**Novel Solid Dispersions of Naphthoquinone Using Di**ff**erent Polymers for Improvement of Antichagasic Activity**

**Verônica da Silva Oliveira <sup>1</sup> , Elen Diana Dantas <sup>1</sup> , Anna Thereza de Sousa Queiroz <sup>1</sup> , Johny Wysllas de Freitas Oliveira <sup>1</sup> , Marcelo de Sousa da Silva 1,2 , Patricia Garcia Ferreira <sup>3</sup> , Fernando de Carvalho da Siva <sup>4</sup> , Vitor Francisco Ferreira <sup>3</sup> and Ádley Antonini Neves de Lima 1,\***


Received: 24 October 2020; Accepted: 20 November 2020; Published: 24 November 2020

**Abstract:** IVS320 (3a,10b-dihydro-1*H*-cyclopenta[*b*]naphtho[2,3-*d*]furan-5,10-dione) is a naphthoquinone that has low solubility in aqueous medium, a physical behavior that limits its biological activities, considering that compounds from this class have several activities. In this work, solid dispersions (SDs) prepared between IVS320 and polymers hydroxypropyl methylcellulose (HPMC), polyethylene glycol (PEG), and polyvinylpyrrolidone (PVP) were developed using physical mixture (PM), kneading (KN), and rotary evaporation (RE) methods. Dispersions were investigated using Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), thermogravimetry (TG), powder X-ray diffraction (PXRD), and scanning electron microscopy (SEM). In addition, in vitro antiparasitic activity in *Trypanosoma cruzi* Y strains was evaluated. Physical-chemical characterization demonstrated the formation of SDs through the interaction of IVS320 with polymeric matrices. SDs of IVS320-polymer presented a significant potentiation of antichagasic activity, with inhibitory growth around 62% (IVS320-HPMC/RE), 55% (IVS320-PEG/RE), and 85% (IVS320-PVP/RE), while pure IVS320 showed a value of 48% for the highest concentrations evaluated (50 µg/mL).

**Keywords:** solid dispersion; delivery system; polymeric carriers; antichagasic activity

#### **1. Introduction**

The parasitic infection caused by *Trypanosoma cruzi*, known as Chagas disease, is included among a list of 20 neglected tropical diseases, affecting around 6 to 8 million people and killing about 15 thousand per year [1]. The main form of transmission requires a vector, with *Triatoma infestans* being the most common insect [2,3]; this type of transmission corresponds to approximately 80% of infections. In addition to this route, *T. cruzi* can be transmitted by blood transfusion, transplant of solid organs, congenital infection, inoculation by accident at work, or orally, through the ingestion of contaminated substances [1].

contaminated substances [1].

In recent years, the interest in studying naphthoquinones has intensified, both for its relevance in vital biochemical processes, as well as for its variety of known pharmacological properties, such as anti-inflammatory [4,5], antitumor [6,7], antibacterial, antifungal [8], antiviral [7,9,10], antimalarial [11], leishmanicidal [12], and trypanocidal activities [13–15]. Among the naphthoquinone compounds included in the quinone family, there is the IVS320 molecule (Figure 1a), chemically named 3a,10b-dihydro-1*H*-cyclopenta[*b*]naphtho[2,3-*d*]furan-5,10-dione, produced synthetically and with proven antifungal [16] and antichagasic activity [17]. In recent years, the interest in studying naphthoquinones has intensified, both for its relevance in vital biochemical processes, as well as for its variety of known pharmacological properties, such as anti-inflammatory [4,5], antitumor [6,7], antibacterial, antifungal [8], antiviral [7,9,10], antimalarial [11], leishmanicidal [12], and trypanocidal activities [13–15]. Among the naphthoquinone compounds included in the quinone family, there is the IVS320 molecule (Figure 1a), chemically named 3a,10bdihydro-1*H*-cyclopenta[*b*]naphtho[2,3-*d*]furan-5,10-dione, produced synthetically and with proven antifungal [16] and antichagasic activity [17]. However, IVS320, like most new drug candidates, presents low solubility in aqueous medium

