The Role and Challenges of Plasma Protein Binding in Oligonucleotide Drug Development

Plasma protein binding refers to the degree drugs bind to proteins within the blood or plasma. In pharmacology, drugs exist in two forms: unbound, or "free," and the bound form. Unbound drug molecules are pharmacologically active, can diffuse across cell membranes, reach their site of action and be eliminated. In contrast, protein-bound drugs largely remain within the vascular compartment, providing a reservable pool of the drug that can be released into circulation as free drug concentrations decline.

The balance between bound and unbound drugs plays a crucial role in pharmacokinetics. These two types of drugs are in equilibrium at steady-state conditions in vivo. Only unbound drugs are usually available for pharmacological interactions, although some exceptions exist. But the extent of protein binding directly influences a drug's distribution volume and clearance rate, impacting its half-life and, ultimately, the dosing regimen. Put simply, the degree of protein binding can significantly impact a drug's efficacy.

Highly protein-bound drugs have a slower elimination rate, potentially leading to prolonged drug action. Lower protein binding, on the other hand, can result in rapid drug elimination, necessitating more frequent dosing to achieve therapeutic levels. Furthermore, alterations in protein binding due to drug-drug interactions or pathophysiological changes can lead to variations in free drug concentration, potentially resulting in sub-therapeutic effects or toxicity.

Oligonucleotides and Plasma Protein Binding

Oligonucleotides, or "oligos," are a popular class of novel therapeutics that present a unique challenge for protein binding. These short DNA or RNA molecules are designed to modulate gene expression, providing targeted treatments for various diseases, including genetic disorders, cancers and viral infections.

However, oligos' large size, negative charge, and the presence of modifications increase their complexity and propensity for nonspecific binding to plasma proteins.

Oligos' properties and anionic nature impede passive diffusion across cell membranes, often a crucial step for reaching intracellular targets. Their negative charge encourages binding to plasma proteins, specifically to positively charged domains, thereby increasing their plasma residence time and potentially limiting target accessibility.

Nonspecific binding represents another obstacle. Oligos can interact with a range of plasma proteins in a nonspecific manner, not dictated by the typical lock-and-key model of molecular interaction. This diversity of binding partners complicates the quantification of relevant, specific binding that directly impacts drug action.

Chemical modifications are often introduced to increase oligos' stability against nucleases or to enhance binding affinity to the target mRNA. However, these modifications can unpredictably alter binding affinity for plasma proteins, introducing another layer of complexity in determining free drug concentrations and, thus, drug efficacy and toxicity.

Modifying oligos to reduce these issues is an ongoing challenge. Chemically modified oligos, like locked nucleic acids or phosphorothioates, may have altered plasma protein binding properties, which can change their pharmacokinetic profiles and therapeutic effectiveness.

The inherent complexities of plasma protein binding in oligos also contribute to difficulties with experimental design and data interpretation. Oligos' intricate binding behavior often defies the assumptions of standard binding assays, potentially leading to inaccurate quantification. Moreover, potential interferences in assays and confounding factors in data analysis make interpretation challenging, complicating the translation of in vitro results to in vivo implications.

The Current State of Plasma Protein Binding Studies

In vitro ADME studies represent the cornerstone of modern drug development, providing essential data to inform pharmacokinetic and pharmacodynamic (PK/PD) modeling. These studies elucidate how a drug behaves within a biological system, influencing its efficacy and safety profile. But these studies face formidable challenges in plasma protein binding, particularly when it comes to oligos. These challenges include:

Technical Challenges

Technical challenges in plasma protein binding studies often arise from the inherent limitations of the assays used. The sensitivity of the assays can be a significant concern. For example, if the drug has a high affinity for proteins, only a tiny fraction will remain unbound. Detecting this small free fraction requires highly sensitive assays, which may not always be available or feasible.

Nonspecific binding represents another technical challenge. Drugs can bind to the materials used in the assay setup (e.g., plasticware), leading to an underestimation of the free drug concentration. Moreover, the diverse range of potential binding sites on a protein can cause drugs to bind nonspecifically, making it difficult to determine the true extent of specific, pharmacologically relevant binding.

Biological Challenges

Biological variables also present significant challenges in studying plasma protein binding. Inter-individual variability due to factors like age, gender, disease state, and genetic factors can influence protein levels and the binding capacity of drugs, leading to varied pharmacokinetic profiles. This makes it challenging to generalize results across different patient populations.

Furthermore, species differences in plasma protein composition and drug affinity to these proteins can complicate the extrapolation of data from preclinical animal studies to humans. For example, albumin is the principal protein responsible for a drug's binding in humans; its binding value may differ in mice since they may have different albumin structures, though the mouse albumin and human albumin are 72% homologous in primary sequence. This difference can lead to disparate binding profiles and requires careful preclinical data interpretation.

Finally, the complexity of plasma protein composition, including numerous binding proteins with multiple binding sites, further complicates the analysis. This complexity can lead to competitive or cooperative binding events, affecting the drug's binding profile in ways that are challenging to predict or interpret.

Interactional Challenges

Predicting drug-drug interactions also poses a significant challenge. If two drugs compete for the same protein binding site, one drug may displace the other, leading to an increased free fraction of the displaced drug. This can enhance its pharmacological effect and potentially cause toxicity. Therefore, studying these interactions in vitro becomes crucial to prevent adverse effects in patients.

And while in vitro studies can help predict potential drug-drug interactions due to displacement from protein binding sites, the clinical relevance of these interactions is often debated. Some argue that such interactions rarely lead to significant changes in free drug concentrations in vivo due to various compensatory mechanisms in the body.

