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. 2017 May;9(9):693-705.
doi: 10.4155/bio-2017-0006. Epub 2017 May 10.

Development of bioanalytical assays for variegin, a peptide-based bivalent direct thrombin inhibitor

Norrapat Shih  1 , Leonardo Pinto de Carvalho  2 , Yie Hou Lee  3 , Mauricio Macario Rocha  4 , Adriano Henrique Pereira Barbosa  4 , José Marconi A de Sousa  4 , Antonio Carlos de C Carvalho  4 , R Manjunatha Kini  1 , Mark Y Chan  2
Affiliations

Development of bioanalytical assays for variegin, a peptide-based bivalent direct thrombin inhibitor

Norrapat Shih et al. Bioanalysis. 2017 May.

Abstract

Aim: Variegin is an anticoagulant peptide that will be tested in porcine models of percutaneous coronary intervention. We developed three bioanalytical assays for variegin quantitation and utilized these methods to evaluate pharmacokinetics of variegin in pigs. Results & methodology: The LC-MS/MS, thrombin amidolytic and modified thrombin time assays had a quantitation range of 21.6-5541.7, 10.8-5541.7 and 5.4-5541.7 nM in human plasma, respectively. The elimination half-lives obtained using the LC-MS/MS, modified thrombin time and thrombin amidolytic assays were 52.3 ± 4.4, 50.4 ± 5.9 and 67.7 ± 6.3 min, respectively.

Conclusion: We developed three bioanalytical assays for a novel direct thrombin inhibitor, variegin. The thrombin time assay is optimized for variegin quantitation during future porcine studies and clinical trials.

Keywords: LC–MS/MS; anticoagulant; bioanalytical assays; thrombin amidolytic assay; thrombin time.

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Conflict of interest statement

Financial & competing interests disclosure

This work was supported by a grant from the NMRC, Singapore (NMRC/CIRG/1399/2014). MY Chan receives salary support from an NMRC Clinician-Scientist Award (CSAINV15may011). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Figures

<b>Figure 1.</b>
Figure 1.. Development of an LC–MS/MS method for variegin quantitation.
(A) Sequence of variegin peptide. IS variegin peptide was labeled with heavy isotopes 13C6, 15N2 on Lys13 (emboldened and underlined). (B) The MS spectrum of variegin in positive ion mode. (C) MS/MS spectra of variegin in positive ion mode with CID at 903.1 m/z. (D) The MS spectrum of variegin-IS in positive ion mode. (E) MS/MS spectra of variegin-IS in positive ion mode with CID at 905.2 m/z. (F) SRM chromatogram of variegin and variegin-IS in plasma. (G) Calibration curve of variegin in plasma (n = 6) with internal standardization. Each data point represents mean ± SD. CID: Collision-induced dissociation; IS: Internal standard; SD: Standard deviation; Variegin-IS: Internal standard variegin peptide labeled with heavy isotopes 13C6, 15N2 on Lys13.
<b>Figure 2.</b>
Figure 2.. Modified thrombin time assay for variegin quantitation in plasma.
(A) Representative absorbance curve of plasma clotting reaction following excess thrombin addition. Thrombin time was taken as the time required to reach half of the maximal absorbance increase from baseline. Calibration curves for variegin plasma concentration using assays containing (B) 7.2, (C) 1.0, (D) 0.15 and (E) 0.037 NIH U/ml thrombin in assay (n = 6). Each data point represents mean ± SD. SD: Standard deviation.
<b>Figure 3.</b>
Figure 3.. Thrombin amidolytic quantitation assay for variegin in plasma.
(A) Representative absorbance curve for extracted-plasma sample with low versus high variegin concentration. Enzyme reaction was monitored at absorbance 405 nm. Calibration curves for variegin plasma concentration (n = 6): (B) enzyme assay with 3.6 NIH U/ml thrombin, 800 μM S2238 and 1 μl extracted-plasma sample; (C) enzyme assay with 0.45 NIH U/ml thrombin, 150 μM S2238 and 2 μl extracted-plasma sample; and (D) enzyme assay with 0.45 NIH U/ml thrombin, 150 μM S2238 and 16 μl extracted-plasma sample. Each data point represents mean ± SD. SD: Standard deviation.
<b>Figure 4.</b>
Figure 4.. Sample processing stability of variegin in citrated plasma and whole blood.
Samples were processed after specified incubation time and variegin quantified by LC–MS/MS (n = 6). (A) Variegin stability in citrated whole blood at room temperature up to 8 h. (B) Variegin stability in citrated plasma at room temperature with or without a protease inhibitor up to 24 h. (C) Variegin stability in lyophilized and nonlyophilized plasma at room temperature up to 1 month. (D) Variegin stability in citrated plasma at -80°C up to 2 months. Each bar represents mean ± SD. Data were tested using the Student’s t-test, where * p < 0.05 in comparison with 0 day or 0 h. SD: Standard deviation.
<b>Figure 5.</b>
Figure 5.. Plasma concentration–time profiles of variegin.
Variegin (1.11 mg/kg) was administered intravenously to pigs (n = 5) and variegin plasma concentration was quantified using (A) LC–MS/MS, (B) modified thrombin time and (C) thrombin amidolytic assays from pig plasma collected postadministration. Each data point represents mean ± SD. SD: Standard deviation.
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References

    1. Miesbach W, Seifried E. New direct oral anticoagulants current therapeutic options and treatment recommendations for bleeding complications. Thromb. Haemost. 2012;108(4):625–632. - PubMed
    1. van Es N, Bleker SM, Buller HR, Coppens M. New developments in parenteral anticoagulation for arterial and venous thromboembolism. Best Pract. Res. Clin. Haematol. 2013;26(2):203–213. - PubMed
    1. Lee GM, Arepally GM. Heparin-induced thrombocytopenia. Hematology Am. Soc. Hematol. Educ. Program. 2013:668–674. 2013. - PMC - PubMed
    1. Hirsh J, Anand SS, Halperin JL, Fuster V American Heart Association. Guide to anticoagulant therapy: heparin: a statement for healthcare professionals from the American Heart Association. Circulation. 2001;103(24):2994–3018. - PubMed
    1. Di Nisio M, Middeldorp S, Büller HR. Direct thrombin inhibitors. N. Engl. J. Med. 2005;353(10):1028–1040. - PubMed

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