Targeting ATMPs to the lung – it’s all in the delivery

Current challenges in developing drug delivery devices for ATMPs

Small molecule APIs have long had the lion’s share of the pulmonary and nasal drug delivery markets, but substantial efforts are being focused on ATMPs, particularly in the regenerative and personalized medicine spaces and oncology, albeit at an early stage of development. At the time of writing, there are no FDA-approved gene or cell therapies for the treatment of pulmonary disease, which speaks to the challenges in developing effective drug delivery products for these therapies.

For example, BNT116 is an mRNA vaccine, developed by BioNTech, to treat advanced or metastasized non-small cell lung cancer (NSCLC), currently undergoing clinical trial in the UK and the USA. The therapy works like the COVID-19 vaccine, conditioning the body’s own immune system to respond to biomarkers present in the membrane of NSCLC cells, but not in the membrane of non-malignant lung cells. BNT116 requires delivery into the circulatory system via needle injection for the vaccine to take effect. However, current thinking is that in many cases, the treatment of pulmonary disease will require a more targeted approach.

Physical challenges associated with ATMP delivery to respiratory pathways

Drug delivery to respiratory pathways is not trivial, especially for the complex formulations required by ATMPs. The physical barriers of the glycocalyx and mucosa serve to keep the pulmonary epithelium free of exogenous macromolecules, viruses and cells, precisely the species of which ATMPs are composed. The highly branching and high surface area structure of the lung also presents challenges to the transport and deposition of high-mass and volume drug products, especially where whole lung administration is advantageous.

To reach and deposit ATMPs in the deep lung where the drug can best access the circulatory system, aerosol particles/droplets must have an aerodynamic diameter of 1 μm–5 μm. Larger particles are lost by deposition in the oropharynx, while smaller particles may be exhaled before they can reach their target site. The quality of aerosols for pulmonary delivery is defined by their particle size distribution, including in vitro measures such as mass median aerodynamic diameter (MMAD), fine particle fraction (FPF) and fine particle dose. Aerosol drug delivery device and formulation developers must optimize for these metrics to effectively overcome factors affecting drug delivery to the lungs and treat disease by the inhalation route.

Compounding these challenges are the complex formulations that are required for ATMPs. Variable concentrations of stabilizers, surfactants and pH required to maintain the integrity of ATMP solutions can significantly affect the physicochemical properties of those solutions, leading to differences in viscosity, flow behavior or temperature sensitivity.

These are not insurmountable challenges, but ATMP delivery requires a holistic approach, considering the drug product, formulation and delivery device together.

Delivery vectors for gene therapies

Gene therapies treat disease by modifying, correcting or supplementing defective genes through editing or replacement. Free-floating oligonucleotides in the body are quickly broken down and reabsorbed, so genetic material must be delivered into the target cells via a delivery vector that protects it during transit and can effectively penetrate cell membranes. Two common approaches to address these challenges are the use of viral vectors or lipid nanoparticles (LNPs).

Viral vectors

Viruses can be considered delivery packages for genetic material, since they already contain the necessary molecular equipment for the penetration of cell membranes and delivery of their contents into the cytoplasm. There are many different virus types that have been used in preclinical and clinical studies, but adeno-associated viruses (AAVs) and lentiviruses (LVs) have become the most common due to their perceived low pathogenicity and broad host range.

Manufacturing viral therapies is complex. Viral genomes are modified to remove the autonomous replication genes and to incorporate the therapeutic genes of interest, then transfected into a bacterial cell line for replication. When enough viruses have been produced, they must be harvested and undergo several rounds of purification and quality control. These steps require specialized processes and equipment, which is part of the reason for the high cost of commercially available viral therapies.

Lipid nanoparticles

Lipid nanoparticles are a type of vesicle popular in both pharmaceutical academia and industry. They can be manufactured with physical processes rather than the complex bioprocessing required for viral vectors and are already FDA approved as carrier particles. They are composed of a small lipid bilayer membrane encapsulating hydrophilic contents (though hydrophobic drugs can also be loaded within the lipid membrane itself). LNPs can encapsulate a wide variety of cargos, exhibit good biocompatibility and can be modified with membrane antibodies to improve cell targeting.

The mechanism of delivery of the genetic payload into the cells from LNPs is more complex than through viral vectors and so requires different approaches to particle design. Vesicles are taken into the cell through endocytosis and encapsulated within endosomes. To release their cargo into the cytoplasm, lipid nanoparticles must be constructed of cationic lipids to disrupt the endosomal membrane sufficiently to allow their contents to escape. Challenges optimizing LNPs for release are significant and despite their many advantages, LNPs are currently considered to have a lower delivery efficiency than viral vectors.

