprehensive map of myelin membrane components in human and mice. Although this study demonstrates many similarities between human and murine myelin, several components have been identified exclusively in each species. Future quantitative validation studies focused on interspecies differences will authenticate the myelin membrane anatomy. The combined lipidome and proteome map presented here can nevertheless be used as a reference library for myelin health and disease.

Myocardial mononuclear cell infiltrates are not associated with increased serum cardiac troponin I in cynomolgus monkeys.

Myocardial mononuclear cell infiltrate is a spontaneous cardiac finding commonly identified in laboratory cynomolgus monkeys. The infiltrates are predominantly composed of macrophages with lesser lymphocytes and are not typically associated with histologically detectable cardiomyocyte degeneration. These infiltrates are of concern because they confound interpretation of test article-related histopathology findings in nonclinical safety toxicology studies. The interpretation of safety studies would be simplified by a biomarker that could identify myocardial infiltrates prior to animal placement on study. We hypothesized that monkeys with myocardial mononuclear cell infiltrates could be identified before necropsy using an ultrasensitive immunoassay for cardiac troponin I (cTnI). Serum cTnI concentrations in monkeys with myocardial infiltrates were not higher than those in monkeys without infiltrates at any of the sampling times before and on the day of necropsy. Increased serum cTnI levels are not suitable for screening monkeys with myocardial mononuclear cell infiltrates before placement in the study.

Label-free visualization of ultrastructural features of artificial synapses via cryo-EM.

The ultrastructural details of presynapses formed between artificial substrates of submicrometer silica beads and hippocampal neurons are visualized via cryo-electron microscopy (cryo-EM). The silica beads are derivatized by poly-d-lysine or lipid bilayers. Molecular features known to exist at presynapses are clearly present at these artificial synapses, as visualized by cryo-EM. Key synaptic features such as the membrane contact area at synaptic junctions, the presynaptic bouton containing presynaptic vesicles, as well as microtubular structures can be identified. This is the first report of the direct, label-free observation of ultrastructural details of artificial synapses.

Isolation of functional presynaptic complexes from CNS neurons: a cell-free preparation for the study of presynaptic compartments In vitro.

The difficulty in developing successful treatments to facilitate nerve regeneration has prompted a number of new in vitro experimental methods. We have recently shown that functional presynaptic boutons can be formed when neuronal cells are cocultured with surface-modified artificial substrates including poly(d-lysine)-coated beads and supported lipid bilayer-coated beads (Lucido(2009) J. Neurosci.29, 12449-12466; Gopalakrishnan(2010) ACS Chem. Neurosci.1, 86-94). We demonstrate here, using confocal microscopy combined with immunocytochemistry, that it is possible to isolate such in vitro presynaptic endings in an exclusive fashion onto glass substrates through a simple "sandwich/lift-off" technique (Perez(2006) Adv. Funct. Mater.16, 306-312). Isolated presynaptic complexes are capable of releasing and recycling neurotransmitter in response to an external chemical trigger. These bead-presynaptic complexes are facile to prepare and are readily dispersible in solution. They are thus compatible with many experimental methods whose focus is the study of the neuronal presynaptic compartment.

Lipid bilayer membrane-triggered presynaptic vesicle assembly.

The formation of functional synapses on artificial substrates is a very important step in the development of engineered in vitro neural networks. Spherical supported bilayer lipid membranes (SS-BLMs) are used here as a novel substrate to demonstrate presynaptic vesicle accumulation at an in vitro synaptic junction. Confocal fluorescence microscopy, cryo-transmission electron microscopy (cryo-TEM), and fluorescence recovery after photobleaching (FRAP) experiments have been used to characterize the SS-BLMs. Conventional immunocytochemistry combined with confocal fluorescence microscopy was used to observe the formation of presynaptic vesicles at the neuron-SS-BLM contacts. These results indicate that lipid phases may play a role in the observed phenomenon, in addition to the chemical and electrostatic interactions between the neurons and SS-BLMs. The biocompatibility of lipid bilayers along with their membrane tunability makes the suggested approach a useful "toolkit" for many neuroengineering applications including artificial synapse formation and synaptogenesis in vivo.

