3.3.5. Transwell® System

The Transwell® system (Figure 5D) consists of an upper (donor compartment) and lower (acceptor compartment) chambers separated by a membrane which is made out of either polyester (PET) or polycarbonate (PC) or collagen coated polytetrafluoroethylene (PTFE). Such systems are used in drug transport studies to characterize the permeability in apical-to-basolateral direction. Due to the lower volume of dissolution medium, the Transwell® system could provide more bio-relevant conditions in comparison to the Franz diffusion cell.

Arora et al., 2010 [20] developed a dissolution method for size classified respirable particles using a Transwell**®** system with limited volumes of stationary aqueous dissolution medium. Aerodynamically dispersed particles were collected on the filter membrane as reported by Davies and Feddah [21] from stage 4 (2.1–3.3 µm) and stage 2 (4.7–5.8 µm) of compendial Andersen cascade impactor. Then, the filter membranes with deposited aerosol powder were placed with particles facing in the downward direction on a semipermeable polyester membrane of the Transwell® insert. Immediately after these inserts were transferred into a receptor compartment containing 1.4 mL of dissolution medium, 0.04 mL of the dissolution medium was placed in the donor compartment to initiate the

particle dissolution. The system was then placed in an incubator maintained at 37 ◦C and an aliquot of 0.5 mL was collected (with replacement) from the receptor compartment at different time. At the end of the experiment, the donor compartment was thoroughly washed to recover the undissolved portion of the drug. 0.04 mL of the dissolution medium was placed in the donor compartment to initiate the particle dissolution. The system was then placed in an incubator maintained at 37 °C and an aliquot of 0.5 mL was collected (with replacement) from the receptor compartment at different time. At the end of the experiment, the donor compartment was thoroughly washed to recover the undissolved portion of the drug.

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 14 of 22

Rohrschneider et al., 2015 [26] evaluated the in vitro dissolution behaviour of orally inhalable products using a commercial Transwell® system with polycarbonate membrane and a modified Transwell® system in which polycarbonate membrane was replaced with a glass microfiber filter. In a modified system, incorporating a more permeable membrane, the drug transfer from donor to acceptor compartment was limited by the dissolution of the particles and not by the diffusion through the membrane. Rohrschneider et al., 2015 [26] evaluated the in vitro dissolution behaviour of orally inhalable products using a commercial Transwell® system with polycarbonate membrane and a modified Transwell® system in which polycarbonate membrane was replaced with a glass microfiber filter. In a modified system, incorporating a more permeable membrane, the drug transfer from donor to acceptor compartment was limited by the dissolution of the particles and not by the diffusion through the membrane.

#### 3.3.6. DissolvIt System 3.3.6. DissolvIt System

Dissolv*It* system (Figure 6) is a recent in vitro dissolution testing method which simultaneously determines the dissolution and absorption of a drug from respirable size dry powder particles [23]. The system consists of a mucus layer (50 µm thick 1.5% *w/v* polyethylene oxide in 0.1 M PBS) on a polycarbonate membrane to mimic the air-blood barrier in the tracheobronchial region of the lung with a blood simulant (0.1 M PBS containing 4% *w/v* albumin) flowing on the other side of the membrane. The fine particle dose (0.99 to 1.20 µg) of respirable size particles was collected on glass coverslips using the PreciseInhale system as discussed in Section 3.2.4. The dissolution behavior of respirable size particles of budesonide and fluticasone propionate was studied by simultaneous observation of particle disappearance under microscope and quantification of drug in the perfusate on the vasular side of the membrane. This new system, in which the blood simulant buffer pumped in singlepass mode through the dissolution cell, enables the generation of in vitro dissolution/absorption curves of drugs from inhaled dry powders. Dissolv*It* system (Figure 6) is a recent in vitro dissolution testing method which simultaneously determines the dissolution and absorption of a drug from respirable size dry powder particles [23]. The system consists of a mucus layer (50 μm thick 1.5% *w/v* polyethylene oxide in 0.1 M PBS) on a polycarbonate membrane to mimic the air-blood barrier in the tracheobronchial region of the lung with a blood simulant (0.1 M PBS containing 4% *w/v* albumin) flowing on the other side of the membrane. The fine particle dose (0.99 to 1.20 μg) of respirable size particles was collected on glass coverslips using the PreciseInhale system as discussed in Section 3.2.4. The dissolution behavior of respirable size particles of budesonide and fluticasone propionate was studied by simultaneous observation of particle disappearance under microscope and quantification of drug in the perfusate on the vasular side of the membrane. This new system, in which the blood simulant buffer pumped in singlepass mode through the dissolution cell, enables the generation of in vitro dissolution/absorption curves of drugs from inhaled dry powders.