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However, IVS320, like most new drug candidates, presents low solubility in aqueous medium [17], causing an impact on the dissolution rate and, consequently, on its bioavailability. There are several ways to get around this problem of solubility and stability, such as inclusion complexes [18] and solid dispersion systems. [17], causing an impact on the dissolution rate and, consequently, on its bioavailability. There are several ways to get around this problem of solubility and stability, such as inclusion complexes [18] and solid dispersion systems. In this case, for the elaboration of solid dispersions (SDs), polymers play a prominent role, as

In this case, for the elaboration of solid dispersions (SDs), polymers play a prominent role, as they can stabilize SDs due to their ability to retain the drug in an amorphous form in the polymeric matrix during storage [19], and can increase the wettability, dispersibility, and solubility of drugs [20]. they can stabilize SDs due to their ability to retain the drug in an amorphous form in the polymeric matrix during storage [19], and can increase the wettability, dispersibility, and solubility of drugs [20]. Some examples of these polymers are polyvinylpyrrolidone (PVP) [21], polyethylene glycol

Some examples of these polymers are polyvinylpyrrolidone (PVP) [21], polyethylene glycol (PEG), and various cellulose derivatives, such as hydroxypropyl methylcellulose (HPMC) [22,23], which are hydrophilic polymer carriers used as excipients in several pharmaceutical formulations, due to (among other characteristics) their low toxicity [24–26]. (PEG), and various cellulose derivatives, such as hydroxypropyl methylcellulose (HPMC) [22,23], which are hydrophilic polymer carriers used as excipients in several pharmaceutical formulations, due to (among other characteristics) their low toxicity [24–26]. In view of the applications of naphthoquinones, pre-preliminary studies of the biological

In view of the applications of naphthoquinones, pre-preliminary studies of the biological activities of compound IVS320, and the need to develop alternatives for the treatment of Chagas disease, this work aimed to use solutions centered on pharmaceutical technology to improve the physicochemical and biological properties of naphthoquinone IVS320, through the preparation of SDs in polymeric matrices. activities of compound IVS320, and the need to develop alternatives for the treatment of Chagas disease, this work aimed to use solutions centered on pharmaceutical technology to improve the physicochemical and biological properties of naphthoquinone IVS320, through the preparation of SDs in polymeric matrices.

**Figure 1.** Chemical structure: (**a**) 3a,10b-dihydro-1*H*-cyclopenta[*b*]naphtho[2,3-*d*]furan-5,10-dione (IVS320), (**b**) hydroxypropyl methylcellulose (HPMC), (**c**) polyethylene glycol (PEG), and (**d**) **Figure 1.** Chemical structure: (**a**) 3a,10b-dihydro-1*H*-cyclopenta[*b*]naphtho[2,3-*d*]furan-5,10-dione (IVS320), (**b**) hydroxypropyl methylcellulose (HPMC), (**c**) polyethylene glycol (PEG), and (**d**) polyvinylpyrrolidone (PVP).

polyvinylpyrrolidone (PVP). In this article, SDs of IVS320 (Figure 1a) were prepared by kneading and rotary evaporation methods with polymers to improve the antichagasic activity of IVS320. Three polymers were selected as carriers: hydroxypropyl methylcellulose (HPMC) (Figure 1b), polyethylene glycol (PEG) (Figure In this article, SDs of IVS320 (Figure 1a) were prepared by kneading and rotary evaporation methods with polymers to improve the antichagasic activity of IVS320. Three polymers were selected as carriers: hydroxypropyl methylcellulose (HPMC) (Figure 1b), polyethylene glycol (PEG) (Figure 1c), and polyvinylpyrrolidone (PVP) (Figure 1d).

1c), and polyvinylpyrrolidone (PVP) (Figure 1d).

#### **2. Materials and Methods**

#### *2.1. Material*

The compound IVS320 (3a,10b-dihydro-1*H*-cyclopenta[*b*]naphtho[2,3-*d*]furan-5,10-dione) is a dihydrofuran fused 1,4-naphthoquinone that was synthesized by the LabSOA (Laboratory of Applied Organic Synthesis) at the Federal University Fluminense [17]. The polymers hydroxypropyl methylcellulose (HPMC), polyethylene glycol 6000 (PEG), and polyvinylpyrrolidone K-30 (PVP) were purchased from Sigma Aldrich Corporation (San Luis, MO, USA), and the solvents used were all analytical grade.

