Alexander R. Harmer

Scientific review and recommendations on preclinical cardiovascular safety evaluation of biologics.

Biological therapeutic agents (biologicals), such as monoclonal antibodies (mAbs), are increasingly important in the treatment of human disease, and many types of biologicals are in clinical development. During preclinical drug development, cardiovascular safety pharmacology studies are performed to assess cardiac safety in accord with the ICH S7A and S7B regulations that guide these studies. The question arises, however, whether or not it is appropriate to apply these guidelines, which were devised primarily to standardize small molecule drug testing, to the cardiovascular evaluation of biologicals. We examined the scientific literature and formed a consensus of scientific opinion to determine if there is a rational basis for conducting an in vitro hERG assay as part of routine preclinical cardiovascular safety testing for biologicals. We conclude that mAb therapeutics have very low potential to interact with the extracellular or intracellular (pore) domains on hERG channel and, therefore, are highly unlikely to inhibit hERG channel activity based on their targeted, specific binding properties. Furthermore, mAb are large molecules (>140,000 Da) that cannot cross plasma membranes and therefore would be unable to access and block the promiscuous inner pore of the hERG channel, in contrast with typical small molecule drugs. Consequently, we recommend that it is not appropriate to conduct an in vitro hERG assay as part of a preclinical strategy for assessing the heart rate corrected QT interval (QTc) prolongation risk of mAbs and other types of biologicals. It is more appropriate to assess QTc risk by integrating cardiovascular endpoints into repeat-dose general toxicology studies performed in an appropriate non-rodent species. These recommendations should help shape future regulatory strategy and discussions for the cardiovascular safety pharmacology testing of mAbs as well as other biologicals and provide guidance for the preclinical cardiovascular evaluation of such agents.

On the relationship between block of the cardiac Na+ channel and drug-induced prolongation of the QRS complex

BACKGROUND AND PURPOSE Inhibition of the human cardiac Na+ channel (hNav1.5) can prolong the QRS complex and has been associated with increased mortality in patients with underlying cardiovascular disease. The safety implications of blocking hNav1.5 channels suggest the need to test for this activity early in drug discovery in order to design out any potential liability. However, interpretation of hNav1.5 blocking potency requires knowledge of how hNav1.5 block translates into prolongation of the QRS complex.

Validation of an in vitro contractility assay using canine ventricular myocytes.

Measurement of cardiac contractility is a logical part of pre-clinical safety assessment in a drug discovery project, particularly if a risk has been identified or is suspected based on the primary- or non-target pharmacology. However, there are limited validated assays available that can be used to screen several compounds in order to identify and eliminate inotropic liability from a chemical series. We have therefore sought to develop an in vitro model with sufficient throughput for this purpose. Dog ventricular myocytes were isolated using a collagenase perfusion technique and placed in a perfused recording chamber on the stage of a microscope at ~36 °C. Myocytes were stimulated to contract at a pacing frequency of 1 Hz and a digital, cell geometry measurement system (IonOptix™) was used to measure sarcomere shortening in single myocytes. After perfusion with vehicle (0.1% DMSO), concentration-effect curves were constructed for each compound in 4-30 myocytes taken from 1 or 2 dog hearts. The validation test-set was 22 negative and 8 positive inotropes, and 21 inactive compounds, as defined by their effect in dog, cynolomolgous monkey or humans. By comparing the outcome of the assay to the known in vivo contractility effects, the assay sensitivity was 81%, specificity was 75%, and accuracy was 78%. With a throughput of 6-8 compounds/week from 1 cell isolation, this assay may be of value to drug discovery projects to screen for direct contractility effects and, if a hazard is identified, help identify inactive compounds.

Assessment of cardiomyocyte contraction in human-induced pluripotent stem cell-derived cardiomyocytes.

Functional changes to cardiomyocytes are a common cause of attrition in preclinical and clinical drug development. Current approaches to assess cardiomyocyte contractility in vitro are limited to low-throughput methods not amenable to early drug discovery. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) were used to assess their suitability to detect drug-induced changes in cardiomyocyte contraction. Application of field stimulation and measurement of cardiac contraction (IonOptix edge detection) and Ca(2+) transients confirmed hiPS-CMs to be a suitable model to investigate drug-induced changes in cardiomyocyte contractility. Using a live cell, fast kinetic fluorescent assay with a Ca(2+) sensitive dye to test 31 inotropic and 20 non-inotropic compounds in vivo, we report that hiPS-CMs provide a high-throughput experimental model to detect changes in cardiomyocyte contraction that is applicable to early drug discovery with a sensitivity and specificity of 87% and 70%, respectively. Moreover, our data provide evidence of the detection of this liability at therapeutically relevant concentrations with throughput amenable to influencing chemical design in drug discovery. Measurement of multiple parameters of the Ca(2+) transient in addition to the number of Ca(2+) transients offered no insight into the mechanism of cardiomyocyte contraction.

The contribution of VEGF signalling to fostamatinib‐induced blood pressure elevation

Fostamatinib is an inhibitor of spleen tyrosine kinase (TK). In patients, fostamatinib treatment was associated with increased BP. Some TK inhibitors cause BP elevation, by inhibiting the VEGF receptor 2 (VEGFR2). Here, we have assessed the mechanistic link between fostamatinib‐induced BP elevation and inhibition of VEGF signalling.

WITHDRAWN: Scientific review and recommendations on preclinical cardiovascular safety evaluation of biologics

Amgen, Inc, Department of Investigative Toxicology, Thousand Oaks, CA, 91320, USA Schering-Plough Research Institute, Kenilworth, NJ, 07033, USA F. Hoffmann-La Roche Ltd., Non-Clinical Safety, Basel, Switzerland Wyeth Research, Chazy, New York, 12921, USA Abbott Laboratories, Department of Integrative Pharmacology, Abbott Park, IL 60064, USA AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, SK10-4TG, U.K. GlaxoSmithKline, Safety Assessment, The Frythe, Welwyn, Herts, AL6 9AR, U.K. Novartis Pharmaceutic Corp., One Health Plaza, East Hanover, NJ, 07936, USA Merck Research Laboratories, Safety Assessment, West Point, PA, 19486, USA Lilly Research Laboratories, Global Safety Pharmacology, Greenfield, IN 46140, USA Johnson & Johnson PR&D, Global Preclinical Toxicology/Pathology, Raritan, NJ, 00869, USA

Quantifying the relationship between inhibition of VEGF receptor 2, drug‐induced blood pressure elevation and hypertension

Several anti‐angiogenic cancer drugs that inhibit VEGF receptor (VEGFR) signalling for efficacy are associated with a 15–60% incidence of hypertension. Tyrosine kinase inhibitors (TKIs) that have off‐target activity at VEGFR‐2 may also cause blood pressure elevation as an undesirable side effect. Therefore, the ability to translate VEGFR‐2 off‐target potency into blood pressure elevation would be useful in development of novel TKIs. Here, we have sought to quantify the relationship between VEGFR‐2 inhibition and blood pressure elevation for a range of kinase inhibitors.