Cell therapies and their use for treatment of damaged or diseased tissues

Cell therapies involve the administration of live cells to a patient to repair, replace or regenerate damaged or diseased tissues. They include stem cell therapies, engineered tissue therapies and immune cell therapies. The most successful cell therapy to date has been chimeric antigen receptor T-cell (CAR-T) therapy, a type of immunotherapy for the treatment of cancer. Here, T-cells are modified to express chimeric proteins composed of chains of immunoglobulins, which serve both as antigen receptor sites outside of the cell and T-cell activation moieties within the cell.

Cell therapies are broadly separated into two classes depending on the origin of the cells: autologous and allogeneic.

Autologous cell therapies

These cell therapies are produced using the patient’s own tissue. Cells are harvested, modified, expanded, then reintroduced to the patient. Autologous therapies are intrinsically personalized and thus carry a low risk of rejection, but care must be taken to ensure that malignant cells are not expanded and reintroduced back to the patient. Despite the advantages of autologous therapies, there are logistical considerations to account for. Currently, extracted cells must be transported to a manufacturing facility for modification before transport back to the clinic. Modern supply chains are effective at transporting delicate and expensive cargo, but any amount of time spent outside the manufacturing process, or the patient, presents a high risk to cell viability.

Allogeneic cell therapies

Allogeneic cell therapies are produced from a donor cell line external to the patient. They are ideal for use in applications where the patient’s own cells make them ineligible for transplant, such as if they suffer from blood cancer. Allogeneic therapies can be produced in bulk, cryopreserved and then shipped to clinics for administration, avoiding much of the logistical and manufacturing challenges that exist for autologous therapies. However, the risk of immunogenicity is larger than when administering autologous cell therapies. As with organ donation, patients and donors must be human leukocyte antigen (HLA) matched to reduce the probability of graft-versus-host disease (GvHD).

Drug delivery device development for solubilized advanced therapies

Drug delivery to the respiratory system is a mature industry with a long history of device development. The focus of the majority of drug delivery device developments to date has been to support the delivery of powdered or solubilized small molecule APIs. Ensuring maximum transmission to the patient and minimizing drug product lost within the device becomes crucially important, and so – soft nanoparticle suspensions and cells require additional considerations for effective delivery.

The following discussion will explore some of the devices being investigated for the delivery of solubilized advanced therapies. Lyophilization (freeze drying) powder is not applicable to all ATMPs and requires significantly different approaches so will be omitted.

Vibrating mesh nebulizers

Vibrating mesh nebulizers (VMN) are the most common choice for delivering solubilized sub-cellular ATMPs. An ultrasonic transducer drives a mesh plate in contact with the drug fluid, forcing the fluid through the mesh to generate a mist of small, respirable droplets. VMN are highly efficient at generating aerosols at low energies, giving narrow droplet size distributions that are tailorable by mesh scale and by vibration frequency.

However, VMNs can struggle to nebulize formulations with viscosities higher than 5 cP, limiting the inclusion of stabilizers and surfactants in those formulations. VMNs also exhibit high shear forces at the mesh during nebulization, which has the potential to damage vesicular particles. Shear stresses across a particle increase with particle size, which limits the diameter of vesicles that can be delivered through a vibrating mesh before a too-high proportion of the vectors begin to break down. This is a lesser concern for viral vectors as protein capsids are considerably more robust than lipid bilayers.

Jet nebulizers

Jet nebulizers (JN) typically work via the Venturi effect. A flow of compressed gas, typically air or oxygen, is accelerated through a nozzle above a liquid reservoir. The gas acceleration induces a pressure differential, which nebulizes the fluid from the reservoir. JNs are inexpensive to operate and maintain, generate minimal heating and can be used with fluid viscosities up to 15 cP.

Despite these benefits, JNs are considered a poor choice for ATMP delivery because they can suffer from poor droplet size distribution control and there is evidence that high shear forces in JNs can cause aggregation and size increases for vesicles, due to transient disruption of their structure during nebulization. The time required to deliver a therapy may also be longer than other nebulizer technologies. While this may be of lesser importance for the delivery of ATMPs than other medications, consideration of patient experience is always important to the success of a therapeutic product. Perhaps most importantly, high residual volumes remain in JN devices after use. This alone may preclude them from advanced therapy applications, which may carry a very high price point per therapy.

Ultrasonic nebulizers

Ultrasonic nebulizers (UN) use an ultrasonic transducer to cause cavitation within a formulation, generating an aerosol. They provide good droplet size distribution control based on the frequency of transduction, lower shear stress than VMN, and can atomize high volumes of formulations to a high flow rate.