#### *2.2. Preparation of the SDs*

Solid dispersions (SDs) were prepared via physical mixture (PM), kneading (KN), and rotary evaporation (RE) methods using the ratio (1:1, *w*/*w*) IVS320:polymer for all methodologies.

The preparation of the physical mixture (PM) of IVS320 and the respective polymer (HPMC, PEG, and PVP) were carried out in a (1:1) ratio, subsequently homogenized with mortar and pestle, and then stored in the dissector.

In the kneading method (KN), the IVS320 and each polymer were weighed, homogenized with mortar and pestle, and then addition a mixture of water/acetone solvent (50:50, *v*/*v*) was added. Afterwards, this solution was dried in an oven at 60 ◦C for 24 h, and the solid obtained was stored in a desiccator.

The rotary evaporation method (RE) consisted of solubilizing the IVS320 and the polymers in a mixture of water/acetone solvents (50:50, *v*/*v*). Then, this solution was subjected to constant stirring at 25 ◦C for 72 h. Finally, the solution was evaporated under vacuum at 75 ◦C, and the solid obtained was stored in a desiccator.

#### *2.3. Characterization of the SDs*

#### 2.3.1. Fourier Transform Infrared Spectroscopy (FTIR)

Infrared spectroscopic analysis was performed using IR Prestige-21 equipment (Shimadzu Corporation, Kyoto, Japan). The analysis was carried out in the 4000–600 cm−<sup>1</sup> region with 15 scans.

#### 2.3.2. Thermal Analysis

Differential scanning calorimetry (DSC) thermal analysis was carried out in a DSC-50 cell Shimadzu® (Tokyo, Japan) using approximately 2 mg of sample in aluminum crucibles under dynamic nitrogen atmosphere (50 mL·min−<sup>1</sup> ) and with a heating rate of 10 ◦C·min−<sup>1</sup> at a temperature range of 30–500 ◦C.

Thermogravimetry (TG) was obtained using a TGA-50 Shimadzu® (Tokyo, Japan) in the temperature range of 30–900 ◦C, using alumina crucibles with approximately 2 mg of samples under dynamic nitrogen atmosphere (50 mL·min−<sup>1</sup> ) and with a heating rate of 10 ◦C·min−<sup>1</sup> .

#### 2.3.3. Powder X-ray Diffraction (PXRD)

PXRD analysis was carried out using a Bruker D2 Phaser (Bruker Corporation, Billerica, MA, USA) with CuKα radiation (λ = 1.54 Å) at a voltage of 30 kV and a current of 15 mA, using a Lynxeye detector (Bruker Corporation, Billerica, MA, USA). Samples were scanned at room temperature for 2 h at a range of 5–40◦ at 0.05◦ s −1 .

#### 2.3.4. Scanning Electronic Microscopy (SEM)

Samples were mounted on aluminum stubs using double-sided adhesive tape. Morphological analysis was performed on a Hitachi TM-3000 Tabletop Microscope (Hitachi Ltd., Tokyo, Japan) at a magnification of 500×. SEM images were obtained at an accelerating potential of 15 kV under reduced pressure.

#### *2.4. Antichagasic Activity*

#### 2.4.1. Parasite

The *T. cruzi* Y strain was grown in LIT (liver infusion tryptose) medium supplemented with 10% SFB (serum fetal bovine) and 5% streptococcus/penicillin antibiotic (100 UI/mL), and kept at 27 ◦C in a BOD (Biochemical Oxygen Demand) oven to obtain the epimastigote forms of the parasite.

