To support clinical development of S-nitrosoglutathione (GSNO) as a therapeutic agent, 28-day toxicology studies in rats and dogs were conducted. Rats (21-25/sex) and dogs (3-5/sex) were exposed for 4 hours or 1 hour, respectively, to inhaled GSNO (0, 3, 9.3, 19, and 28 mg/kg per d in rats and 0, 4.6, 9.0, and 16.2 mg/kg per d in dogs) or vehicle daily via a nebulizer. Animals were monitored throughout the 28-day dosing period and during a postexposure recovery period. Complete necropsy and tissue examinations were performed. Experimental end points included clinical pathology, toxicokinetics, and immunotoxicology. No biologically significant adverse findings were noted in either species, and the no observed adverse effect levels (NOAELs) under these conditions were the highest achieved doses (28 and 16.2 mg/kg per d in rats and dogs, respectively). These data demonstrate that GSNO is well tolerated in rodents and dogs and predict a favorable toxicity profile in humans, thus supporting future clinical development of GSNO or closely related compounds.

Import of DNA from the cytoplasm into the mitochondrial matrix is an obligatory step for an in organello site-directed mutagenesis or gene therapy approach on mitochondrial DNA diseases. In this context, we have developed an artificial DNA translocation vector that is composed of the mitochondrial signal peptide of the ornithine transcarbamylase (OTC) and a DNA moiety. While this vector is capable of directing attached passenger molecules to the mitochondrial matrix, the recognition of this artificial molecule by the endogenous mitochondrial signal peptide processing machinery as well as the cleavage of the peptide plays a pivotal role in the release of the attached DNA. To study the proteolytic processing of the artificial vector, various signal peptide-DNA-conjugates were treated with purified mitochondrial intermediate peptidase. When the leader peptide is directly linked to the DNA moiety without an intervening spacer, MIP processing is prevented. Cleavage of the peptide can be restored, however, when the first ten amino acid residues of the mature part of OTC are appended at the carboxy-terminal end of the signal peptide. Our results show that artificial peptide-DNA-conjugates are recognized by the mitochondrial proteolytic machinery, and therefore an interference of the peptide with the DNA function can be excluded.

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