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Research ArticleArticle

Plasma and Liver Protein Binding of N-Acetylgalactosamine–Conjugated Small Interfering RNA

Sara C. Humphreys, Mai B. Thayer, Julie M. Lade, Bin Wu, Kelvin Sham, Babak Basiri, Yue Hao, Xin Huang, Richard Smith and Brooke M. Rock
Drug Metabolism and Disposition October 2019, 47 (10) 1174-1182; DOI: https://doi.org/10.1124/dmd.119.086967
Sara C. Humphreys
Pharmacokinetics and Drug Metabolism Department, Amgen Research, South San Francisco, California (S.C.H., M.B.T., J.M.L., B.B., R.S., B.M.R.); Hybrid Modality Engineering Department, Amgen Research, Thousand Oaks, California (B.W., K.S.); and Molecular Engineering Department, Amgen Research, Cambridge, Massachusetts (Y.H., X.H.)
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  • ORCID record for Sara C. Humphreys
Mai B. Thayer
Pharmacokinetics and Drug Metabolism Department, Amgen Research, South San Francisco, California (S.C.H., M.B.T., J.M.L., B.B., R.S., B.M.R.); Hybrid Modality Engineering Department, Amgen Research, Thousand Oaks, California (B.W., K.S.); and Molecular Engineering Department, Amgen Research, Cambridge, Massachusetts (Y.H., X.H.)
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Julie M. Lade
Pharmacokinetics and Drug Metabolism Department, Amgen Research, South San Francisco, California (S.C.H., M.B.T., J.M.L., B.B., R.S., B.M.R.); Hybrid Modality Engineering Department, Amgen Research, Thousand Oaks, California (B.W., K.S.); and Molecular Engineering Department, Amgen Research, Cambridge, Massachusetts (Y.H., X.H.)
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Bin Wu
Pharmacokinetics and Drug Metabolism Department, Amgen Research, South San Francisco, California (S.C.H., M.B.T., J.M.L., B.B., R.S., B.M.R.); Hybrid Modality Engineering Department, Amgen Research, Thousand Oaks, California (B.W., K.S.); and Molecular Engineering Department, Amgen Research, Cambridge, Massachusetts (Y.H., X.H.)
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Kelvin Sham
Pharmacokinetics and Drug Metabolism Department, Amgen Research, South San Francisco, California (S.C.H., M.B.T., J.M.L., B.B., R.S., B.M.R.); Hybrid Modality Engineering Department, Amgen Research, Thousand Oaks, California (B.W., K.S.); and Molecular Engineering Department, Amgen Research, Cambridge, Massachusetts (Y.H., X.H.)
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Babak Basiri
Pharmacokinetics and Drug Metabolism Department, Amgen Research, South San Francisco, California (S.C.H., M.B.T., J.M.L., B.B., R.S., B.M.R.); Hybrid Modality Engineering Department, Amgen Research, Thousand Oaks, California (B.W., K.S.); and Molecular Engineering Department, Amgen Research, Cambridge, Massachusetts (Y.H., X.H.)
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Yue Hao
Pharmacokinetics and Drug Metabolism Department, Amgen Research, South San Francisco, California (S.C.H., M.B.T., J.M.L., B.B., R.S., B.M.R.); Hybrid Modality Engineering Department, Amgen Research, Thousand Oaks, California (B.W., K.S.); and Molecular Engineering Department, Amgen Research, Cambridge, Massachusetts (Y.H., X.H.)
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Xin Huang
Pharmacokinetics and Drug Metabolism Department, Amgen Research, South San Francisco, California (S.C.H., M.B.T., J.M.L., B.B., R.S., B.M.R.); Hybrid Modality Engineering Department, Amgen Research, Thousand Oaks, California (B.W., K.S.); and Molecular Engineering Department, Amgen Research, Cambridge, Massachusetts (Y.H., X.H.)
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Richard Smith
Pharmacokinetics and Drug Metabolism Department, Amgen Research, South San Francisco, California (S.C.H., M.B.T., J.M.L., B.B., R.S., B.M.R.); Hybrid Modality Engineering Department, Amgen Research, Thousand Oaks, California (B.W., K.S.); and Molecular Engineering Department, Amgen Research, Cambridge, Massachusetts (Y.H., X.H.)
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Brooke M. Rock
Pharmacokinetics and Drug Metabolism Department, Amgen Research, South San Francisco, California (S.C.H., M.B.T., J.M.L., B.B., R.S., B.M.R.); Hybrid Modality Engineering Department, Amgen Research, Thousand Oaks, California (B.W., K.S.); and Molecular Engineering Department, Amgen Research, Cambridge, Massachusetts (Y.H., X.H.)
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    Fig. 1.

    Representative time to equilibrium of siRNA binding to total human plasma. Reference tip-subtracted sensorgrams depicting a titration of total human plasma interacting with biotinylated siRNA on streptavidin tips. Top plasma concentration is 3.3% (v/v) in buffer A followed by a 1:2 dilution series.

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    Fig. 2.

