**4. Dry powder inhalers**

### **4.1 Powder formulation**

Although nebulization has been the method of choice for phage delivery in treating lung infections in clinical settings, dry powder formulations are preferred to liquid formulations in terms of storage, transportation and administration [34]. Compared to nebulizers, dry powder inhalers (DPIs) are easier to handle without the need of a power source, fewer cleaning requirements and quick delivery [35]. Current research on pharmaceutical development of inhaled phage dry powder mainly focuses on formulation optimization for sufficient powder dispersibility to deliver phage to the lung and storage stability. The choice of excipients plays a key role among all the techniques to produce phage dry powder. Zhang et al. published a comprehensive review to discuss how the choice of excipients affecting the stability of phage in the solid-state [36]. Overall, sucrose, lactose and trehalose are the most popular disaccharides in phage powder formulations. Freeze drying (FD), spray drying (SD) and spray freeze drying (SFD) have been used to generate inhalable phage dry powders with these excipients.

FD is a commonly employed technique to stabilize drugs in solid state [37]. Puapermpoonsiri et al. used FD to generate dry powder of phage-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres designed for pulmonary delivery [38]. Although phages were successfully incorporated into the PLGA microparticles, the poor shelf-life of the encapsulated phage which completely deactivated within 7 days either stored at 4 °C or 22 °C was discouraging. In their follow-up study, they investigated the feasibility of using a high concentration of sucrose (0.5 M) or PEG6000 (5%) to stabilize the FD phage cake [39]. Although rapid phage reduction was still noted over the first 7–14 days, phage remained relatively stable in the powder formulations thereafter. Since then, a number of studies have studied the impacts of various excipients on the production loss and storage stability of FD phages [40–43]. Among all excipients examined, sucrose and trehalose were identified as the most promising stabilizers to preserve phage viability upon the dehydration in the drying process and upon storage. The residual moisture content was found to play an important role in maintaining phage stability. Similar to other protein therapeutics, a 3–6% moisture content of the powder cake was found to be optimal for phage preservation [39, 41]. Although the mechanisms of phage stabilization in dry powder by these sugars are still unclear. Two most acceptable hypotheses for the stabilization of proteins in the solid state by sugars are water replacement and vitrification, which may also be applicable to phages because they are mostly composed of proteins.

In general, FD powder is not respirable, and a separate milling step is required to reduce the particle size to <5 μm, suitable for pulmonary delivery. However, the high-energy milling may cause additional phage loss due to the generation of heat and mechanical stresses. Golshahi et al. prepared FD formulations of KS4-M and ΦKZ phages with 60% lactose and 40% lactoferrin suitable for pulmonary delivery without milling [44]. The size of the phage powder was within the inhalable range (< 5 μm) and acceptable aerosol performance with a fine particle dose of >106 pfu using an Aerolizer was achieved. The production loss was 1–2 log which was not desirable, but the FD phage powders were stable with negligible titer reduction within 3 months storing either at 4 °C or 22 °C.

### *Potential of Inhaled Bacteriophage Therapy for Bacterial Lung Infection DOI: http://dx.doi.org/10.5772/intechopen.96660*

SD is a well-established single-step technique employed for the production of many inhaled pharmaceutical products [45]. Matinkhoo et al. were among the first to study the feasibility of using SD to produce inhalable phage powders comprising trehalose and leucine with or without a third excipients (a surfactant or casein sodium salt) [46]. In these formulations, trehalose was used to protect phage against dehydration; leucine forming a crystalline shell at the particle surface was used to enhance the dispersibility of powders; and a surfactant was employed to reduce aggregation of phage during the drying process. Due to the thermal sensitivity of phage, a low drying temperature was used to produce SD powders with acceptable production loss (0.4–0.8 log) and phage lung dose (7–8 log pfu). Trehalose-alone formulation was employed by Vandenheuvel et al., but the production loss was found to be phage dependent [47]. On the other hand, trehaloseleucine and lactose-leucine systems could stabilize a panel of *Pseudomonas* phage upon the SD process [48–51]. Since the SD trehalose and lactose is amorphous, Chang et al. demonstrated that the addition of a sufficient amount of leucine (at least 20%) was critical to stabilize phage by minimizing recrystallization of trehalose/lactose during powder production process [48]. Despite a low production loss was achieved, particle merging was still significant for formulation containing 80% sugar and 20% leucine due to moisture sorption upon handling. Therefore, higher leucine content and the addition of mannitol to the excipient system was attempted to improve the morphology and reduce the moisture sorption capacity of the phage powders during handling and storage (**Figure 1a-c**) [49, 50]. Although these approaches significantly reduce the problem of particle merging and make powder handling easier, they failed to stop the recrystallization of the amorphous content at high humidity conditions (RH > 50%). Therefore, storing the SD powders at low humidity conditions (RH ≤ 20%) was generally recommended [48, 49, 52, 53]. The storage temperature was also reported to be important on phage dry powder stability. It is generally recommended to store phage drug powder at a temperature at least 50 °C below the glass transition temperature (Tg) of the powders [54].

### **Figure 1.**

*Representative scanning electron microscopy images of phage powders produced by spray drying (a-c) and spray freeze drying (d). (a) 80% trehalose+20% leucine; (b) 60% trehalose +20% mannitol +20% leucine; (c) 70% trehalose +30% leucine and (d) 60% trehalose+20% mannitol and 20% leucine.*

Overall, SD phage powders composed of trehalose/lactose not less than 40% of the total solid content together with leucine and mannitol was able to stabilize phage in powder form with sufficient long shelf-life (≤ 1 log titer loss in 12 months) under refrigeration or room temperature at RH < 20% and yield acceptable lung dose (105 –107 pfu) [46–50, 53]. While leucine is a commonly employed surface active agent to improve the powder dispersity of inhaled pharmaceuticals, trileucine has also been increasingly used to improve aerosol performance and stability of SD powders for inhalation. Recently, Carrigy et al. demonstrated the effectiveness of a trileucine and trehalose system in preserving an anti-Campylobacter phage, CP30A, in powder form for long-distance ambient temperature transportation [55, 56].

SFD is a relatively new drying technique to produce inhalable dry powders. The produced powders are superior to those prepared by traditional FD in terms of structure, quality, and the retention of volatiles and bioactive compounds [57]. The suitability of SFD porous mannitol carriers for pulmonary delivery of drug nanoparticles and biologics have been demonstrated [58–60]. Leung et al. produced SFD phage powder and compared their differences of powder properties with the SD phage powders (**Figure 1d**). With the use of a high frequency of ultrasonic nozzle in the SFD process, a significant titer reduction (>2 log) was noted in the spraying process, making the overall production loss inferior compared with the SD process [53]. Nonetheless, the larger porous carrier provided a larger extent of protection of the embedded phage during aerosolization with a higher recovery of viable phage compared with the SD counterparts. The conventional SFD process is a two-step manufacturing process, which hinders scaling up. Ly et al. used an atmospheric spray freeze-drying (ASFD) technique, which is a single step process, to prepare D29 phage powder [61]. An acceptable titer loss (~0.6 log) was noted due to the use of a twin-fluid nozzle and improved mass and heat transfer rates.
