A highly sensitive canine telemetry model for detection of QT interval prolongation: studies with moxifloxacin, haloperidol and MK-499.

INTRODUCTION: Preclinical evaluation of delayed ventricular repolarization manifests electrocardiographically as QT interval prolongation and is routinely used as an indicator of potential risk for pro-arrhythmia (potential to cause Torsades de Pointes) of novel human pharmaceuticals. In accordance with ICH S7A and S7B guidelines we evaluated the sensitivity and validity of the beagle dog telemetry (Integrated Telemetry Services (ITS)) model as a preclinical predictor of QT interval prolongation in humans. METHODS: Cardiovascular monitoring was conducted for 2 h pre-dose and 24 h post-dosing with moxifloxacin (MOX), haloperidol (HAL), and MK-499, with a toxicokinetic (TK) evaluation in a separate group of dogs. In both cardiovascular and TK studies, MOX (0, 10, 30 and 100 mg/kg), HAL (0, 0.3, 1, 3 mg/kg) and MK-499 (0, 0.03, 0.3 and 3 mg/kg) were administered orally by gavage in 0.5% methylcellulose. Each dog received all 4 doses using a dose-escalation paradigm. Inherent variability of the model was assessed with administration of vehicle (0.5% methylcellulose) alone for 4 days. RESULTS: Significant increases in QT(c) were evident with 10, 30 and 100 mg/kg of MOX (C(max)< or =40 microM), 0.3, 1 and 3 mg/kg of HAL (C(max)< or =0.36 microM) and 0.3 and 3 mg/kg of MK-499 (C(max)< or =825 nM) with peak increases of 45 (20%), 31 (13%), and 45 (19%) ms, respectively (p< or =0.05). DISCUSSION: In conclusion, we have demonstrated that the ITS-telemetry beagle dog exhibits low inherent intra-animal variability and high sensitivity to detect small but significant increases in QT/QT(c) interval ( approximately 3-6%) with MOX, HAL and MK-499 in the same range of therapeutic plasma concentrations attained in humans. Therefore, this dog telemetry model should be considered an important preclinical predictor of QT prolongation of novel human pharmaceuticals.

The sequences of heat shock protein 40 (DnaJ) homologs provide evidence for a close evolutionary relationship between the Deinococcus-thermus group and cyanobacteria.

The genes encoding for heat shock protein 40 (Hsp40 or DnaJ) homologs were cloned and sequenced from the archaebacterium Halobacterium cutirubrum and the eubacterium Deinococcus proteolyticus to add to sequences from the gene banks. These genes were identified downstream of the Hsp70 (or DnaK) genes in genomic fragments spanning this region and, as in other prokaryotic species, Hsp70-Hsp40 genes are likely part of the same operon. The Hsp40 homolog from D. proteolyticus was found to be lacking a central 204 base pair region present in H. cutirubrum that encodes for the four cysteine-rich domains of the repeat consensus sequence CxxCxGxG (where x is any amino acid), present in most Hsp40 homologs. The available sequences from various archaebacteria, eubacteria, and eukaryotes show that the same deletion is also present in the homologs from Thermus aquaticus and two cyanobacteria, but in no other species tested. This unique deletion and the clustering of homologs from the Deinococcus-Thermus group and cyanobacterial species in the Hsp40 phylogenetic trees suggest a close evolutionary relationship between these groups as was also shown recently for Hsp70 sequences (R.S. Gupta et al., J Bacteriol 179:345-357, 1997). Sequence comparisons indicate that the Hsp40 homologs are not as conserved as the Hsp70 sequences. Phylogenetic analysis provides no reliable information concerning evolutionary relationship between prokaryotes and eukaryotes and their usefulness in this regard is limited. However, in phylogenetic trees based on Hsp40 sequences, the two archaebacterial homologs showed a polyphyletic branching within Gram-positive bacteria, similar to that seen with Hsp70 sequences.

Sequencing of heat shock protein 70 (DnaK) homologs from Deinococcus proteolyticus and Thermomicrobium roseum and their integration in a protein-based phylogeny of prokaryotes.

The 70-kDa heat shock protein (hsp70) sequences define one of the most conserved proteins known to date. The hsp70 genes from Deinococcus proteolyticus and Thermomicrobium roseum, which were chosen as representatives of two of the most deeply branching divisions in the 16S rRNA trees, were cloned and sequenced. hsp70 from both these species as well as Thermus aquaticus contained a large insert in the N-terminal quadrant, which has been observed before as a unique characteristic of gram-negative eubacteria and eukaryotes and is not found in any gram-positive bacteria or archaebacteria. Phylogenetic analysis of hsp70 sequences shows that all of the gram-negative eubacterial species examined to date (which includes members from the genera Deinococcus and Thermus, green nonsulfur bacteria, cyanobacteria, chlamydiae, spirochetes, and alpha-, beta-, and gamma-subdivisions of proteobacteria) form a monophyletic group (excluding eukaryotic homologs which are derived from this group via endosybitic means) strongly supported by the bootstrap scores. A closer affinity of the Deinococcus and Thermus species to the cyanobacteria than to the other available gram-negative sequences is also observed in the present work. In the hsp7O trees, D. proteolyticus and T. aquaticus were found to be the most deeply branching species within the gram-negative eubacteria. The hsp70 homologs from gram-positive bacteria branched separately from gram-negative bacteria and exhibited a closer relationship to and shared sequence signatures with the archaebacteria. A polyphyletic branching of archaebacteria within gram-positive bacteria is strongly favored by different phylogenetic methods. These observations differ from the rRNA-based phylogenies where both gram-negative and gram-positive species are indicated to be polyphyletic. While it remains unclear whether parts of the genome may have variant evolutionary histories, these results call into question the general validity of the currently favored three-domain dogma.