Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease.

Niemann-Pick type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT) was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients.

Improved neuroprotection using miglustat, curcumin and ibuprofen as a triple combination therapy in Niemann-Pick disease type C1 mice.

OBJECTIVES: Niemann-Pick disease type C (NPC) is a neurodegenerative lysosomal storage disorder characterised by the storage of multiple lipids, reduced lysosomal calcium levels, impaired late endosome:lysosome fusion and neuroinflammation. NPC is caused by mutations in either of the two genes, NPC1 or NPC2, which are believed to function in a common cellular pathway, the function of which remains unclear. The complexity of the pathogenic cascade in NPC disease provides a number of potential clinical intervention points. To date, drugs that target pivotal stages in the pathogenic cascade have been tested as monotherapies or in combination with a second agent, showing additive or synergistic benefit. In this study, we have investigated whether we can achieve greater therapeutic benefit in the Npc1(-/-) mouse by combining three therapies that each targets unique aspects of the pathogenic cascade. METHODS: We have treated Npc1(-/-) mice with miglustat that targets sphingolipid synthesis and storage, curcumin that compensates for the lysosomal calcium defect by elevating cytosolic calcium, and the non-steroidal anti-inflammatory drug ibuprofen to reduce central nervous system inflammation. RESULTS/INTERPRETATION: We have found that triple combination therapy has a greater neuroprotective benefit compared with single and dual therapies, increasing the time period that Npc1(-/-) mice maintained body weight and motor function and maximally delaying the onset of Purkinje cell loss. In addition, ibuprofen selectively reduced microglial activation, while curcumin had no anti-inflammatory effects, indicating differential mechanisms of action for these two therapies. When taken together, these results demonstrate that targeting multiple unique steps in the pathogenic cascade maximises the clinical benefit in a mouse model of NPC1 disease.

Early glial activation, synaptic changes and axonal pathology in the thalamocortical system of Niemann-Pick type C1 mice.

Niemann-Pick disease type C (NPC) is an inherited lysosomal storage disease characterised by accumulation of cholesterol and glycosphingolipids. NPC patients suffer a progressive neurodegenerative phenotype presenting with motor dysfunction, mental retardation and cognitive decline. To examine the onset and progression of neuropathological insults in NPC we have systematically examined the CNS of a mouse model of NPC1 (Npc1(-/-) mice) at different stages of the disease course. This revealed a specific spatial and temporal pattern of neuropathology in Npc1(-/-) mice, highlighting that sensory thalamic pathways are particularly vulnerable to loss of NPC1 resulting in neurodegeneration in Npc1(-/-) mice. Examination of markers of astrocytosis and microglial activation revealed a particularly pronounced reactive gliosis in the thalamus early in the disease, which subsequently also occurred in interconnected cortical laminae at later ages. Our examination of the precise staging of events demonstrate that the relationship between glia and neurons varies between brain regions in Npc1(-/-) mice, suggesting that the cues causing glial reactivity may differ between brain regions. In addition, aggregations of pre-synaptic markers are apparent in white matter tracts and the thalamus and are likely to be formed within axonal spheroids. Our data provide a new perspective, revealing a number of events that occur prior to and alongside neuron loss and highlighting that these occur in a pathway dependent manner.

Pemetrexed in relapsed small-cell lung cancer and the impact of shortened vitamin supplementation lead-in time: results of a phase II trial.