Drug-drug interactions due to inhibition and induction of drug metabolizing enzymes and transporters are also important and challenging parts of drug discovery and development. Since protein binding values are critical parameters in drug interaction models, measuring fraction unbound to make accurate predictions is essential.

The gold standard for plasma protein binding studies is equilibrium dialysis. This method involves placing the drug in a chamber separated from a buffer solution by a semi-permeable membrane. Over time, the drug reaches equilibrium, diffusing across the membrane until the free-drug concentration is equal on both sides. The ratio of drug concentrations in the buffer and plasma compartments provides an estimate of the fraction of the unbound drug.

The drawbacks to equilibrium dialysis are that it is time-consuming, labor-intensive, and requires large amounts of test compound. It is also susceptible to nonspecific binding to apparatus materials, particularly for highly lipophilic compounds (including oligos).

Despite clear challenges, in vitro ADME studies remain critical to successful drug design and development. Their predictive power also helps avoid late-stage drug development failures, saving sponsors and developers time and resources. Overcoming these hurdles will be instrumental in improving ADME studies' predictability and applicability in the future.

New Methods for Studying Plasma Protein Binding

We know that high-throughput screening (HTS) in ADME studies allows for rapid, simultaneous testing of multiple oligo candidates under varying conditions. These tests enhance the efficiency and breadth of data collection, accelerating the early stages of drug development. However, the accuracy of HTS has been criticized due to the potential for false positives and negatives, not to mention the question of how well HTS reflects the complexities of the human body.

However, technological advancements and improved test designs have the potential to revolutionize in vitro ADME studies, paving the way for more accurate and comprehensive plasma protein binding analysis. For example, microscale thermophoresis (MST) is an alternative plasma protein binding analysis approach. This method tracks the movement of molecules in a temperature gradient, enabling precise quantification of protein-drug interactions without the need for chemical modifications to the oligo.

Liquid chromatography-mass spectrometry (LC-MS) is a highly sensitive and specific tool that can separate components and deliver data on each compound's molecular mass. This technology provides detailed insight into the protein binding assay, integrated with ultrafiltration or ultracentrifugation to separate unbound oligos from plasma.

Plasma Protein Binding in Oligos

Considering the unique challenges associated with studying plasma protein binding in oligos, specific methods tailored to these molecules are gaining traction, too. Advanced techniques include using capillary electrophoresis and ultrafiltration coupled with mass spectrometry. Electrophoretic mobility shift assays are also reported for the characterization of siRNA PPB. These methods are designed to handle oligos' size, charge, and binding characteristics, providing more reliable and accurate results.

Furthermore, computational modeling and simulation hold immense potential for studying plasma protein binding in oligos. By leveraging in silico models—including physiologically based pharmacokinetic (PBPK) models—we can simulate complex interactions between oligos and plasma proteins at a molecular level, bypassing some experimental constraints. These models incorporate diverse parameters from the drug's characteristics to patient physiology, facilitating the prediction of tissue drug concentrations over time. This allows us to anticipate binding sites, affinities, and potential off-target interactions, thus enriching our understanding of oligo pharmacokinetics and aiding in developing safer and more effective therapeutics.

Refining these technologies—and continuing to develop new ones— will undoubtedly provide deeper insights into plasma protein binding and help scientists overcome the inherent challenges in studying oligo drugs. Continued advancements in this area are essential to harness the full potential of oligo therapeutics and to develop more effective and safer drugs.

Regulatory Considerations in Plasma Protein Binding Studies

From a regulatory perspective, in vitro plasma protein binding studies are essential to the preclinical evaluation of a new drug. Regulatory agencies like the U.S. Food & Drug Administration (FDA) and the European Medicines Agency (EMA) have issued guidance on these studies.

Generally speaking, regulators expect detailed information about a drug's plasma protein binding properties, including:

• The percentage of drug bound;

• The identity of the primary binding proteins;

• The influence of disease states on drug binding.

That said, regulatory guidance often lacks specific protocols for conducting these studies, leading to variations in study design and interpretation across different labs. There's a need for harmonized guidelines and best practices for conducting plasma protein binding studies, particularly for novel therapeutics like oligos.

Protein binding is a multi-faceted process with significant drug disposition and efficacy implications. Given the increasing complexity of new therapeutic entities, rigorous in vitro studies coupled with in silico models and careful consideration of pathophysiological influences are necessary for predicting in vivo behavior accurately. Further refinement and harmonization of regulatory guidelines could clarify and promote consistency in these vital studies.

A Final Word

Plasma protein binding constitutes a critical component of drug pharmacokinetics, heavily influencing a drug's distribution, clearance rate, and efficacy. This is particularly pertinent for oligonucleotides, a class of therapeutics whose large size, negative charge and chemical modifications intensify the complexity of their interaction with plasma proteins—however, technological advancements and experimental design promise to overcome these hurdles.

MST and LC-MS have the potential to enhance our understanding of protein-drug interactions. At the same time, computational modeling and simulation may prove invaluable in exploring molecular-level interactions and anticipating drug behavior. These advancements and the harmonization of regulatory guidelines could pave the way toward developing safer, more effective oligo therapies and ultimately improved patient outcomes.

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Authors: Jie Wang

Committed to accelerating drug discovery and development, we offer a full range of discovery screening, preclinical development, clinical drug metabolism, and pharmacokinetic (DMPK) platforms and services. With research facilities in the United States (New Jersey) and China (Shanghai, Suzhou, Nanjing, and Nantong), 1,000+ scientists, and over fifteen years of experience in Investigational New Drug (IND) application, our DMPK team at WuXi AppTec are serving 1,500+ global clients, and have successfully supported 1,200+ IND applications.