Preclinical pharmacologic studies of the new antitumor agent carmethizole (NSC-602668) in the mouse and beagle dog

Summary The chemical breakdown of carmethizole [1-methyl-2-methylthio-4,5-bis-(hydroxymethyl)imidazole-4,5-bis(N-methylcarbamate)hydrochloride] and its pharmacokinetics in the mouse and beagle dog were studied. Carmethizole was relatively unstable in aqueous media, having a half-life of 1 h in 0.9% sodium chloride, human whole blood, human plasma, and dog urine at 37°C. Its major breakdown product in 0.9% sodium chloride and pH 5.0 sodium phosphate buffer was carmethizole diol. When carmethizole was added to pH 7.0 or pH 9.0 sodium phosphate buffer, the major breakdown product was carmethizole diol-4-monophosphate. Carmethizole reacted directly with glutathione at pH 8.0, forming a glutathione adduct of carmethizole monocarbamate. Elimination of the drug from the plasma of the beagle dog following i.v. bolus doses of 22.4 and 4.3 mg/kg was biphasic. At these doses the terminal half-life was 39 and 46 min, respectively, and the respective total body clearance was 4.6 and 7.7 ml/min per kg. The 22.4 mg/kg dose was lethal to the beagle dog by day 4. Elimination of carmethizole from the plasma of mice following an i. v. bolus dose of 115 mg/kg was monoexponential, with a half-life of 11.6 min and a total body plasma clearance of 43.6 ml/min per kg. When the drug was infused at 230 mg/kg over 8 h into mice, the total body clearance was 40.8 ml/min per kg. Following the i.v. bolus administration of carmethizole to mice, 30% of the total dose was excreted in urine over 3 h as carmethizole diol, 10%, as carmethizole diol-sulfate, 3.4%, as carmethizole 4-monocarbamate, and 2.4%, as unchanged drug.     

Deletion of Brca2 exon 27 causes hypersensitivity to DNA crosslinks,chromosomal instability,and reduced life span in mice

The Brca2 tumor-suppressor gene contributes to genomic stability, at least in part by a role in homologous recombinational repair. BRCA2 protein is presumed to function in homologous recombination through interactions with RAD51. Both exons 11 and 27 of Brca2 code for domains that interact with RAD51; exon 11 encodes eight BRC motifs, whereas exon 27 encodes a single, distinct interaction domain. Deletion of all RAD51-interacting domains causes embryonic lethality in mice. A less severe phenotype is seen with BRAC2 truncations that preserve some, but not all, of the BRC motifs. These mice can survive beyond weaning, but are runted and infertile, and die very young from cancer. Cells from such mice show hypersensitivity to some genotoxic agents and chromosomal instability. Here, we have analyzed mice and cells with a deletion of only the RAD51-interacting region encoded by exon 27. Mice homozygous for this mutation (called brca2(lex1)) have a shorter life span than that of control littermates, possibly because of early onsets of cancer and sepsis. No other phenotype was observed in these animals; therefore, the brca2(lex1) mutation is less severe than truncations that delete some BRC motifs. However, at the cellular level, the brca2(lex1) mutation causes reduced viability, hypersensitivity to the DNA interstrand crosslinking agent mitomycin C, and gross chromosomal instability, much like more severe truncations. Thus, the extreme carboxy-terminal region encoded by exon 27 is important for BRCA2 function, probably because it is required for a fully functional interaction between BRCA2 and RAD51.     

Holt-Oram syndrome associated with the hypoplastic left heart syndrome

We present the first case of Holt-Oram syndrome associated with the lethal congenital heart defect of hypoplastic left heart syndrome. The possible pathophysiological link is explored and the need for careful genetic and cardiologic evaluation in these patients is reiterated.     

Physical Deletion of the p53 Gene in Bladder Cancer: Detection by Fluorescence in Situ Hybridization

To understand better the role of physical p53 deletion in bladder cancer, 106 formalin-fixed and 45 unfixed bladder tumors were examined using fluorescence in situ hybridization. Probes for centromere 17 and the p53 locus were hybridized simultaneously to interphase tumor cells to analyze p53 and chromosome 17 copy number on a cell by cell basis. 17p deletion was found in four of 43 pTa tumors, 18 of 43 pT1 tumors and 29 of 58 pT2-4 tumors (P = 0.0001). 17p deletion was also highly correlated with grade (P = 0.0001) and with p53 immunostaining (P = 0.0005). Chromosome 17 polysomy was associated with stage, grade, 17p deletions, and p53 immunostaining (P = 0.0001). The strong difference in centromere 17 copy number and 17p deletions between pTa and pT1 tumors supports a relevant biological distinction between pTa and pT1 tumors.     

Differential Effects of Clostridium difficile Toxins on Tissue-Cultured Cells

Two immunologically distinct Clostridium difficile toxins elicited similar morphological changes on cultured cells, although there were differences in both toxin potency and cell sensitivities.     

Use of subtractive hybridization to identify a diagnostic probe for a cystic fibrosis epidemic strain of Pseudomonas aeruginosa

A multiresistant strain of Pseudomonas aeruginosa is widespread among cystic fibrosis (CF) patients attending clinics in Liverpool, United Kingdom. Suppression subtractive hybridization was used to identify sequences present in the Liverpool CF epidemic strain but absent from strain PAO1. Using dot blot and PCR amplification assays, the prevalence of such sequences among a panel of CF isolates was determined. Several sequences were found only in the Liverpool epidemic strain. Some sequences were present in the Liverpool epidemic strain and in a minority of other isolates, including sequences with homology to genes implicated in O6 serotype and siderophore production. The Liverpool epidemic strain and 81% of nonepidemic isolates contained a sequence identified as part of the PAGI-1 genomic island. Other strains implicated in epidemic spread, which were from Manchester, United Kingdom, and Melbourne, Australia, were also screened. None of the sequences identified was present in the Manchester strain. However, one of two Melbourne strains contained some of the sequences found in the Liverpool epidemic strain. All isolates implicated in epidemic spread and 76% of sporadic isolates contained the exoS gene. A sequence present in all isolates of the Liverpool epidemic strain was used to develop a diagnostic PCR test for identification of the strain from colonies or directly from sputum samples.