#### 2.4.2. In Vitro Antichagasic Evaluation

The parasites were diluted and counted with the aid of a Neubauer improved camera (New optics, El Centro, CA, USA) and used at a concentration of 1 <sup>×</sup> <sup>10</sup><sup>7</sup> parasites/mL. The IVS320 compound and the IVS320-polymer systems were solubilized in dimethylsulfoxide (DMSO) with a maximum concentration of 1% in 96-well plates. Briefly, a stock solution was prepared for each of the evaluated systems, and subsequently diluted in the culture medium, obtaining concentrations in the range of 50 µg/mL to 2.5 µg/mL for the pure IVS320 and the concentration of the IVS320 in the SDs. Then cultures of the epimastigote form of *T. cruzi* were dispensed in 96-well plates and incubated for 24 h. The positive control contained only medium and strain; negative controls (medium and drug) and solvent control (contained 1% DMSO, medium and strain) were also obtained to ensure that the solvent did not influence the assay. The inhibition of *T. cruzi* was evaluated by resazurin reduction assay (Sigma Aldrich). After 24 h, a reading was performed at 570 nm and 600 nm using a microplate reader (Epoch, BioTek Instruments, Winooski, VT, USA). Experiments were performed in triplicate, and benznidazole was used as a trypanocidal reference drug. The inhibition percentage was based on the following formula:

$$\% \text{ Inhibition} = 100 - ((\text{A}\_{570t} - (\text{A}\_{600t} \times \text{R}\_0)) \text{(A}\_{570c} - (\text{A}\_{600c} \times \text{R}\_0))) \times 100 \tag{1}$$

A570t = Absorption of the treatment at 570 nm wavelength.

A600t = Absorption of the treatment at 600 nm wavelength.

A570c = Absorption of control at 570 nm wavelength.

A600c = Absorption of the control at 600 nm wavelength.

R<sup>0</sup> = Medium correction factor interacting with resazurin.

$$\mathbf{R}\_0 = \mathbf{A}\mathbf{m}\_{570} / \mathbf{A}\mathbf{m}\_{600} \tag{2}$$

Am<sup>570</sup> = Absorbance of the medium at a wavelength of 570 nm. Am<sup>600</sup> = Absorbance of the medium at a wavelength of 600 nm.

#### 2.4.3. Statistical Analysis

Data analysis was performed using GraphPad Prism software version 5.0 (La Jolla, CA, USA). The results are presented as mean ± standard error of the mean (S.E.M.). A one-way analysis of variance (ANOVA) was performed to determine the significant differences between the groups, followed by Dunnett's *t*-test for multiple comparisons. Values of *p* < 0.05 were considered significant.

#### **3. Results and Discussion**

#### *3.1. Fourier Transform Infrared Spectroscopy (FTIR)*

The infrared spectrum of IVS320 showed vibrational modes referring to νC=O asymmetrical and symmetrical, respectively, at 1678 and 1644 cm−<sup>1</sup> , characteristic of the carbonyl group, corroborating with the stretching of 1,4-naphthoquinone, lawsone, and lapachol [27–29], as well as bands at 1614, 1591, and 1568 cm−<sup>1</sup> , attributed to C=C stretching vibration present in the benzene ring and cyclopentene in the structure of the IVS320. Other bands were observed in the region from 1300 to 1020 cm−<sup>1</sup> , corresponding to C-O-C and C-O (aromatic ether) stretching.

Analyzing the polymers spectra, HPMC exhibited bands at 3457 cm−<sup>1</sup> attributed to the O-H stretching and at 1053 cm−<sup>1</sup> corresponding to C-O-C stretching [20]. In the PEG, a broad band at 3477 cm−<sup>1</sup> (νO-H) and a peak at 1096 cm−<sup>1</sup> (νC-O) were observed, characteristic of ether. The PVP exhibited a broad band in the region of 3412 cm−<sup>1</sup> , referring to the presence of water and corroborating with the DSC results of the polymer. Additionally, bands were also observed for PVP at 2933 cm−<sup>1</sup> , 1648 cm−<sup>1</sup> , and 1287 cm−<sup>1</sup> , which were attributed to C-H, C=O (pyrrolidone group), and C-N stretching, respectively [30].

The SDs exhibited vibrational modes characteristic of the IVS320 molecule and isolated polymers, as well as a shift in the stretching vibration and changes in the profiles of some bands. Some changes were observed in the range of 1400–1170 cm−<sup>1</sup> , corresponding to the vibrational modes of various C-C, C-O, and C-O-C bonds, and C-H and O-H deformations.