INTRODUCTION: We undertook a phase II trial to assess the efficacy and safety of single-agent pemetrexed (P) in relapsed small-cell lung cancer (SCLC) patients. METHODS: Patients had limited- or extensive-stage SCLC, performance status 0 to 2, and one prior chemotherapy regimen. Initial P dose was 500 mg/m every 21 days. Planned sample sizes were 36 sensitive (S) patients in a two-stage sequential fashion with early stopping rule, and 25 refractory (R) patients in a single-stage design without stopping rule. Patients received folic acid and Vitamin B12 prior to P, and B12 could be given up until P treatment. Primary outcome measure was response rate. RESULTS: Enrollment occurred from July 2004 to March 2006. The stopping rule was invoked when <3 of 14 S patients responded. The protocol was amended to evaluate P 900 mg/m in cohorts of 40 S and 40 R patients. Overall, 121 patients were enrolled, with 116 patients treated. S (n = 53) and R (n = 63) patients were analyzed separately at both dose levels. Across the 4 treatment groups (S500, S900, R500, R900), 1 patient (2.63%) in the S900 group had a partial response. Overall, 18 patients (16%) had stable disease. Eighty-seven patients (75%) had progressive disease. Responses were not evaluable in 10 patients (8.6%). Overall response rate was 0.9%. Across treatment groups, disease control rates (partial response + stable disease) were 20%, 15.8%, 21.7%, and 12.5%, respectively. Median time to progression ranged from 1.2 to 1.5 months, median survival times ranged from 2.5 to 6.1 months, and 1-year survival rates ranged from 5.6 to 25.8%. Common grade 3/4 hematologic toxicities (at 500 and 900 mg/m) were neutropenia (16%; 9%) and leukopenia (11%; 8%), and nonhematologic toxicities were dyspnea (11%; 10%) and fatigue (16%; 9%). Retrospective analysis of cycle 1 events by timing of B12 administration showed no trend toward more frequent serious toxicities when B12 was given <7 days prior to P. CONCLUSIONS: Single-agent P 500 mg/m shows minimal activity in relapsed SCLC patients. P can be given at 900 mg/m without significant increase in serious toxicities, but does not seem to increase efficacy. B12 given <7 days before P does not seem to be associated with increased serious toxicities.

Beneficial effects of substrate reduction therapy in a mouse model of GM1 gangliosidosis.

GM1 gangliosidosis is an inherited neurodegenerative disorder caused by lysosomal beta-galactosidase deficiency, resulting in the storage of GM1 and GA1, primarily in the central nervous system. This disease typically afflicts infants and young children and there is currently no effective therapy. Substrate reduction therapy (SRT) could be of potential benefit. The imino sugars N-butyldeoxynojirimycin (NB-DNJ, miglustat, Zavesca) and N-butyldeoxygalactonojirimycin (NB-DGJ) used for SRT inhibit glucosylceramide synthase (GlcCerS) that catalyses the first committed step in glycosphingolipid biosynthesis. We have compared the efficacy and tolerability of NB-DNJ and NB-DGJ in the beta-galactosidase knockout mouse. NB-DGJ was better tolerated than NB-DNJ, due to intrinsic gastrointestinal tract dysfunction that was exacerbated by NB-DNJ. However, functional improvement was greatest with NB-DNJ treatment which may potentially be caused by novel anti-inflammatory properties of NB-DNJ.

NSAIDs increase survival in the Sandhoff disease mouse: synergy with N-butyldeoxynojirimycin.

The GM2 gangliosidoses are caused by incomplete catabolism of GM2 ganglioside in the lysosome, leading to progressive storage and a neurodegenerative clinical course. An inflammatory response (microglial activation, macrophage infiltration, oxidative damage) has been found to be a consequence of GM2 storage in the brain, although it remains unclear whether this contributes to pathogenesis or disease progression. In this study, we treated Sandhoff disease mice with nonsteroidal antiinflammatory drugs (indomethacin, aspirin, and ibuprofen) and antioxidants (L-ascorbic acid and alpha-tocopherol acetate). The treated mice lived significantly longer than untreated littermates (12-23%, p <0.0001) and showed a slower rate of disease progression (p <0.001). When aspirin treatment was combined with substrate reduction therapy, synergy resulted (11%, p <0.05) with a maximum improvement of 73% in survival (p <0.00001). This study demonstrates that inflammation contributes to disease progression and identifies antiinflammatory and antioxidant therapies as a potential adjunctive approach to slow the clinical course of this and related disorders.