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Plasma Protein Binding (PPB) evaluation for GalNAc-conjugated small interfering RNA (siRNA)

The plasma protein binding (PPB) evaluation for GalNAc-conjugated small interfering RNA (siRNA) is an important parameter in understanding its pharmacokinetics (PK), distribution, and clearance from the body. GalNAc-siRNAs are designed for targeted delivery to hepatocytes via the asialoglycoprotein receptor (ASGPR), but their interaction with plasma proteins can impact their bioavailability and efficacy.

Key Considerations in PPB Evaluation of GalNAc-siRNA

  1. Purpose of Plasma Protein Binding (PPB) Evaluation

    • Distribution: Determines the extent to which the siRNA is free in plasma vs. bound to proteins, impacting its availability for hepatocyte targeting.
    • Clearance: High protein binding often reduces renal clearance, prolonging circulation time, while low protein binding may lead to rapid clearance.
    • Efficacy and Safety: Protein binding influences the siRNA’s pharmacodynamic (PD) effect by modulating its free fraction available for receptor binding.

  2. Methodology for PPB Evaluation

    • Equilibrium Dialysis or Ultrafiltration: These techniques are used to assess the free (unbound) fraction of siRNA in plasma. Both methods are widely accepted, but each has distinct benefits:
      • Equilibrium Dialysis: This technique is highly effective for small molecules and siRNAs with lower protein binding. It involves separating free drug from bound drug across a semipermeable membrane.
      • Ultrafiltration: Useful for GalNAc-siRNAs if they exhibit high protein binding, as it allows quick separation of bound and unbound fractions.
    • Surface Plasmon Resonance (SPR) or Isothermal Titration Calorimetry (ITC): These are advanced methods used to directly characterize binding kinetics and affinity with specific plasma proteins, such as albumin.

  3. Common Plasma Proteins Binding GalNAc-siRNA

    • Albumin: Major protein in plasma, often binds GalNAc-conjugated molecules, which can stabilize and prolong circulation.
    • α-1 Acid Glycoprotein and Lipoproteins: Although they bind at lower affinities than albumin, they still contribute to overall PPB, especially in cases of variable protein levels (e.g., disease states).
    • Binding Affinity to ASGPR: Specific to GalNAc conjugates, which are designed for receptor-mediated endocytosis into hepatocytes, ensuring targeted liver delivery.

  4. Interpreting Results and Impact on PK/PD

    • High Protein Binding: When protein binding exceeds 95%, it suggests a smaller fraction of free siRNA in circulation, potentially reducing hepatocyte uptake and ASGPR interaction.
    • Moderate to Low Protein Binding: Greater free fraction may increase clearance and hepatocyte uptake, potentially enhancing therapeutic efficacy but reducing circulation time.
    • Target Organ Delivery: For GalNAc-siRNA, moderate PPB that allows selective ASGPR binding without excessive clearance is optimal for targeting hepatocytes.

  5. Challenges in PPB Evaluation of siRNA

    • Non-covalent Interactions: siRNA can have dynamic interactions with various plasma proteins that may vary based on formulation, conjugation, and physicochemical properties.
    • Stability: GalNAc-siRNAs must be stable in plasma for accurate PPB measurements, as degradation can skew results.
    • Method Sensitivity: As siRNAs are relatively large and complex molecules, sensitivity in detecting free vs. bound fractions is crucial to avoid underestimating protein binding.

Summary

The PPB evaluation of GalNAc-siRNAs is essential in understanding their distribution, clearance, and efficacy in hepatocyte targeting. Utilizing methods like equilibrium dialysis and ultrafiltration provides insights into binding dynamics, while interpreting binding data helps optimize dosing, therapeutic window, and targeted delivery to the liver 

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