In the spectrum of SDs obtained with HPMC (Figure 2a), the presence of νO-H and νC-H was found to correspond with HPMC. In addition to a 1053 cm−<sup>1</sup> band referring to C-O-C stretching, in the region from 1115 to 1016 cm−<sup>1</sup> in the same spectrum, changes were observed in the profile of the three bands corresponding to the IVS320, located at 1102, 1069, and 1035 cm−<sup>1</sup> , due to interactions between the IVS320 and the polymer, suggesting the formation of dispersions. By analyzing the spectra (Figure 2a), the physical mixture proved to be less effective at showing the IVS320-HPMC interaction, considering that the characteristic bands of the polymer were not very evident.

**Figure 2.** Infrared spectrum of systems: (**a**) IVS320-HPMC, (**b**) IVS320-PEG, and (**c**) IVS320-PVP.

IVS320-PEG SDs showed changes in the profile of the bands in the range of 1510 to 1213 cm−<sup>1</sup> . In IVS320-PEG/KN and IVS320-PEG/RE SDs, the band displayed at 1248 cm−<sup>1</sup> corresponding to the IVS320 underwent a small shift to 1250 cm−<sup>1</sup> (C-O stretching). The presence of PEG in the dispersions was evidenced by the vibrational modes corresponding to this polymer, with small variations. In the SDs, bands were found at 1102 cm−<sup>1</sup> referring to C-O deformation characteristic of ether, while in the PEG, it was at 1096 cm−<sup>1</sup> .

1

In the range of 1678 to 1407 cm−<sup>1</sup> in the spectrum of the IVS320-PVP SDs (Figure 2c), modifications were observed in the ratio of the intensity of the bands referring to C=O and C=C stretching, showing that such changes may be due to interactions between the IVS320 molecule and the polymeric chain through these functional groups, thus changing the profile of these bands. While in the spectrum of the physical mixture (IVS320-PVP/PM), the profile of this set of bands remained practically unchanged. Additionally, in the dispersions, it was possible to verify the O-H stretching referring to the presence of the polymer, showing small displacements when compared to the isolated polymer, with a value of 3412 cm−<sup>1</sup> , and 3401 cm−<sup>1</sup> in the PVP.

IVS320-HPMC SDs exhibited peak shifts for the O-H stretching (from 3457 cm−<sup>1</sup> to 3434 cm−<sup>1</sup> ), C-O stretching (from 1248 cm−<sup>1</sup> to 1250 cm−<sup>1</sup> ), and C-O-C stretching (from 1194 cm−<sup>1</sup> to 1197 cm−<sup>1</sup> ). Similar shifts were observed for IVS320-PEG SDs (Figure 2b). In addition, in the IVS320-PEG SDs, a shift was observed regarding the C-O stretching of the PEG (from 1096 to 1102 cm−<sup>1</sup> ). These changes suggest the possibility of hydrogen bonding between the hydroxyl of HPMC and the carbonyl group of IVS320.

Hydrogen bonds between the carbonyl group (C=O) of the IVS320 and the hydroxyl of the HPMC or PEG polymers would be expected. However, spectral changes were observed, mainly in relation to the O-H, C-O, and C-O-C groups, which may suggest that interactions occur between the ether group of the IVS320 and the hydroxyl of the polymers. On the other hand, in the spectrum of the IVS320-PVP system, the main changes were found in the profile of the bands corresponding to C=O and C=C stretching, possibly attributed to the interactions between the C=O group of the IVS320 and polymer.

#### *3.2. Di*ff*erential Scanning Calorimetry (DSC)*

The DSC curve of the IVS320 showed two events (Figure 3a): the first, an endothermic event peaking at 189 ◦C, corresponding to its melting point; the second, an exothermic event at 203 ◦C, attributed to possible changes in the crystalline form of the IVS320.

