Differentiating multichannel block on the guinea pig ECG: Use of Tpeak-Tend and J-Tpeak.

INTRODUCTION: The anaesthetised guinea pig is a well characterised assay for early assessment of drug effects on ventricular repolarisation and risk of Torsade de Pointes (TdP). We assessed whether a selective hERG blocker with known TdP risk could be differentiated from lower risk, balanced ion channel blockers in the guinea pig, using corrected QT (QTc) interval alongside novel electrocardiogram (ECG) biomarkers J-Tpeakc and Tpeak-Tend. Effects were compared with previous clinical investigations at similar plasma concentrations and with another index of TdP risk, the electromechanical window (EMW). METHODS: Twenty-two Dunkin Hartley guinea pigs anaesthetised with sodium pentobarbitone were instrumented for haemodynamic measurement and ECG recording. Three ascending doses of vehicle (n = 6), dofetilide (2, 6 or 20 µg/kg; n = 7), ranolazine (2, 6 or 20 mg/kg; n = 5) or verapamil (0.1, 0.3 or 1.0 mg/kg; n = 4) were administered intravenously. RESULTS: As reported in previous clinical studies, dofetilide induced dose-dependent increases in QTc interval, with increases in both J-TpeakC or Tpeak-Tend, while verapamil caused no significant increase in QTc interval, J-TpeakC or Tpeak-Tend. Ranolazine caused dose-dependent increases in QTc interval and corrected J-Tpeakc, but had no effect on Tpeak-Tend, which is in contrast to the effects reported in humans at similar concentrations. Only dofetilide caused a clear, dose-related decrease in the EMW. DISCUSSION: These findings suggest that measurements of J-Tpeakc and Tpeak-Tend in addition to QT interval, may help differentiate pure hERG channel blockers with high risk of TdP from lower risk, multichannel blockers.

177Lu-Lilotomab Satetraxetan Has the Potential to Counteract Resistance to Rituximab in Non-Hodgkin Lymphoma.

Patients with non-Hodgkin lymphoma (NHL) who are treated with rituximab may develop resistant disease, often associated with changes in expression of CD20. The next-generation ß-particle-emitting radioimmunoconjugate 177Lu-lilotomab-satetraxetan (Betalutin) was shown to up-regulate CD20 expression in different rituximab-sensitive NHL cell lines and to act synergistically with rituximab in a rituximab-sensitive NHL animal model. We hypothesized that 177Lu-lilotomab-satetraxetan may be used to reverse rituximab resistance in NHL. Methods: The rituximab-resistant Raji2R and the parental Raji cell lines were used. CD20 expression was measured by flow cytometry. Antibody-dependent cellular cytotoxicity (ADCC) was measured by a bioluminescence reporter assay. The efficacies of combined treatments with 177Lu-lilotomab-satetraxetan (150 or 350 MBq/kg) and rituximab (4 × 10 mg/kg) were compared with those of single agents or phosphate-buffered saline in a Raji2R-xenograft model. Cox regression and the Bliss independence model were used to assess synergism. Results: Rituximab binding in Raji2R cells was 36% ± 5% of that in the rituximab-sensitive Raji cells. 177Lu-lilotomab-satetraxetan treatment of Raji2R cells increased the binding to 53% ± 3% of the parental cell line. Rituximab ADCC induction in Raji2R cells was 20% ± 2% of that induced in Raji cells, whereas treatment with 177Lu-lilotomab-satetraxetan increased the ADCC induction to 30% ± 3% of that in Raji cells, representing a 50% increase (P < 0.05). The combination of rituximab with 350 MBq/kg 177Lu-lilotomab-satetraxetan synergistically suppressed Raji2R tumor growth in athymic Foxn1nu mice. Conclusion:177Lu-lilotomab-satetraxetan has the potential to reverse rituximab resistance; it can increase rituximab binding and ADCC activity in vitro and can synergistically improve antitumor efficacy in vivo.

Social-housing and use of double-decker cages in rat telemetry studies.

