Antivenoms: potency or median effective dose, which to use?

LETTER TO THE EDITOR

Antivenoms: potency or median effective dose, which to use?

Morais V^I; Ifran S^I; Berasain P^{I, II}; Massaldi H^{I, III}

^IDepartment of Biotechnology and Production, Hygiene Institute, School of Medicine, University of the Republic, Montevideo, Uruguay

^IIUnit of Parasite Biology, Hygiene Institute, School of Sciences, University of the Republic, Montevideo, Uruguay

^IIINational Council of Scientific and Technological Research, CONICET, Ministry of Science, Technology and Productive Innovation, Argentina

^{Correspondence to}

Dear Editor:

In the field of envenomations by poisonous animals, the ability of antivenoms to neutralize the lethal effects of venoms is estimated by a biological assay in which mice are inoculated with a range of venom/antivenom concentrations and the survival/death ratio is recorded. A statistical technique (e.g., Probit) is employed to estimate the amount of antivenom that protects 50% of the animals. This quantity is called median effective dose or effective dose 50 (ED₅₀) and is normally expressed in volume units (i.e., mL or μL). The ED₅₀ is used in an expression for the assessment of the potency (P) of the antivenom, as follows (1, 2):

where "LD₅₀" is the median lethal dose (mass of venom that kills 50% of mice), and "n" is the number of LD₅₀s used in the assay. "P" is the amount of venom, expressed in mass units or number of median lethal doses, that is completely neutralized per unit volume of antivenom (the expression "(n - 1) LD₅₀" is used instead of the total amount of venom, nLD₅₀, because at the endpoint of the neutralization assay, one LD₅₀ remains unneutralized and causes the death of 50% of mice). However, in the literature, it is very frequent to find that the same term, ED₅₀, is utilized to represent the neutralization capacity of antivenoms, typically as "the ratio of the total amount of venom used in the assay to the volume dose of antivenom that keeps alive 50% of the animals", in the same units as potency. The use of this term as a measure of the antivenom neutralization capacity is ambiguous and can generate severe errors, as shown below. To avoid confusion, let us call ER₅₀ ("median effective ratio") the ED₅₀ (mass/volume) magnitude so that:

Now, by opening the parentheses in equation [1] and substituting equation [2] we obtain:

which clearly shows that ER₅₀ is larger than P, precisely on the account of the contribution of the last term on the right side of the equation, which stems from the lethal dose that remains unneutralized at the endpoint of the assay. Equation [3] also indicates that P is a property of the antivenom that represents 100% protection. Therefore, in a set of assays where the venom challenge dose is changed, P can be expected to remain constant.

So, to show the behavior of ER₅₀ under these conditions, we have carried out simulated assays where when the potency is fixed at 1 LD₅₀/mL; the venom dose challenge, n, changes between 2 and 20 and the ED₅₀ and ER₅₀ are estimated from equations [1] and [2], respectively. The results of the assays are presented in Table 1 in increasing values of n. Notice that for low values of n (2 or 3), as frequently used in the literature, ER₅₀ can differ from P by as much as 100%. Thereafter, ER₅₀ decreases with increasing n and, for larger values of n, ER₅₀ and P tend to the same value. On the other hand, for n = 5, the value recommended in the Pharmacopoeia (1), there is still a 25% difference between P and ER₅₀.

In conclusion, it can be stated that ER₅₀ varies substantially with the venom challenge dose at lower values of n. On the other hand, since P is independent of n, and the calculation uses the same experimental information, it should be the parameter of choice to report neutralization results.

ACKNOWLEDGEMENTS

The support from ANII and CSIC are gratefully acknowledged.

Received: January 6, 2010

Accepted: January 6, 2010

Full paper published online: May 30, 2010

• 1. European Directorate for the Quality of Medicines (EDQM). European viper venom antiserum. In: European pharmacopoeia. Strasbourg: EDQM; 1997. p. 1712-3.
• 2. Araujo HP, Bourguignon SC, Boller MAA, Dias AASO, Lucas EPR, Santos IC, Delgado IF. Potency evaluation of antivenoms in Brazil: The national control laboratory experience between 2000 and 2006. Toxicon. 2008;51(4):502-14.

Publication Dates

• Publication in this collection
  07 June 2010
• Date of issue
  2010