The thermogram of the HPMC and PVP polymers (Figure 3a) showed a broad endothermic peak, as a result of dehydration, in the range of 75 to 135 ◦C [30], while no peak corresponding to the melting point was found for these polymers, considering their amorphous structure [20]. However, PEG revealed an endothermic peak at a temperature around 63.9 ◦C, corresponding to its melting point, according to literature reports [20,31].

In the IVS320-HPMC/KN SD, an endothermic event at 184 ◦C and another exothermic at 200 ◦C were observed, both attributed to the IVS320 present in the polymeric matrix, while in IVS320-HPMC/RE SD, these same events were recorded at 181 ◦C and 195 ◦C. In the DSC thermograms (Figure 3b), characteristic fusion peaks of the IVS320 still existed, indicating the microcrystalline form of the IVS320 in the HPMC matrix, according to results reported in the 1:1 ratio (drug:polymer) [32].

In the DSC curve of the IVS320-PEG system, the process related to the melting point of the PEG showed variations that were observed at 65.2 ◦C in IVS320-PEG/KN SD and 62.7 ◦C in IVS320-PEG/RE SD. The endothermic events related to the IVS320 were not observed, while the exothermic events were observed with temperature variations, with values of 200 ◦C (IVS320-PEG/KN) and 198 ◦C (IVS320-PEG/RE) for SDs (Figure 3c).

There was also no endothermic event related to IVS320 in the IVS320-PVP SDs (Figure 3d); this indicates that the IVS320 was converted into an amorphous state, thus justifying its dispersion in the polymeric carrier. However, the exothermic event was found at 200 ◦C in the IVS320-PVP/KN SD and at 198 ◦C in the IVS320-PVP/RE SD [33].

Analyzing DSC data, it was found that in all dispersions, the exothermic event referring to the IVS320 underwent small temperature variations when compared to the isolated IVS320, implying that such changes occurred due to possible modifications in the crystalline form of the IVS320, thus suggesting its interaction in the polymeric matrices.

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**Figure 3.** Differential scanning calorimetry (DSC) curve of (**a**) IVS320 and polymers, (**b**) solid dispersions (SDs) of IVS320-HPMC, (**c**) SDs of IVS320-PEG, and (**d**) SDs of IVS320-PVP. RE: rotary evaporation; KN: kneading. **Figure 3.** Differential scanning calorimetry (DSC) curve of (**a**) IVS320 and polymers, (**b**) solid dispersions (SDs) of IVS320-HPMC, (**c**) SDs of IVS320-PEG, and (**d**) SDs of IVS320-PVP. RE: rotary evaporation; KN: kneading.

#### *3.3. Thermogravimetric Analysis (TG) 3.3. Thermogravimetric Analysis (TG)*

The TG curve of the IVS320 shows three mass loss events (Figure 4a). The first occurred in the range of 150 to 162 °C, with approximately 5% mass loss. The second variation occurred in the temperature range from 262 to 315 °C, with about 6.25% mass loss. Lastly, the third event was found in the range of 315 to 900 °C, with a reduction of 18.73%, as reported by Dantas et al. [17]. This high thermal resistance of the IVS320 is consistent with some substances of the naphthoquinone class [34]. The TG curve of the IVS320 shows three mass loss events (Figure 4a). The first occurred in the range of 150 to 162 ◦C, with approximately 5% mass loss. The second variation occurred in the temperature range from 262 to 315 ◦C, with about 6.25% mass loss. Lastly, the third event was found in the range of 315 to 900 ◦C, with a reduction of 18.73%, as reported by Dantas et al. [17]. This high thermal resistance of the IVS320 is consistent with some substances of the naphthoquinone class [34].

