Peter DeMarsh

Identification of Unique Truncated KC/GROβ Chemokines with Potent Hematopoietic and Anti-Infective Activities

SK&F 107647, a previously described synthetic immunomodulatory peptide, indirectly stimulates bone marrow progenitor cells and phagocytic cells, and enhances host defense effector mechanisms in bacterial and fungal infection models in vivo. In vitro, SK&F 107647 induces the production of a soluble mediator that augments colony forming cell (CFU-GM) formation in the presence of CSFs. In this paper we purified and sequenced the stromal cell-derived hematopoietic synergistic factors (HSF) secreted from both murine and human cell lines stimulated with SK&F 107647. Murine HSF is an N-terminal 4-aa truncated form of the CXC chemokine, KC, while human HSF was identified as an N-terminal 4-aa truncated form of the CXC chemokine, GROβ. In comparison to their full-length forms, truncated KC and truncated GROβ were 10 million times more potent as synergistic growth stimulants for CFU-GM. Enhanced potency of these novel truncated chemokines relative to their full-length forms was also demonstrated in respiratory burst assays, CD11b Ag expression, and intracellular killing of the opportunistic pathogen, Candida albicans. Administration of truncated KC significantly enhanced survival of mice lethally infected with C. albicans. The results reported herein delineate the biological mechanism of action of SK&F 107647, which functions via the induction of unique specific truncated forms of the chemokines KC and GROβ. To our knowledge, this represents the first example where any form of KC or GROβ were purified from marrow stromal cells. Additionally, this is the first demonstration of in vivo efficacy of a CXC chemokine in an animal infectious fungal disease model.

Staphylococcus aureus Formyl-Methionyl Transferase Mutants Demonstrate Reduced Virulence Factor Production and Pathogenicity

ABSTRACT Inhibitors of peptide deformylase (PDF) represent a new class of antibacterial agents with a novel mechanism of action. Mutations that inactivate formyl methionyl transferase (FMT), the enzyme that formylates initiator methionyl-tRNA, lead to an alternative initiation of protein synthesis that does not require deformylation and are the predominant cause of resistance to PDF inhibitors in Staphylococcus aureus. Here, we report that loss-of-function mutations in FMT impart pleiotropic effects that include a reduced growth rate, a nonhemolytic phenotype, and a drastic reduction in production of multiple extracellular proteins, including key virulence factors, such as α-hemolysin and Panton-Valentine leukocidin (PVL), that have been associated with S. aureus pathogenicity. Consequently, S. aureus FMT mutants are greatly attenuated in neutropenic and nonneutropenic murine pyelonephritis infection models and show very high survival rates compared with wild-type S. aureus. These newly discovered effects on extracellular virulence factor production demonstrate that FMT-null mutants have a more severe fitness cost than previously anticipated, leading to a substantial loss of pathogenicity and a restricted ability to produce an invasive infection.

Pharmacokinetics/Pharmacodynamics of Peptide Deformylase Inhibitor GSK1322322 against Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus in Rodent Models of Infection

ABSTRACT GSK1322322 is a novel inhibitor of peptide deformylase (PDF) with good in vitro activity against bacteria associated with community-acquired pneumonia and skin infections. We have characterized the in vivo pharmacodynamics (PD) of GSK1322322 in immunocompetent animal models of infection with Streptococcus pneumoniae and Haemophilus influenzae (mouse lung model) and with Staphylococcus aureus (rat abscess model) and determined the pharmacokinetic (PK)/PD index that best correlates with efficacy and its magnitude. Oral PK studies with both models showed slightly higher-than-dose-proportional exposure, with 3-fold increases in area under the concentration-time curve (AUC) with doubling doses. GSK1322322 exhibited dose-dependent in vivo efficacy against multiple isolates of S. pneumoniae, H. influenzae, and S. aureus. Dose fractionation studies with two S. pneumoniae and S. aureus isolates showed that therapeutic outcome correlated best with the free AUC/MIC (fAUC/MIC) index in S. pneumoniae (R2, 0.83), whereas fAUC/MIC and free maximum drug concentration (fCmax)/MIC were the best efficacy predictors for S. aureus (R2, 0.9 and 0.91, respectively). Median daily fAUC/MIC values required for stasis and for a 1-log10 reduction in bacterial burden were 8.1 and 14.4 for 11 S. pneumoniae isolates (R2, 0.62) and 7.2 and 13.0 for five H. influenzae isolates (R2, 0.93). The data showed that for eight S. aureus isolates, fAUC correlated better with efficacy than fAUC/MIC (R2, 0.91 and 0.76, respectively), as efficacious AUCs were similar for all isolates, independent of their GSK1322322 MIC (range, 0.5 to 4 μg/ml). Median fAUCs of 2.1 and 6.3 μg · h/ml were associated with stasis and 1-log10 reductions, respectively, for S. aureus.

Novel peptidomimetic hematoregulatory compounds

The activity of a novel series of peptidomimetic hematoregulatory compounds, designed based on a pharmacophore model inferred from the structure activity relationships of a peptide SK&F 107647 (1), is reported. These compounds induce a hematopoietic synergistic factor (HSF) which in turn modulates host defense. The compounds may represent novel therapeutic agents in the area of hematoregulation.

Reducing antibacterial development risk for GSK1322322 by exploring potential human dose regimens in nonclinical efficacy studies using immunocompetent rats

ABSTRACT Directly testing proposed clinical dosing regimens in nonclinical studies can reduce the risk during the development of novel antibacterial agents. Optimal dosing regimens can be identified in animal models by testing recreated human pharmacokinetic profiles. An example of this approach using continuous intravenous infusions of GSK1322322 in immunocompetent rats to evaluate recreated human exposures from phase I trials in pneumonia models with Streptococcus pneumoniae and Haemophilus influenzae and an abscess model with Staphylococcus aureus is presented. GSK1322322 was administered via continuous intravenous infusion to recreate 1,000- or 1,500-mg oral doses every 12 h in humans. Significant reductions (P ≤ 0.05 for all comparisons) in bacterial numbers compared with those for the baseline controls were observed for S. pneumoniae and H. influenzae (mean log10 reductions, 1.6 to ≥2.7 and 1.8 to 3.3 CFU/lungs, respectively) with the recreated 1,000-mg oral dose. This profile was also efficacious against S. aureus (mean log10 reduction, 1.9 to 2.4 CFU/abscess). There was a nonsignificant trend for improved efficacy against S. aureus with the 1,500-mg oral dose (mean log10 reduction, 2.4 to 3.1 CFU/abscess). These results demonstrate that the human oral 1,000- or 1,500-mg exposure profiles of GSK1322322 recreated in rats were effective against representative community-associated pathogens and supported selection of the 1,500-mg oral dose given every 12 h for a phase II clinical skin infection study. Furthermore, this work exemplifies how the testing of recreated human pharmacokinetic profiles can be incorporated into the development process and serve as an aid for selecting optimal dosing regimens prior to conducting large-scale clinical studies.