**Figure 6.** Schematic diagram of DissolvIt® system. Reproduced with permission from Börjel et al., 2014 [59], Respiratory Drug Delivery 2014, Virginia Commonwealth University. **Figure 6.** Schematic diagram of DissolvIt® system. Reproduced with permission from Börjel et al., 2014 [59], Respiratory Drug Delivery 2014, Virginia Commonwealth University.

In a recent study, Dissolv*It* system was used to assess the impact of dissolution medium on dissolution of fluticasone proprionate aerosol particles. A synthetic simulated lung lining fluid, 1.5% poly(ethylene oxide) + 0.4% L-alphaphosphatidyl choline and In a recent study, Dissolv*It* system was used to assess the impact of dissolution medium on dissolution of fluticasone proprionate aerosol particles. A synthetic simulated lung lining fluid, 1.5% poly(ethylene oxide) + 0.4% L-alphaphosphatidyl choline and Survanta were three different media used in the Dissolv*It* chamber. It was illustrated that biorelevant dissolution studies can generate input parameters for physiologically based pharmacokinetic modeling of inhaled drug products [87].

#### 3.3.7. Custom Made Flow Perfusion Cell

Eedara et al. [58] custom made a flow perfusion cell which resembles an air-blood perfusion model to evaluate the dissolution behaviour of respirable size particles. The flow perfusion cell was connected to a syringe pump (100 DM syringe pump, Teledyne ISCO, Lincoln, NE, USA) to collecte the perfusate and an optical microscope equipped with a digital camera (OPTIKA SRL, Ponteranica BG, Italy) to capture the images of respirable size particle dissolution [18,58,62,62]. Using this apparatus, the dissolution behaviours of fine particle doses (collected using mTSI) of moxifloxacin and ethionamide in 25 µL of mucus simulant were evaluated. The respirable size particles of moxifloxacin dissolved quickly (<30 min) compared to the ethionamide.

Similarly, Saha et al. conducted in vitro experiments using custom made flow perfusion cell and showed that ~68% of ivermectin got permeated in 30 h from dry powder formulation. The dissolution medium was polyethylene oxide (1.5% *w*/*w*) + Curosurf® (0.4% *w*/*w*) in phosphate-buffered saline (PBS) and Tween 80 (0.2% *w*/*v*) in PBS was perfusate. 50 µL of the dissolution medium was loaded on the apparatus and the flow rate of the perfusate was fixed at 0.05 mL/min [88].

A major drawback of the dialysis bag, Franz-type diffusion cell, Transwell®, Dissolv*It*® and custom made flow perfusion cell methods is that the mass of the drug released into the donor compartment is limited. The advantages and disadvantages/limitations of all the above methods are summarized in Table 3. Even though various dissolution apparatus has been developed for inhaled dry powder particles, maintaining very limited volumes of the dissolution media to simulate the lung conditions is still a challenge. Therefore, the development of a standardized in vitro dissolution method for dry powder particles is still an interesting topic to research.


**Table 3.** Advantages and disadvantages/limitations of the apparatus used to evaluate the dissolution behaviour of the inhaled products. Reproduced with permissions from Eedara et al., 2019 [58], Elsevier.


**Table 3.** *Cont*.

It has always been fascinating to explore the interactions of inhaled drugs and components of RTLF that ultimately affect their dissolution and absorption in the lungs. For instance, Langmuir monolayer technique enables the formation of a lipid film on the water subphase and facilitates characterization of lipid–water, lipid–lipid or lipid–drug interactions [89]. However, understanding the interactions of inhaled drug molecules and RTLF components is out of the scope of this current review.

#### **4. Models for Pulmonary Drug Absorption**

In vitro, ex vivo and in vivo models are commonly used to study absorption of inhaled drug particles. Table 4 summarizes the various models used to study pulmonary drug absorption.

**Table 4.** Summary of various models for pulmonary drug absorption.


In vitro air-to-blood barrier is reconstructed using cell models in the Transwell or Snapwell system under cell culture [96]. Table 5 summarizes different types of cells used for in vitro lung barrier models. Stem cell-derived lung epithelial cells and "lung-on-a-chip" models have grabbed the interest of many researchers. Most importantly, differentiation of human embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) to alveolar epithelial type II-like cells facilitates large-scale alveolar epithelial cell production. Air-liquid interface (ALI) culture can induce differentiation further to alveolar epithelial type I-like cells. Furthermore, a microfluidic device, "lung-on-a-chip" has been developed as lung model to study biological development and pathogenic responses of lungs. The utility of a unique six-well "lung-on-a-chip" prototype that can integrate an in vitro aerosol deposition system is currently being examined. This attempt looks interesting as it includes the presence of air, media flow and breathing-like stretching that resembles the movement of lungs [96].