The TG curve of the HPMC exhibited a degradation process, in the temperature range of 250 to 395 °C (peak 355 °C), with about 61% mass loss (Δm). The SDs formed between the IVS320 and the HPMC exhibited, in the range 210 to 390 °C (peak 345 °C), a mass loss of 58.12% and 57.41% for the dispersions IVS320-HPMC/KN and IVS320-HPMC/RE, respectively. In the range of 390 to 700 °C, a gradual mass loss of around 10.9% (IVS320-HPMC/KN) and 9.81% (IVS320-HPMC/RE) was observed, while in the TG curve of HPMC, a mass loss of around 7.41% was observed (Figure 4b). The TG curve of the HPMC exhibited a degradation process, in the temperature range of 250 to 395 ◦C (peak 355 ◦C), with about 61% mass loss (∆m). The SDs formed between the IVS320 and the HPMC exhibited, in the range 210 to 390 ◦C (peak 345 ◦C), a mass loss of 58.12% and 57.41% for the dispersions IVS320-HPMC/KN and IVS320-HPMC/RE, respectively. In the range of 390 to 700 ◦C, a gradual mass loss of around 10.9% (IVS320-HPMC/KN) and 9.81% (IVS320-HPMC/RE) was observed, while in the TG curve of HPMC, a mass loss of around 7.41% was observed (Figure 4b).

The PEG polymer showed major mass loss, with a mass loss of approximately 95.3% in the range of 290 to 450 °C (Figure 4c). The IVS320-PEG SDs showed variations in mass loss at temperatures from 220 to 315 °C, with mass losses of 9.27% (IVS320-PEG/KN) and 6.36% (IVS320-PEG/RE). On the other hand, in the range of 315 to 440 °C, higher mass losses were observed, with values of 52.3% for the SD of IVS320-PEG/KN and about 49.17% for the SD of IVS320-PEG/RE. The PEG polymer showed major mass loss, with a mass loss of approximately 95.3% in the range of 290 to 450 ◦C (Figure 4c). The IVS320-PEG SDs showed variations in mass loss at temperatures from 220 to 315 ◦C, with mass losses of 9.27% (IVS320-PEG/KN) and 6.36% (IVS320-PEG/RE). On the other hand, in the range of 315 to 440 ◦C, higher mass losses were observed, with values of 52.3% for the SD of IVS320-PEG/KN and about 49.17% for the SD of IVS320-PEG/RE.

The TG curve of the PVP exhibited two mass losses: the first, in the range of 30 to 100 °C, with a mass variation of 8.77%, corresponding to the presence of water; the second, in the range of 357 to 480 °C, with a mass loss of 83.16%, attributed to the process of thermal polymer degradation, corroborating the literature [35,36]. For SDs with PVP, in the range of 137 to 245 °C, mass variations The TG curve of the PVP exhibited two mass losses: the first, in the range of 30 to 100 ◦C, with a mass variation of 8.77%, corresponding to the presence of water; the second, in the range of 357 to 480 ◦C, with a mass loss of 83.16%, attributed to the process of thermal polymer degradation, corroborating the literature [35,36]. For SDs with PVP, in the range of 137 to 245 ◦C, mass variations of

of 3.53% (IVS320-PVP/RE) and 1% (IVS320-PVP/KN) were found. In turn, in the range of 245 to 480

3.53% (IVS320-PVP/RE) and 1% (IVS320-PVP/KN) were found. In turn, in the range of 245 to 480 ◦C, mass losses of 49.26% and 39.75% were obtained, respectively, for the SDs of IVS320-PVP/RE and IVS320-PVP/KN (Figure 4d). *Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 8 of 15 °C, mass losses of 49.26% and 39.75% were obtained, respectively, for the SDs of IVS320-PVP/RE and IVS320-PVP/KN (Figure 4d).

Analyzing the percentages of mass loss among all dispersions, those obtained with the PVP polymer presented the smallest variations in mass, mainly the IVS320-PVP/KN SD, suggesting that at higher temperatures, there was an increase in the thermal stability of the dispersion. Analyzing the percentages of mass loss among all dispersions, those obtained with the PVP polymer presented the smallest variations in mass, mainly the IVS320-PVP/KN SD, suggesting that at higher temperatures, there was an increase in the thermal stability of the dispersion.

According to the TG and DSC curves of the IVS320-polymer systems, the materials obtained have a stable thermal profile, showing that the presence of IVS320 does not interfere with the thermal profile of the polymeric carriers, which is behavior that strongly suggests a good interface between compound and polymers [37]. According to the TG and DSC curves of the IVS320-polymer systems, the materials obtained have a stable thermal profile, showing that the presence of IVS320 does not interfere with the thermal profile of the polymeric carriers, which is behavior that strongly suggests a good interface between compound and polymers [37].

