Biopharmaceutics of Nanomedicines Revisited: COVID and Beyond
Today’s world is much more complex than we could imagine 40 years ago. Drug products are developed by international research teams and involve biotechnological and conventional production processes. During the COVID19 pandemic, we all learned about the vulnerability of the pharmaceutical supply chains, providing raw materials for the manufacture of drug products. The first lockdown in Wuhan resulted in shortages in medical supplies globally.
Few technologies have challenged both, the scientific community and the pharmaceutical industry, in a way nanomedicines did. These tiny dosage forms are difficult to manufacture, difficult to characterize, and, in consequence, difficult to understand. More than for other drug products, the raw materials play a key role in their therapeutic performance.
In recent years, evolving technologies such as microfluidics or nanomilling have made it much easier to manufacture nanocarrier delivery systems at high quality. Still, we do not fully understand how their properties affect the in vivo performance. While the technological progress provided us with suitable tools to define narrow specification ranges in production, for example with regards to particle size and size distribution, the interactions with the in vivo system are hardly predictable [1].
Our current research is focusing on the biopharmaceutical behavior of nanomedicines. This is closely related to the development of in vitro performance assays elucidating their interactions with the physiological microenvironment [2]. Rather than investigating cellular interactions at high resolution, we aim at a direct correlation of kinetic in vitro measurements to the in vivo data. Similarities and differences in pharmacokinetics between drug formulations, subjects, or patient populations sometimes are the missing puzzle piece that indicates a particular mechanistic relationship, for example between the drug release and the clearence of nanomedicines. They provide evidence for physicochemical features that quantitatively affect their bioavailability.
Performance testing
Traditionally, the kinetics of drug dissolution plays an eminent role in the in vivo performance of peroral dosage forms. It was successfully correlated to pharmacokinetics and bioavailability. Nevertheless, the scientific term “in vitro performance assay” signifies the broadly inclusive approach that is often required in drug delivery. Although dissolution remains one of the most important mechanisms affecting the performance of nanomedicines, it is not the only process that has to be taken into consideration. The presence of nanomedicines in blood circulation enables multiple interactions with the living system all of which can contribute to the pharmacological and toxicological effects.
Focusing on nanomedicines for intravenous administration, the absence of an initial permeation step makes the drug release the sole mechanism responsible for the availability of the free drug in the blood plasma. This indicates how much we depend on the clinical data used in the evaluation of the therapeutic performance.
While most cellular interactions occur in the “invisible space” of the cellular fraction [3], the plasma concentration-time profile provides an indirect measure of all processes eliminating the drug from blood circulation. To a certain extent, the release assay should reflect this change in the concentration gradient as well. Similar observations can be made for the subcutaneous route of administration where several overlapping transport mechanisms contribute to pharmacokinetics [4, 5].
Performance testing of nanomedicines requires assays with high sensitivity and, equally important, high selectivity for the particle population under investigation. Consequently, we evaluated several of the existing techniques with regards to their capability to discriminate between different nanoformulations [6, 7]. While dialysis membranes have pore sizes optimized for the separation of particle populations, the sensitivity widely depends on the membrane permeation rate. We developed a dynamic dialysis process that enables a meaningful time-resolved quantification of the drug release. In 2015, we filed a patent for the dispersion releaser (DR) technology (Figure 1), an adapter to be used with the standard dissolution apparatus II of the United States Pharmacopeia (USP) [8]. Since 2018, the PT-DR (Pharma Test, Hainburg, Germany) is commercially available.
Also, some of the more recent alternatives such as asymmetric flow field-flow fraction (AF4) hold great promise in the separation of particle populations at lowered shear forces. However, as for all kinetic measurements, release testing also requires short separation times without changing the release rate [2]. Therefore, a careful evaluation of the effect of the dilution and the overall separation time in the flow channel will be required. So far, this aspect has not been sufficiently addressed and the overwhelmingly positive responses in literature [9, 10] must be sustained by hard evidence. These aspects will be discussed in more detail in a stimuli article of the Nanomaterial Working Group of the USP expert panel on New Advancements in In-Vitro Performance Testing soon.
