Reversal of neurobehavioral social deficits in dystrophic mice using inhibitors of phosphodiesterases PDE5A and PDE9A.

Duchenne muscular dystrophy is caused by mutations in the DYSTROPHIN gene. Although primarily associated with muscle wasting, a significant portion of patients (approximately 25%) are also diagnosed with autism spectrum disorder. We describe social behavioral deficits in dystrophin-deficient mice and present evidence of cerebellar deficits in cGMP production. We demonstrate therapeutic potential for selective inhibitors of the cGMP-specific PDE5A and PDE9A enzymes to restore social behaviors in dystrophin-deficient mice.

Preclinical characterization of selective phosphodiesterase 10A inhibitors: a new therapeutic approach to the treatment of schizophrenia.

We have recently proposed the hypothesis that inhibition of the cyclic nucleotide phosphodiesterase (PDE) 10A may represent a new pharmacological approach to the treatment of schizophrenia (Curr Opin Invest Drug 8:54-59, 2007). PDE10A is highly expressed in the medium spiny neurons of the mammalian striatum (Brain Res 985:113-126, 2003; J Histochem Cytochem 54:1205-1213, 2006; Neuroscience 139:597-607, 2006), where the enzyme is hypothesized to regulate both cAMP and cGMP signaling cascades to impact early signal processing in the corticostriatothalamic circuit (Neuropharmacology 51:374-385, 2006; Neuropharmacology 51:386-396, 2006). Our current understanding of the physiological role of PDE10A and the therapeutic utility of PDE10A inhibitors derives in part from studies with papaverine, the only pharmacological tool for this target extensively profiled to date. However, this agent has significant limitations in this regard, namely, relatively poor potency and selectivity and a very short exposure half-life after systemic administration. In the present report, we describe the discovery of a new class of PDE10A inhibitors exemplified by TP-10 (2-{4-[-pyridin-4-yl-1-(2,2,2-trifluoro-ethyl)-1H-pyrazol-3-yl]-phenoxymethyl}-quinoline succinic acid), an agent with greatly improved potency, selectivity, and pharmaceutical properties. These new pharmacological tools enabled studies that provide further evidence that inhibition of PDE10A represents an important new target for the treatment of schizophrenia and related disorders of basal ganglia function.

Analysis of mechanisms underlying BRMS1 suppression of metastasis.

Introduction of normal, neomycin-tagged human chromosome 11 (neo11) reduces the metastatic capacity of MDA-MB-435 human breast carcinoma cells by 70-90% without affecting tumorigenicity. Differential display comparing MDA-MB-435 and neo11/435 led to the discovery of a human breast carcinoma metastasis suppressor gene, BRMS1, which maps to chromosome 11q13.1-q13.2. Stable transfectants of MDA-MB-435 and MDA-MB-231 breast carcinoma cells with BRMS1 cDNA still form progressively growing, locally invasive tumors when injected in mammary fat pads of athymic mice but exhibit significantly lower metastatic potential (50-90% inhibition) to lungs and regional lymph nodes. To begin elucidating the mechanism(s) of action, we measured the ability of BRMS1 to perturb individual steps of the metastatic cascade modeled in vitro. Consistent differences were not observed for adhesion to extracellular matrix components (laminin, fibronectin, type IV collagen, type I collagen, Matrigel); growth rates in vitro or in vivo; expression of matrix metalloproteinases, heparanase, or invasion. Likewise. BRMS1 expression did not up regulate expression of other metastasis suppressors, such as NM23, Kai1, KiSS1 or E-cadherin. Motility of BRMS1 transfectants was modestly inhibited (30-60%) compared to parental and vector-only transfectants. Ability to grow in soft agar was also decreased in MDA-MB-435 cells by 80-89%, but the decrease for MDA-MB-231 was less (13-15% reduction). Also, transfection and re-expression of BRMS1 restored the ability of human breast carcinoma cells to form functional homotypic gap junctions. Collectively, these data suggest that BRMS1 suppresses metastasis of human breast carcinoma by complex, atypical mechanisms.

Metastasis-suppressed C8161 melanoma cells arrest in lung but fail to proliferate.

The incidence of melanoma continues to increase at a rapid rate. As for most cancers, it is melanoma metastases, rather than the primary malignancy, that is the principal cause of death. We previously showed that the introduction of a normal copy of chromosome 6 into the metastatic human melanoma cell line C8161 suppresses metastasis at a step subsequent to tumor cells entering the bloodstream. To better define the step(s) in metastasis blocked by the addition of chromosome 6 we engineered cells that constitutively express green fluorescent protein (GFP). When these tagged, chromosome 6 hybrid cells were injected intravenously into athymic mice, grossly detectable metastases did not form. However, fluorescence microscopy revealed micro-metastases (single cells or clusters of <10 cells) in the lungs, suggesting that these cells lodged in the lungs but failed to proliferate. Cells isolated from lung up to 60 days post-injection grew in culture and/or formed tumors when injected into the skin, indicating that they were still viable, but dormant. This result implies that the gene(s) on chromosome 6 interfere specifically with growth regulatory response in the lung, but not in the skin. Thus, the gene(s) responsible for metastasis suppression represents a new class of metastasis inhibitors acting at the final stages of the metastatic cascade--that is, affecting the ability of the cells to survive and proliferate at a specific secondary site.

A use-dependent sodium channel antagonist, 619C89, in reduction of ischemic brain damage and glutamate release after acute subdural hematoma in the rat.

Acute subdural hematoma kills or disables more severely head injured patients than any other complication of cranial trauma. The main pathological factor involved is ischemic neuronal damage, which is caused by raised intracranial pressure and local effect. The authors have evaluated the hypothesis that a novel use-dependent sodium channel antagonist, 619C89, could reduce ischemic brain damage in the rat subdural hematoma model. Because previous studies have shown that focal neuronal damage may be mediated by "excitotoxic" mechanisms, and because excitatory amino acid levels have been shown to be markedly elevated after brain trauma in humans, the authors have measured levels of glutamate, aspartate, and threonine within the cortex underneath the hematoma, using in vivo microdialysis before and after induction of hematoma, in both vehicle- and drug-treated rats. Postinjury treatment with 619C89 (30 mg/kg) significantly reduced the volume of hemispheric ischemic damage produced by subdural hematoma, from 99.77 +/- 7.51 mm3 in vehicle-treated control rats to 46.07 +/- 11.06 mm3 (p = 0.0007) in drug-treated animals. In the vehicle-treated animals, induction of subdural hematoma led to a fourfold increase in glutamate in the first 30 minutes after subdural hematoma occurred. The mean extracellular glutamate concentration in these animals remained 2- to 2.6-fold increased over the following 2.5 hours. In the 619C89-treated animals, the release of glutamate from the cortex underneath the hematoma was significantly attenuated. In these rats, induction of subdural hematoma led to a 2.7-fold increase in the first 30-minute sample, but extracellular glutamate concentration returned to near-basal levels thereafter, compared with vehicle-treated animals (p < 0.05). These results show that 619C89 is highly neuroprotective in this model and that its effects may, in part, be mediated by the inhibition of glutamate release from the ischemic cortex underneath the hematoma.