Pharmaceutical Outsourcing Q3 2023 - 19

ANALYTICAL TESTINGSECTION TITLE
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.
pharmoutsourcing.com | 19 | July/August/September 2023
http://www.pharmoutsourcing.com
Pharmaceutical Outsourcing Q3 2023

Table of Contents for the Digital Edition of Pharmaceutical Outsourcing Q3 2023