    (A) Workflow of determination of siRNA fu via ultrafiltration. Step 1: pretreat filter with detergent-containing buffer [we found that PBST (Table 1) and PBS+CHAPS (Supplemental Fig. 2) provided good recovery with a 50 kDa MWCO filter]. Step 2: add pre-equilibrated siRNA-spiked matrix into donor compartment of filter and centrifuge. Step 3: collect flow-through and quantify siRNA fu. (B) Depiction of siRNA-X Rh based on the crystal structure.

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    Fig. 3.

    PPB and liver protein binding of siRNA-X. (A) Cross-species comparison of fu,plasma across a range of therapeutically relevant siRNA concentrations. There was a significant increase in fu,plasma with concentration (P < 0.01) that was not dependent on species (determined by two-way ANOVA, GraphPad Prism). (B) siRNA-X fu,liver across a range of therapeutically relevant concentrations. Plasma measurements were performed in triplicate; liver measurements were performed in duplicate.

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    Fig. 4.

    Effect of chemical modifications on fu,plasma at 1 μM siRNA concentration. (A) Constructs tested with the sense strand depicted at the top (5′-3′) and the complementary antisense strand at the bottom. The RNA base sequence was constant across constructs, with only the conjugated ligand(s) (GalNAc and biotin), the ribose (2′-OMe, 2′-F), and the backbone (PS or phosphodiester) changing. (B) fu,plasma for each of the constructs in plasma; siRNA-X was also measured in serum. Results were compared using an ordinary one-way ANOVA with multiple comparisons in GraphPad Prism. Significant differences between the test article, siRNA-X in plasma, and the other constructs are reported as *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001.

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    Fig. 5.

    BLI sensorgrams of positive screening hits for select human plasma proteins binding to biotinylated siRNA-X ± GalNAc. A side-by-side comparison of anti-siRNA pAb (positive control), α-2-macroglobulin, α-thrombin, fibrinogen, and fibronectin binding to bn-siRNA-X with (left column) or without (right column) GalNAc conjugation. Titrations were 1:2 dilutions with a top concentration of 0.5 μM (except pAb = 0.2 μM).

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    Fig. 6.

    The relationship between Rh and MW for globular proteins does not hold for the dsRNA linear polymer according to Rh prediction based on dsRNA helical rise and the “siRNA rigid rod” assumption. siRNA is marked in red. GalNAc was not included in the Rh calculation. Linear regression was performed on the protein subset to estimation of the MW for a protein with an equivalent Rh to siRNA. The equation of the line obtained (with siRNA omitted) was y = 0.03509x + 1.233, and this was used to calculate the siRNA-protein MW equivalence value of 48 kDa (GraphPad Prism; calculations provided in Supplemental Calculations 2).

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    TABLE 1

    Comparison of percentage of siRNA-X recovery using different fu isolation techniques

    fu Isolation MethodMWCO% Recovery
    kDa
    Ultracentrifugationn/a0.0062 ± 0.0003
    Equilibrium dialysis205.53 ± 7.13
    Ultrafiltration3024.7 ± 1.40
    Ultrafiltration5092.4 ± 11.6
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    TABLE 2

    Validation of 50 kDa MWMCO ultrafiltration method for determination of fu,plasma and fu,liver using well characterized small molecules

    SMMatrix (Human)fu (Measured)fu (Reference)Reference Method
    AntipyrinePlasma0.867 ± 0.1100.9Ultracentrifugation
    TimololPlasma0.347 ± 0.0740.4Ultracentrifugation
    WarfarinPlasma0.043 ± 0.0070.01aUltracentrifugation
    RosuvastatinLiver homogenate0.14 ± 0.030.23bEquilibrium dialysis
    • SM, small molecule.

    • ↵a O’Reilly (1972).

    • ↵b Pfeifer et al. (2013); Yoshikado et al. (2017).

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    • Supplemental Data -

      Supplemental methods and 6 figures. 

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Drug Metabolism and Disposition: 47 (10)
Drug Metabolism and Disposition
Vol. 47, Issue 10
1 Oct 2019
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Research ArticleArticle

Plasma and Liver Protein Binding of GalNAc-Conjugated siRNA

Sara C. Humphreys, Mai B. Thayer, Julie M. Lade, Bin Wu, Kelvin Sham, Babak Basiri, Yue Hao, Xin Huang, Richard Smith and Brooke M. Rock
Drug Metabolism and Disposition October 1, 2019, 47 (10) 1174-1182; DOI: https://doi.org/10.1124/dmd.119.086967

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Research ArticleArticle

Plasma and Liver Protein Binding of GalNAc-Conjugated siRNA

Sara C. Humphreys, Mai B. Thayer, Julie M. Lade, Bin Wu, Kelvin Sham, Babak Basiri, Yue Hao, Xin Huang, Richard Smith and Brooke M. Rock
Drug Metabolism and Disposition October 1, 2019, 47 (10) 1174-1182; DOI: https://doi.org/10.1124/dmd.119.086967
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