Rapid assembly of functional presynaptic boutons triggered by adhesive contacts.

CNS synapse assembly typically follows after stable contacts between "appropriate" axonal and dendritic membranes are made. We show that presynaptic boutons selectively form de novo following neuronal fiber adhesion to beads coated with poly-d-lysine (PDL), an artificial cationic polypeptide. As demonstrated by atomic force and live confocal microscopy, functional presynaptic boutons self-assemble as rapidly as 1 h after bead contact, and are found to contain a variety of proteins characteristic of presynaptic endings. Interestingly, presynaptic compartment assembly does not depend on the presence of a biological postsynaptic membrane surface. Rather, heparan sulfate proteoglycans, including syndecan-2, as well as others possibly adsorbed onto the bead matrix or expressed on the axon surface, are required for assembly to proceed by a mechanism dependent on the dynamic reorganization of F-actin. Our results indicate that certain (but not all) nonspecific cationic molecules like PDL, with presumably electrostatically mediated adhesive properties, can effectively bypass cognate and natural postsynaptic ligands to trigger presynaptic assembly in the absence of specific target recognition. In contrast, we find that postsynaptic compartment assembly depends on the prior presence of a mature presynaptic ending.

Supported bilayers formed from different phospholipids on spherical silica substrates.

Spherical supported bilayer membranes (SS-BLMs) are very attractive candidates in modern bioanalytics and biorecognition studies. A uniform, facile method of preparing different SS-BLMs on silica beads is reported. Confocal fluorescence microscopy and cryo-TEM imaging have been used to characterize these SS-BLMs. Thermal analysis data and FRAP experiments show that the bilayer properties of the SS-BLM are consistent with those of lipid vesicles from which they are formed. The possibility of modulating the size, lipid type and functionality, and mechanical stability makes these rigid liposomes very attractive candidates in biosensors, drug screening, and gene delivery-related applications. This is especially true in work with native vesicle membranes derived from living cells because the existing methods can only accommodate anionic membranes to a limited extent.

Osteocartilaginous metaplasia in the right atrial myocardium of healthy adult sheep.

Among 172 hearts from clinically normal adult sheep aged 1.5 to 7 years evaluated for the presence of cartilage and/or bone in the right atrial myocardium, 3.49% (6/172) had palpable evidence of osteocartilaginous foci. An additional 8% prevalence was estimated based on radiographs of hearts that contained < or =1 mm foci of nonpalpable, radiographically dense bone. Microscopically, the nodules in the atria were characterized by mature lamellar bone enclosing adipose tissue, with occasional new bone formation by endochondral ossification. No degenerative changes were evident in the affected atrial myocardium, suggesting that these lesions were clinically insignificant background changes.

Congenital lobar emphysema and tension pneumothorax in a dog.

Congenital lobar emphysema (CLE) and tension pneumothorax (TPT) are rarely reported in dogs. A case of CLE of the right middle lung lobe predisposing to air trapping, alveolar hyperinflation, and pleural rupture resulting in fatal spontaneous TPT in a 6-month-old mixed breed dog is described. The unique alteration of "bloat line" was observed in this case in addition to compressive atelectasis of all other lung lobes and lack of negative pressure within the thoracic cavity, signifying markedly elevated intrathoracic pressure. Bronchial cartilage hypoplasia and bronchiectasis were confirmed microscopically, which likely led to abnormal dynamic collapse of bronchi during expiration, consequentially leading to increased intrapulmonary pressure, bullous emphysema, and pleural rupture resulting in TPT. TPT consequent to CLE may therefore be considered one of the potential causes of sudden death in young dogs without overt clinical illness.