Duchenne muscular dystrophy: current knowledge,treatment, and future prospects

The cloning of the dystrophin gene has led to major advances in the understanding of the molecular genetic basis of Duchenne, Becker, and other muscular dystrophies associated with mutations in genes encoding members of the dystrophin-associated glycoprotein complex. The recent introduction of pharmaceutical agents such as prednisone has shown great promise in delaying the progression of Duchenne muscular dystrophy but there remains a need to develop more long-term therapeutic interventions. Knowledge of the nature of the dystrophin gene and the glycoprotein complex has led many researchers to think that somatic gene replacement represents the most promising approach to treatment. The potential use of this strategy has been shown in the mdx mouse model of Duchenne muscular dystrophy, where germ line gene transfer of either a full-length or a smaller Becker-type dystrophin minigene prevents necrosis and restores normal muscle function.     

Small intestinal bacterial overgrowth: a possible risk factor for metabolic bone disease

Small intestinal bacterial overgrowth (SIBO) is one of the causes of malabsorption syndromes. The prevalence of metabolic bone disease in patients with SIBO is unknown, but a recent prospective case-control study indicated significant contribution of SIBO to the development of metabolic bone disease. We review this and other reports in the literature and discuss the possible mechanisms causing metabolic bone disease in patients with SIBO.     

Heat-directed suicide gene therapy for breast cancer

Adjuvant hyperthermia can improve treatment outcome for locally recurrent breast cancer (LRBC). Previously, we demonstrated that infection of human breast cancer cells with a recombinant adenovirus expressing beta-galactosidase from the human hsp70b gene promoter (Ad.70b.betagal) results in 50- to 800-fold increases in reporter gene expression following heat treatment (30 minutes at 43 degrees C). Here, we describe a heat-directed suicide gene therapy strategy based on an adenoviral vector (Ad.70b.CDTK) in which expression of the dual prodrug-activating E. coli cytosine deaminase/herpes simplex virus thymidine kinase (CDTK) fusion gene is under the control of the hsp70b promoter. Treatment of T47D and MCF-7 breast cancer cells with mild hyperthermia (43 degrees C/30 minutes) and prodrugs (100 microg/ml 5-fluorocytosine and 10 microg/ml ganciclovir) following infection with Ad.70b.CDTK (10-100 PFU/cell) resulted in 30- to 60-fold decreases in clonogenic survival relative to control cultures treated with heat or prodrugs alone. Clonogenic survival declined even further (up to 240-fold) following heat treatment at 41.5 degrees C for 120 minutes. A decreased clonogenic survival was accompanied by tumor cell apoptosis. These results demonstrate that this combined treatment strategy can be highly effective against heat- and radiation-resistant breast tumor cells and supports the continued development of heat-directed CDTK suicide gene therapy strategies for LRBC.     

Novel therapeutic approaches to gastric and duodenal ulcers: an update

Over the last 25 years, a remarkable revolution in the pathophysiology and treatment of gastric and duodenal ulcers has occurred. Effective therapies were developed not only to heal ulcers, but also to cure most patients. The two principal causes for gastric and duodenal ulcers are either infection with Helicobacter pylori or the use of non-steroidal anti-inflammatory drugs (NSAIDs). With H. pylori eradication, gastric and duodenal ulcers are rapidly becoming historical diseases. This communication reviews the salient pharmacology of the novel anti-ulcer drugs currently in development, with particular emphasis on the treatment of gastric and duodenal ulcers. Intense research is currently focused on the development of proton pump inhibitors primarily for the treatment and prevention of gastroesophageal reflux disease. The older proton pump inhibitors, omeprazole and lansoprazole, are effective in healing gastric and duodenal ulcers. Furthermore, both drugs are effective in eradicating H. pylori when given with various antibiotics. Pantoprazole, rabeprazole and esomeprazole are new proton pump inhibitors, which appear to have comparable therapeutic profiles with omeprazole and lansoprazole. Rebamipide is a new mucosal protective drug, which is effective in healing gastric ulcers. Polaprezinc and nocloprost are also mucosal protective drugs, which are in clinical development. However, none of these three cytoprotective drugs have been evaluated for their efficacy in eradicating H. pylori when given in combination with antibiotics. Likewise, no published literature exists on the use of these drugs for preventing NSAID-induced ulcers. With the rapid eradication of H. pylori currently happening in the developed world, the therapeutic challenge is now directed toward preventing NSAID-associated ulcer. Significant reduction of NSAID-induced ulcers is achieved by using continuous prophylactic anti-ulcer therapy (misoprostol or omeprazole) or by using NSAIDs possessing selective COX-2 inhibitory activity. However, outcome clinical studies are needed to compare the adjuvant anti-ulcer therapies given with COX-1 inhibitors versus the selective COX-2 inhibitors given alone.     