Rat telemetry is widely used for biomedical research purposes and is used routinely in early pre-clinical drug development to screen for the potential cardiovascular risk of candidate drugs. Historically, these studies have been conducted in individually housed conditions which can impact significantly on an animal's welfare. Here we present data from a survey of pharmaceutical companies and contract research organisations to define current industry practices relating to the housing of rats during telemetry studies and to expand and complement a similar project in non-rodents. Results of the survey showed that 75% of respondents socially house rats on non-recording days of telemetry studies, whereas on recording days only 46% of respondents socially house the animals. When social housing is used on rat telemetry studies, rats are usually housed with an unrecorded companion animal. We also present and compare data from a telemetry study in standard individually ventilated cages (IVCs) with a study using new double-decker IVCs, both conducted using a companion animal approach. Telemetry signals were successfully collected from the double-decker IVCs without a loss of signal quality whilst offering a more spacious environment that allowed the animals to exhibit natural behaviours including full upright posture. Cardiovascular responses following pharmacological intervention with verapamil were similar when assessed in the standard and double-decker cages. Power analysis was conducted on pooled data from the studies in socially housed animals with preliminary results showing the power of detection of drug-induced effects is equivalent to previously published data in individually housed rats. This illustrates that telemetry recordings can be made from rats in socially housed conditions within standard or larger double-decker cages for the for the collection of cardiovascular telemetry data.

Determination of the safety and efficacy of therapeutic neutralization of tumor necrosis factor-α (TNF-α) using AZD9773, an anti-TNF-α immune Fab, in murine CLP sepsis.

OBJECTIVE AND DESIGN: TNF-α neutralization is associated with increased mortality in mouse cecal ligation puncture (CLP) models. AZD9773 is an ovine polyclonal human TNF-α immune Fab, with pharmacological properties that differ from previously studied anti-TNF-α agents. We explored the safety and efficacy of therapeutically administered AZD9773 in mouse CLP sepsis. METHODS: A moderate/severe-grade CLP model resulting in 20-30 % 5-day survival and a mild-grade CLP model resulting in ~70 % 5-day survival were established in human TNF-α transgene/murine TNF null (Tg1278/-/-) mice. TREATMENT: Mice received saline resuscitation and imipenem administration every 12 h (0-72 h post-CLP). AZD9773 (or DigiFab control) was dosed 24, 36, 48 and 60 h post-CLP. RESULTS: Therapeutic dosing of AZD9773 in moderate/severe-grade CLP resulted in significantly increased survival (>70 %) compared with DigiFab (27 %, P < 0.05). Therapeutic dosing of AZD9773 in mild-grade CLP did not significantly affect survival outcome compared with DigiFab or imipenem alone (~60-70 % survival). CONCLUSIONS: These data demonstrate that TNF-α neutralization can improve survival in moderate/severe CLP sepsis. TNF-α suppression in mild-grade models was not associated with survival benefit and did not increase 5-day mortality. These findings suggest that therapeutic benefit following TNF-α attenuation in models of sepsis may depend on model severity.

Characterization of the complex formed between a potent neutralizing ovine-derived polyclonal anti-TNFα Fab fragment and human TNFα.

TNFα (tumour necrosis factor α) is an early mediator in the systemic inflammatory response to infection and is therefore a therapeutic target in sepsis. AZD9773 is an ovine-derived, polyclonal anti-TNFα Fab fragment derived from a pool of serum and currently being developed as a treatment for severe sepsis and septic shock. In the present study, we show that although AZD9773 has a modest affinity for TNFα in a binding assay, the Ki in a cell-based assay is approximately four orders of magnitude lower. We show using SEC (size exclusion chromatography) that the maximum size of the complex between AZD9773 and TNFα is consistent with approximately 12 Fabs binding to one TNFα trimer. A number of approaches were taken to map the epitopes recognized by AZD9773. These revealed that a number of different regions on TNFα are involved in binding to the polyclonal Fab. The data suggest that there are probably three epitopes per monomer that are responsible for most of the inhibition by AZD9773 and that all three can be occupied at the same time in the complex. We conclude that AZD9773 is clearly demonstrated to bind to multiple epitopes on TNFα and suggest that the polyclonal nature may account, at least in part, for the very high potency observed in cell-based assays.

In search of preclinical robustness.

Systematic engagement of statisticians in preclinical research could help address the weaknesses that are undermining the likelihood of subsequent success in drug discovery and development.