Heating is a major consideration when using ultrasonic nebulizers, and while modern UNs may only heat a formulation by 10°C, it still may be enough to denature proteins and damage or reduce the efficacy of delicate molecules like RNA. UNs struggle to nebulize suspensions of greater than 1 μm diameter particles and have traditionally been considered unsuitable for delivery of such formulations. It was thought that UNs could similarly suffer when nebulizing nanosuspensions like LNPs, but advances in colloid and formulation science have led to improvements in particle stability under ultrasonication, and researchers are now reconsidering their use.

However, it is still difficult to make the case for using UNs over other nebulizers, since their delivery efficiency is much lower, but they do continue to present a significant advantage in cases where an advanced therapy must be delivered in high volume over a short period of time.

Soft mist inhalers

Soft mist inhalers (SMIs) use impinging jets to generate a low-velocity droplet mist and have been less explored as ATMP delivery devices compared to vibrating mesh and jet nebulizers. The low droplet velocity aids droplet transport to the deep lung while maintaining the appropriate droplet size distribution. SMIs also exhibit lower shear stresses and heating during nebulization than most nebulizer technologies, which may help maintain the integrity of vesicular drug products during delivery.

At the date of writing, there is only one commercially available SMI, the Respimat, developed by Boehringer Ingelheim, though several others are undergoing clinical trials.

The development of formulations to work with soft mist inhalers is complex. SMIs have a viscosity limit of 2-3 cP but operate best at 1-2 cP, a viscosity the same as, or lower than water. Foaming at the nozzle can also be an issue with SMIs and thus the balance of surfactants and formulation surface tension, together with viscosity, is a considerable challenge. Optimizations are being investigated to try to realize the benefits that SMIs may bring for advanced therapies, but these studies remain exploratory.

New approaches to delivering cell therapies to the lung

There are no devices currently demonstrated to be capable of delivering cells to the deep lung by inhalation. At a fundamental level, cells are too large to be delivered by most commercially available liquid inhalers. The droplet sizes emitted by liquid inhalers need to be 5 μm or less to ensure good airway transport, but even small T-lymphocytes are 8-10 μm in size and stem cells can be as large as 15-30 μm, all in the non-respirable range. The shear stresses present at nebulizer nozzles are a risk to the integrity of cells, particularly for vibrating mesh and jet nebulizers, which can cause ruptures in a cell membrane, destruction of the cells and expulsion of its contents, potentially causing an inflammatory response. Given these limitations, researchers have been investigating new approaches for delivering cell therapies beyond the first few generations of airways.

Bronchoscopic or intratracheal instillation

Bronchoscopic instillation uses a bronchoscope to bypass the upper airways, delivering therapeutic formulations with a syringe via the working channel. This is conceptually the simplest approach to delivering cell therapies beyond the upper airways, creating a bolus at the distal tip. An oxygen stream through the working channel can then push the formulation down an airway to administer beyond the distal tip of the bronchoscope, coating the airways as it does so. Cell therapy formulations must contain appropriate surfactant concentrations for this approach to promote adhesion to the airway walls for effective coverage.

However, the application of formulations through the working channel of a bronchoscope is difficult to control. Fluid in the lung may collect at its lowest point under gravity, both reducing the effect of the therapy at its intended site and increasing the risk of immunogenicity.

Endoscopic atomization

Endoscopic atomization attempts to combine the precise positioning of bronchoscopy with the even distribution of liquid nebulizers. The bronchoscope is distally modified with a nebulizing catheter, which sprays a target site with cell therapy solution. This avoids the 5 μm or less droplet size requirement as transport through the upper lung is accomplished by the bronchoscope and smaller droplet sizes promote airway wall adhesion compared to bronchoscopic instillation.

Obviously, these approaches are considerably more invasive than the use of oral inhalers and preclude self-administration. Many bronchoscopic procedures must be performed under general anesthesia in a clinic by medical professionals. Both of these technologies are also in relatively early development, especially compared to the maturity of liquid inhalers, and to the best of my knowledge, no devices for bronchoscopic instillation or endoscopic atomization of cell therapies have progressed to clinical trial, globally.

The future of ATMPs and drug delivery to treat respiratory diseases

Successfully developing an advanced therapy medicinal product for the treatment of respiratory disease will require a complex interplay between delivery vector, drug formulation and delivery device. This therapy area is in relative infancy compared to existing pulmonary drug delivery technologies and there remain many challenges to address before the benefits of these ATMPs can be fully realized. Overcoming these challenges will require careful integration of therapy, formulation and delivery device, but there are encouraging signs that promise to provide very effective outcomes for patients accessing advanced therapies.