For excipients intended for injectable use, an in vitro hemolysis study could be performed at the intended concentration for I.V. administration (bolus and/or infusion) to determine the hemolytic potential.
FDA Guidance for Industry: Nonclinical Studies for the Safety Evaluation of Pharmaceutical Excipients (May 2005)
|Test System||Anticoagulant treated whole blood|
|Species||Human and rat (other species available)|
|Incubation Time||45 min at 37° C|
|Test Article Concentration||8 concentrations up to 250 µM or the highest concentration at which the compound is soluble; two-fold dilutions|
|Number of Replicates||N=3 per concentration|
|Quality Controls||Vehicle (negative control)
Triton X-100 (positive control)
|Test Article Requirements||50 µL of 20 mM solution|
|Analysis Method||Spectrophotometry at 540 nm|
|Data Delivery||Degree of lysis at each concentration
|Compound||Concentration (%, v/v)||% Lysis|
Please provide an overview of the in vitro hemolysis assay.
Serial dilutions of the compound are mixed with human blood and incubated for 45 minutes at 37°C. The cells are centrifuged and the absorbance of the supernatant, which includes plasma and lysed erythrocytes, is measured. Percent lysis is calculated from a standard curve of lysed erythrocytes.
Why is it important to investigate toxic hemolysis by drugs?
Exposure to high concentrations of drugs can cause hemolysis even in normal adults, but in individuals who are genetically predisposed to hemolysis, this can occur at lower concentrations. Neutropenia, thrombocytopenia, hemolytic anemia, aplastic anemia, and macrocytic anemia are known to occur in some patients treated with drugs3. Drugs, their metabolites, or excipients used in formulation can cause toxic hemolysis; therefore the FDA recommends testing for hemolytic potential2.
What controls are included in the in vitro hemolysis assay?
Positive control is Triton X-100, a known hemolytic agent4, 1% v/v and 0.1% v/v. Negative control is the vehicle (typically 0.5% DMSO).
How does in vitro hemolysis relate to in vivo drug-induced toxic hemolysis?
Cyprotex’s in vitro toxic hemolysis assay is a sensitive and accurate method for predicting toxic hemolysis of a drug. However, it should be noted that hemolysis sometimes occurs when blood is drawn. This should be taken into account when performing the assay. Also, colored drugs that absorb light at the same wavelength at which the assay is measured (540 nm) can lead to false positives.
Learn more about toxicology in our popular Mechanisms of Drug-Induced Toxicity guide
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