In vitro Toxicology

In vitro Hemolysis

Background Information Protocol

• Drug-induced hemolysis is a relatively rare Test System but serious toxicity liability. Anticoagulant treated whole blood It occurs by two mechanisms1: Species • Toxic hemolysis — direct toxicity of the Human and rat (other species available) drug, its metabolite, or an excipient in the formulation. Incubation Time • Allergic hemolysis — toxicity caused by 45 min at 37ºC an immunological reaction in patients previously sensitised to a drug. Test Article Concentration 8 concentrations up to 250 µM or Cyprotex’s in vitro hemolysis testing is the highest concentration at which the ‘For excipients intended for designed to screen for toxic hemolysis as compound is soluble; two-fold dilutions injectable use, an in vitro recommended by the US FDA2. hemolysis study could be • Although the majority of normal Number of Replicates performed at the intended N=3 per concentration concentration for I.V. individuals may suffer toxic hemolysis at sufficiently high concentrations of administration (bolus and/ hemolytic drugs, for most drugs toxic Quality Controls or infusion) to determine the hemolysis involves lower doses given Vehicle (negative control) hemolytic potential.’ to individuals who are genetically Triton X-100 (positive control) predisposed to hemolysis1. FDA Guidance for Industry: Test Article Requirements Nonclinical Studies for the Safety 50 µL of 20 mM solution • The US FDA recommends that for Evaluation of Pharmaceutical excipients intended for injectible use, Analysis Method Excipients (May 2005) an in vitro hemolysis study should be Spectophotometry at 540 nm performed at the intended concentration for IV administration to test for hemolytic Data Delivery potential2. Degree of lysis at each concentration IC • The in vitro hemolysis assay evaluates 50 hemoglobin release in the plasma (as an indicator of red blood cell lysis) following test agent exposure.

To find out more [email protected] Hemoglobin released into plasma can be assessed as a measure of red blood cell lysis following drug/chemical exposure.

Table 1 Figure 1 Hemolysis by Control Compounds Hemolysis by Hemolysin in Human and Rabbit Erythrocytes

100%

Concentration 90% Compound (%, v/v) % Lysis 80%

70% Triton X-100 1% 70% 60% Triton X-100 0.1% 14%

50%

Triton X-100 0.01% 2% 40% % Lysis SDS 1% 82% 30%

SDS 0.1% 18% 20% 10% SDS 0.01% 7% 0% Saponin 1% 57% 1 10 100 1000 10000 Hemolysin Concentration (U/well) Saponin 0.1% 19%

Human RBCs Rabbit RBCs

Human erythrocytes were treated with agents known to Rabbit erythrocytes are much more susceptible to hemolysis by the bacterial cause hemolysis. The percent lysis increased with increasing exotoxin, hemolysin, than human erythrocytes, demonstrating the need to use concentrations of the agents. human erythrocytes in hemolysis experiments.

References 1 Dausset J and Contu L (1967) Ann Rev Med 18; 55-70 2 FDA Guidance for Industry - Nonclinical Studies for the Safety Evaluation of Pharmaceutical Excipients (May 2005) 3 Lubran MM (1989) Ann Lab Clin Sci 19; 114-21 4 Deibler GE (1959) J Appl Physiol 14; 133-6