**Table 5.** In vitro models for absorption of inhaled drug particles [33].


When in vivo or in vitro models cannot clearly explain the mechanism of drug transport or lung disposition kinetics, ex vivo tissue models are used. Isolated perfused lung (IPL) is one of the most used method, where the lung is isolated from the body and kept in an artificial system maintaining certain experimental conditions. This separates distribution, metabolism and elimination from lung specific assessments. Architecture and functionality of the tissue is closely maintained in an isolated organ experiment enhancing its resemblance to in vivo state in comparison to in vitro monolayer models from a single cell type. An IPL prepared from small rodents has commonly been employed for lung disposition studies [97].

In vivo studies in intact animal models are used for investigating the absorption, distribution, and pharmacodynamics of inhaled drug particles. In such models, formulations are administered to conscious or anesthetized animals using different types of delivery devices with or without surgical intervention. Small animals such as mice and rats have been commonly used to study pulmonary pharmacokinetics. However, because of higher cost and logistics required for handling and housing, the use of larger animals such as guinea pigs, rabbits, dogs, sheep and monkeys are limited [98]. In larger animals, regional delivery/distribution of drugs can be achieved by the appropriate selection of aerosol size and inspiratory manoeuvres facilitating the study of region dependent lung absorption and disposition [96].

#### **5. Future Perspectives**

We have summarized various instruments and methods used for dissolution studies of inhaled drug particles, however there is no standard method that can be recommended for routine studies. Therefore, there is a need for sophisticated instrument for testing inhalable formulations. Moreover, currently available small volume dissolution apparatus only accounts for dissolution studies in stagnant medium (simulated RTLF) ignoring the fact that mucociliary clearance occurs in the upper airways and breathing results in the movement of alveoli and air sacs of the lungs. Therefore, small volume dissolution instruments should be developed or upgraded in such a way to incorporate fluid movement in them [30]. In addition, in vitro cell-based models that are being used for absorption studies are inconvenient to conduct routine testing of formulations. Therefore, automated cell free systems are always preferred over them.

On the other hand, various simulated RTLFs (dissolution media) [99] that are being used for in vitro dissolution studies do not closely resemble the human RTLF [30]. Composition and thickness of RTLF vary regionally from one region of respiratory tract to another and individually from healthy to diseased. RTLF is rich in mucus in upper respiratory tract whereas, it is rich in surfactant in lower respiratory tract [30]. For instance, in pulmonary disease like cystic fibrosis (CF), patients have highly tenacious (adhesive and cohesive) sputum. Along with mucin (regular component of normal mucus), CF sputum contains large amounts of DNA and filamentous actin [100] in comparison to RTLF of healthy person. Therefore, there is a need for region-specific and disease-specific simulated RTLFs. Determination of absolute concentration of components of RTLF is a must to mimic them but it is a challenging task. Therefore, there is a need for sophisticated method (technology) to accurately determine them. Moreover, components of simulated RTLF should always be chosen keeping in mind about their cost and availability that in turn will help commercialization in future [30].

#### **6. Conclusions**

In vitro dissolution testing is a well-established quality control test in characterizing the performance of a solid oral dosage form. However, no approved methods are available for evaluating the dissolution behaviour of inhaled dry powder particles even though many studies proved the relationship between dissolution and pharmacokinetics of inhaled drugs. The complex nature of the lungs with anatomical and physiological differences in the tracheobronchial region and alveolar region make a great challenge in the development of an in vitro dissolution method which mimics the lung conditions.

In this review, we summarized various dissolution methods and absorption models developed for the evaluation of dissolution and absorption behaviour of inhaled drug particles. Even though the recent methods used a small volume of dissolution medium, it represents only a particular region of the lung. A further improvement in the dissolution methods which mimic the different regions of the lungs is necessary.

**Author Contributions:** Invitation received, S.C.D.; conceptualization, B.B.E. and S.C.D.; writing and original draft preparation, B.B.E. and R.B.; writing, reviewing, and editing, B.B.E., R.B. and S.C.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by School of Pharmacy's Marsden Fund near miss grant.

**Acknowledgments:** Basanth Babu Eedara would like to acknowledge the University of Otago, Dunedin, New Zealand for a doctoral scholarship.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