The DR technology was designed to control the balance between separation time and the shear forces applied to the formulation [2, 8]. The donor chamber, a “cage” in the center of the dissolution vessel, is agitated by a small paddle stirrer that accelerates the membrane transport [8]. The surrounding vessel represents the acceptor compartment and is stirred at the same rate. Although the membrane transport is significantly altered as compared to conventional dialysis methods [8], biorelevant studies still mandate the determination of the analytical error due to the impact of the membrane permeation rate [11, 12]. Mathematical models such as the four-step model were developed to carry out a more detailed analysis followed by the calculation of a normalized release rate [13].
Another aspect is the selectivity of the method for a particular size fraction. The release of intravenously administered nanomedicines is not followed by a permeation step before the drug becomes available in the blood plasma. Still it plays a major role with regards to the availability of the drug at the target site. Differences in tissue permeation widely depend on the changes, the drug molecule undergoes in the microenvironment of the carrier. This “gained permeability” can, for example, be influenced by the kinetics of the plasma protein binding [14]. This process, the direct transfer of drug molecules from the carrier to proteins, was termed the drug-protein transfer [15, 16].
Model-informed development
To understand the implications of the molecular changes on pharmacokinetics and bioavailability of nanomedicines, computational models can be applied [16]. Importantly, many of the assumptions made during the analysis of clinical data are based on experiences with drug molecules. Going back to the fundamentals of drug delivery, nanomedicines have been a game-changer blurring the line between drug discovery and formulation science [17]. While conventional therapies change the liberation and absorption of the drug, nanomedicines impact distribution, metabolism, and elimination due to the presence of a second non-degradable and non-excretable fraction of the drug. This encapsulated fraction can improve the selectivity of treatments, but also lead to new formulation-related side effects.
The paradigm shift associated with the clinical application of nanomedicines underlines their importance for the future of drug delivery. While there seems to be an imbalance between the tremendous research activities in this area and the niche markets addressed by nanomaterial-related drug products [1, 18], the huge medical need for more personalized treatments and enhanced selectivity continues to raise the same scientific questions and challenges.
One of the challenges lies, for example, in the limitations of the existing in vivo data. During the preclinical and clinical studies, drug concentrations are determined after centrifugation of blood samples, separating the cellular fraction from the blood plasma. This separation step has a strong impact on the outcome of the pharmacokinetic study. Accordingly, to enhance the resolution of the data, recent guidelines of the United States Food and Drug Administration (US-FDA) suggest the quantification of the encapsulated and the non-encapsulated fraction of the drug [13]. More recently, our group developed a smart algorithm to analyze the pharmacokinetic profiles of nanomedicines. The physiologically-based nanocarrier biopharmaceutics (PBNB) model offers a model-based deconvolution of the total plasma concentration into the encapsulated and the non-encapsulated fraction [3, 19]. The model allows retrospective analysis of studies from the 1990s and 2000s, expanding the existing knowledge base. It is an important starting point for model-informed decision-making in the development of nanomedicines.
In addition to the most common pharmacokinetic parameters, the targeting capability (Ftarget) and mean circulation time (MCT) of the carrier fraction are calculated. They provide a simple measure of the expected nanomaterial-related effects due to the new formulation approach.
Currently, nanomedicine experiences a ‘renaissance’ due to the challenges associated with the stability and delivery of RNA molecules. Some of the most successful vaccines developed in response to the COVID-19 pandemic use lipid nanoparticle technology. The methods applied in the manufacture of these particles are more than 10 years old and there is a long list of literature dealing with the protection of nucleic acids from early degradation [20-24]. Thinking beyond the current crisis, the influx of biotechnological drug products into the global healthcare market will continue and increase the demand for smart formulation approaches. Therefore, it is only a matter of time before the new applications will lead us back to the unresolved questions of the past decade of nanomedicine research.
Conclusion
Nanomedicine has widened the spectrum of drug delivery by altering the biodistribution of compounds. Although many hurdles have been limiting their commercial success, the concept of personalized medicine will confront us with similar challenges. Also, understanding the clinical performance of nanomedicines requires a change in perspective rather than knowledge extrapolation from drug discovery to formulation science. While the technological progress has gifted us with suitable tools to manufacture and analyze nanomedicines at high resolution, we rarely question the fundamental theories underlying drug development. With the next generation of nanomedicines, the extracellular vesicles, at the starting line, there is a certain risk of repeating the same mistakes over and over. Therefore, we have to challenge our prior knowledge and carefully analyze successes and failures of the past.
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