advanced pharmacology and toxicology

1. Advance Pharmacology & Toxicology
Evaluation seminar on
Direct Peptide Reactivity
Assay(DPRA)
Presented By: Ragini Singh
1st M.Pharm(Pharmacology)
PES College of Pharmacy
08.12.2018

2. Introduction
Direct Peptide Reactivity Assay (DPRA)
 The Direct Peptide Reactivity Assay (DPRA) is an in
chemico method used to predict epidermal protein
binding.
 Binding of epidermal proteins is the molecular initiating
event on the Adverse Outcome Pathway. The DPRA uses
HPLC to measure the depletion of synthetic peptides in
solution following exposure to test chemicals.
 The DPRA test allows for a yes/no answer in terms of
sensitization potential and further more allows
quantification of a chemical’s reactivity.

3. Objectives
• The DPRA is a chemistry-based assay exploiting the fact that
most chemical allergens have electrophilic properties and are
therefore able to react with the nucleophilic side chains of
amino acids to form covalent bonds.
• The underlying rationale of the assay is that if a chemical is
capable of reacting with proteins then it has the potential to act
as a sensitizer.
• To find out the endpoint in the assay by getting the percentage
depletion over time of two synthetic peptides (containing
respectively a cysteine and a lysine amino acid).
• The percentage of peptide depletion is calculated by High
Performance Liquid Chromatography using ultra-violet
detection.

4. Purpose
• Due to the complexity of the mechanisms underlying skin
sensitisation, it is likely that information from different methods (in
silico, in chemico, in vitro) is needed to reduce or replace the need
for animal testing, both for hazard identification and potency
characterisation purposes.
• The DPRA is a reliable test method that provides information on
peptide reactivity, which is considered to be the molecular initiating
event of skin sensitisation .
• Therefore, peptide depletion values generated with the DPRA could
be used to support read-across from chemical analogues or
combined with information from other non-animal methods in the
context of a Weight of Evidence (WoE) approach or Integrated
Testing Strategy (ITS).

5. Structural View

6. DPRA -procedure
Stock solutions of cysteine and lysine containing synthetic peptides of >85% purity are
freshly prepared just prior to incubation with the test item. The final concentration of
the cysteine peptide is 0.667mM in pH7.5 phosphate buffer and the final concentration
of the lysine peptide is 0.667mM in pH10.2 ammonium acetate buffer.
Solubility of the test item in an appropriate solvent (acetonitrile) is assessed in advance
and stock solutions of 100mM concentration are prepared just before the test.
The DPRA measured the reaction of 10 reference chemicals of known sensitization
potential with synthetic peptides containing Cystine (Ac-RFAACAA-COOH) and
Lysine (Ac-RFAAKAA-COOH).

7. Contd.....
Peptide depletion is monitored by High Performance Liquid Chromatography (HPLC),
using a reversed-phase column, gradient elution and UV detection at 220nm.
A standard calibration curve is generated for both the cysteine and lysine peptides. Six
standards are prepared using serial dilutions of the 0.667mM stock solution, to cover
the final concentration range from 0.534mM to 0.0167mM. A blank of the dilution
buffer is also included and calibration curves must have an r2>0.99.
Cysteine and lysine peptide solutions are incubated in glass autosampler vials with the
test item at 1:10 and 1:50 ratio respectively. The reaction solution is left in the dark at
25±2.5°C for 24±2 hours before running the HPLC analysis.
The positive control is 100mM cinnamic aldehyde in acetonitrile. Reference controls
(containing only the peptide in the appropriate solvent) are also included and are used
to calculate the percentage peptide depletion for each test item. In addition, a co-
elution control (containing only the test item in the appropriate solvent) is included in
the HPLC run to detect possible co-elution of the test item with either the cysteine or
lysine peptide.

8. Contd...
% Peptide depletion = 100 – Peptide peak area in replicate depletion samples (x 100)
Mean Peptide peak area of reference control sample
The amount of remaining unreacted peptide was measured and peptide depletion
measured.
A range of acceptance criteria must be satisfied in order for the test to be considered
valid.
The percentage peptide depletion is determined by measuring the peak area and dividing
it by the mean peak area of the relevant reference controls

9. Work flow of DPRA

10. Advantages
• Quick results
• ECVAM validated protocol
• Cost efficient method for pre-screening and modifying
new formulations.
• The Direct Peptide Reactivity Assay was recently
validated by the European Center for the Validation of
Alternative Methods and was endorsed by their scientific
advisory group as a useful tool for early decision making
during product screening and as a component in a weight-
of-evidence approach or integrated testing strategy for
safety/hazard assessment.

11. Precautions
• In any case a negative DPRA result should be interpreted
with care, taking into consideration the possibility of false
negatives due to:
(1) Possible reactivity with amino acid residues other than
cysteine and lysine,
(2)The lack of metabolic capacity of the assay leading to
possible misclassification of pro-haptens.
(3) The uncertain capacity of the DPRA to correctly pick up
pre-haptens. For hazard assessment purposes, possible
uses of DPRA data in the context of a WoE or ITS have
been reported in several scientific publications (Ball et al.,
2011; Bauch et al., 2012).

12. References
• 1.Chin Lin Wong. “In vitro methods for hazard
assessment of industrial chemicals –
opportunities and challenges”. Front.
Pharmacol. 2015;18(10);338-9
• 2. Gerberick. “Direct Peptide Reactivity Assay
(DPRA) Method”. Tox. Sci. 2004;9(81);332-
43

13. Thank You