Calmodulin levels in developing muscle tissues and primary cultures of normal and dystrophic (UM-X7.1) hamsters

Calmodulin levels have been assessed in whole muscle and primary culture extracts in order to examine the relationship between calmodulin and the accumulation of calcium in dystrophic hamster muscle tissues. Significant decreases in both normal and dystrophic skeletal muscle, tongue, and heart calmodulin levels were observed between 2 and 12 weeks of age. Dystrophic values, however, tended to be somewhat higher than normal, especially in 12-week-old skeletal muscle total and soluble extracts (normal 29.7 and 0.6 and dystrophic 117.0 and 3.1 micrograms/g wet weight, respectively). No significant differences were observed in dystrophic myoblast (total 2.22-2.78, soluble 2.85-3.26 micrograms/mg protein) or fibroblast (total 2.64-2.94, soluble 2.54-3.60 micrograms/mg protein) calmodulin levels, except for a significant decrease in dystrophic fibroblast levels (total 1.97, soluble 2.18 micrograms/mg protein) at 7 days in culture. Elevated calmodulin levels in dystrophic muscle are discussed in terms of increases in intracellular Ca2+ concentrations and immature regenerating fibers.     

Powdered diatrizoic acid for radiography of the respiratory tract. Part I. Experimental investigation

Powdered diatrizoic acid as a contrast medium administered by inhalation and insufflation for visualization of the airways was tested in vitro and in 21 dogs. Good radiopacity of the contrast medium and its antibacterial activity was found in vitro. In the majority of animal experiments visualization of the bronchial tree down to segmental and partially to subsegmental bronchi with only minimal agglomeration of contrast medium and with good or very good demonstration of anatomic details was achieved. In the majority of dogs contrast medium was eliminated from the lungs within 18 hours. Arterial blood gases tested on 5 dogs showed only unimportant changes after contrast medium administration. No adverse reactions were observed. Histologic and ultrastructural examinations after contrast studies showed phagocytic reaction to diatrizoic acid, transient impairment of production of surfactant, reactive changes of bronchial mucosa, and no fibrotic changes in the long-term observation.     

Phase I and pharmacologic study of the human DNA methyltransferase antisense oligodeoxynucleotide MG98 given as a 21-day continuous infusion every 4 weeks

Purpose: MG98 is a second generation phosphorothioate antisense oligodeoxynucleotide which is a highly specific inhibitor of translation of the mRNA for human DNA MeTase I (DNMT 1). This phase I study examined the toxicity and pharmacologic profile of MG98 administered as a continuous 21-day intravenous infusion every 4 weeks. Patients and methods: Fourteen patients with solid cancers received a total of 25 cycles of MG98 at doses ranging from 40 to 240 mg/m²/day. Steady-state concentrations of MG98 were measured as were several pharmacodynamic assessments including mRNA of the target gene, DNMT1, in PBMC. In addition, other potential surrogate markers of drug effects were explored, including hemoglobin F, Vimentin and GADD45. Results: Dose limiting effects were drug-related reversible transaminase elevation and fatigue seen at doses of 240, 200 and 160 mg/m²/day. The dose level of 80 mg/m²/day was felt to be safe and tolerable when delivered on this schedule. No evidence of antitumor activity was observed. Although pharmacokinetic analysis revealed that at the higher dose levels, mean Css values of MG98 were approximately 10-fold times the IC₅₀ values associated with target inhibition in vitro, the extent of MG98 penetration into target tumors in this trial was not determined. No consistent, dose-related changes in correlative markers including DNMT1 mRNA, hemoglobin F, Vimentin and GADD45, were observed. Conclusions: This schedule of MG98 given as a 21-day continuous intravenous infusion every 4 weeks was poorly tolerated in the highest doses; therefore, further disease-site specific evaluation of the efficacy of this agent will utilize a more favorable, intermittent dosing schedule. Pharmacodynamic evaluations undertaken in an attempt to explore and validate the biological mechanisms of MG98 did not show dose-related effects.     

Assessment of the relationship between genotypic status of a DT-diaphorase point mutation and enzymatic activity

DT-diaphorase, a cytosolic reductase, has been implicated as an activator of chemotherapeutic prodrugs and a detoxifier of certain potentially carcinogenic xenobiotics. A common C to T nucleotide 609 substitution in DT-diaphorase cDNA has been associated with protein instability and reduced catalytic activity. The degree to which the allelic status of the substitution correlates with enzymatic activity was assessed in 45 normal human skin fibroblast strains using a PCR-RFLP assay. Included in this study was the 3437T strain, which is unique in that it is heterozygous for the polymorphism yet contains undetectable enzymatic activity. An allele-specific RT-PCR-RFLP technique attributed this phenomenon to exclusive DT-diaphorase mRNA expression from the variant allele. Overlap in activities was observed between individual strains homozygous for the wild-type allele and heterozygotes, but the former group displayed enzymatic activity that was on average 2-fold higher. Western blot analysis of the two strains in this panel that are homozygous for the variant allele revealed that they express relatively low amounts of DT-diaphorase protein, consistent with the role of the substitution in protein instability. This work confirms that genotypic status is a reliable initial estimate of DT-diaphorase activity.