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Given the relatively low molecular weight and multiple potential pharmacophores present within the compound though 5 ml fml forte with amex, it seems likely that other biological targets of hal- ofuginone will be discovered 5 ml fml forte mastercard, which may contribute to its pharmacological effects. Furthermore, this knowledge will allow further possible toxicological effects to be anticipated and tested proactively. A critical consideration with any reproling approach is availability of adened target hypothesis and accompanying assay system to test the compounds. Alignment of both the optimal compound set with a suitable test system with disease relevance is therefore essential. The more established approaches to drug discovery undertaken within the pharmaceutical industry are as valid with neuromuscular disease as with the study of any other disease class, and indeed, a distinct paradigm shi has occurred in recent years, effectively amounting to a rediscovery (or reinvention) of phenotypic screening as an effective means to both validate disease targets and identify novel compounds,56 either through de novo screening or reproling strategies. Given the encouraging results seen using the mdx mouse, results in patients will be of great interest to the scientic community. Following completion of the study, there was no evidence of efficacy with either the cyclosporin as a monotherapy, nor in combination with prednisone in providing improvement in the muscle strength of trial participants. Whether or not any further investigation of this agent is View Online 274 Chapter 11 undertaken remains to be seen. Following 15 months of once daily oral dosing to mdx mice, striking effects on both muscle structure and function were seen, although the mechanism through which it acted was not clear. Importantly, signicant improvements were also seen in both the diaphragm and cardiac muscles, which oen prove refractory to experi- mental therapeutics. Alternative therapeutic modalities other than small molecules have been the subject of much investigation. Gene therapy, whereby the missing or damaged genetic material is introduced using an View Online Drug Discovery Approaches for Rare Neuromuscular Diseases 275 Figure 11. Results from the Phase 1 clinical evaluation of a dystrophin mini-gene have been published,68,69 and the prospects for the approach recently reviewed. Although not observed in all treated patients, this phenomenon appeared to be associated with the exons downstream from the missing sections of the patient’s dystrophin gene. Aside from the fact that this agent required direct injection to the site of action, and issues therefore remain over delivery to the heart and diaphragm muscle, it high- lights an important additional advantage for small-molecule therapeutic approaches such as those described elsewhere in this section. Aside from some cases involving compounds that form covalent bonds with biological targets (i. Further compounding the hereditary aspects of the disease is the fact that dystrophin is the largest gene in the human genome, and therefore the chances of spontaneous mutations occurring is far more likely than in most other genes. It is also for this reason that genetic screening of adults is unlikely to completely eliminate occurrence of the disease. Morpholino, and other related compounds (‘oligos’), are chemically more stable derivatives of oligonucleotides, and have been extensively investigated as agents for promoting exon skipping. Indeed, several have demonstrated efficacy using in vivo models such as the mdx mouse. Hence they can be viewed as a development from anti-sense gene silencing strategies. While progress has been encouraging, with activity of morpholinos seen in clinical trials, a number of challenging technological questions remain, and have hampered the wider applicability of the strategy. Foremost amongst them relates to how to achieve the most effective and systematically widespread delivery of drug to patients, because the compounds evaluated clinically to date are not orally available molecules and thus far have had to be injected directly to the site of the affected muscle in both pre-clinical experiments using animals,80 as well as in clinical studies. This also suggests that there will be limited opportunity to treat cardiac muscle using this strategy, although one can imagine combination therapy with other therapeutic paradigms being explored. Micro- encapsulated derivatives have shown encouraging data, but nonetheless wider systematic bioavailability is still a problem. While it is theoretically possible that these agents could provoke an immune response or even unintended exon skipping (resulting in a faulty dystrophin protein in a healthy patient), the risk of this actually occurring is View Online 278 Chapter 11 unknown at this time, and so will have to be monitored carefully during clinical evaluation. Ultimately, exon-skipping strategies should allow production of functional dystrophin protein in individuals who carry the mutation in that particular exon, although importantly the dystrophin protein produced is shorter. In an extension of this strategy, a screening approach was employed by Miceli and co-workers to search for small molecules that could enhance exon skipping. View Online Drug Discovery Approaches for Rare Neuromuscular Diseases 279 Data from both in vitro assays (mdx and human muscle cells), and func- tional tests in mdx mice indicated the effects of anti-sense were augmented by dantrolene, and although the precise mechanisms in play are not clear at this time, the results were encouraging in their own right and provide precedent for the use of drug combinations. Encouraging increases in dystrophin levels were seen in patient biopsies, and no series safety issues were noted. Increases in dystrophin protein have been seen in preclinical87 and clinical studies. Results from Phase 1 and 2 clinical trials for this agent were published recently. The advantage of the class was that the drugs had already been evaluated in humans as antibiotics (which was in fact where the ground-breaking observation of their stop codon read-through ability was originally made),95 and therefore had a known toxicological prole.

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