**Figure 4.** TG curves of (**a**) IVS320, (**b**) SDs of IVS320-HPMC, (**c**) SDs of IVS320-PEG, and (**d**) SDs of IVS320-PVP under N2 atmosphere. **Figure 4.** TG curves of (**a**) IVS320, (**b**) SDs of IVS320-HPMC, (**c**) SDs of IVS320-PEG, and (**d**) SDs of IVS320-PVP under N<sup>2</sup> atmosphere.

#### *3.4. Powder X-ray Diffraction (PXRD) 3.4. Powder X-ray Di*ff*raction (PXRD)*

The X-ray diffractograms of IVS320 presented high intensity peaks at 10.50°, 14.34°, 24.38°, and 28.22°, followed by a series of secondary reflections, indicating a crystalline profile, as already reported by Dantas et al. [17]. The X-ray diffractograms of IVS320 presented high intensity peaks at 10.50◦ , 14.34◦ , 24.38◦ , and 28.22◦ , followed by a series of secondary reflections, indicating a crystalline profile, as already reported by Dantas et al. [17].

The PEG showed two intense crystalline reflections at 19.30° and 23.43° (Figure 5b), while HPMC and PVP showed an amorphous pattern, with no peak in the diffractogram [30,35]. The PEG showed two intense crystalline reflections at 19.30◦ and 23.43◦ (Figure 5b), while HPMC and PVP showed an amorphous pattern, with no peak in the diffractogram [30,35].

For all IVS320-polymer systems, a reduction in the intensity of the standard peaks was observed, indicating a change in the amorphous state, both in the dispersions obtained by the kneading (KN) and rotary evaporation (RE) methods. Evidence of less intense peaks in polymeric systems may indicate the achievement of partially amorphous shapes due to the influence of polymers. For all IVS320-polymer systems, a reduction in the intensity of the standard peaks was observed, indicating a change in the amorphous state, both in the dispersions obtained by the kneading (KN) and rotary evaporation (RE) methods. Evidence of less intense peaks in polymeric systems may indicate the achievement of partially amorphous shapes due to the influence of polymers.

The physical mixtures showed peaks of the individual species, showing IVS320 crystallinity characteristics with a decrease in its intensity, which is attributed to the dilution and surface coverage The physical mixtures showed peaks of the individual species, showing IVS320 crystallinity characteristics with a decrease in its intensity, which is attributed to the dilution and surface coverage

of the IVS320 particles by the polymers [20]. On the other hand, the PXRD profile of the SDs showed

of the IVS320 particles by the polymers [20]. On the other hand, the PXRD profile of the SDs showed a more marked reduction in the peaks, suggesting the formation of amorphous solid dispersions. *Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 9 of 15

In SDs with HPMC, a more pronounced reduction in peaks was observed, particularly in the range of 20◦ to 30◦ (Figure 5a), indicating a change in the amorphous characteristic state of pure HPMC, thus suggesting that the polymer inhibits IVS320 crystallization, mainly in the SD of IVS320-HPMC/RE. HPMC, thus suggesting that the polymer inhibits IVS320 crystallization, mainly in the SD of IVS320- HPMC/RE. On the other hand, the SDs obtained with PEG were more crystalline than the IVS320-HPMC

On the other hand, the SDs obtained with PEG were more crystalline than the IVS320-HPMC and IVS320-PVP (Figure 5c), possibly suggesting a recrystallization of the IVS320 during the process of preparing its dispersions, after evaporation of the solvent. This can be confirmed by the small displacements of the peaks corresponding to the PEG, bearing in mind that in the diffractogram, in relation to the polymer, crystalline reflections appeared at 19.30◦ and 23.43◦ , while in the SDs they were found at 18.95◦ and 23.07◦ . and IVS320-PVP (Figure 5c), possibly suggesting a recrystallization of the IVS320 during the process of preparing its dispersions, after evaporation of the solvent. This can be confirmed by the small displacements of the peaks corresponding to the PEG, bearing in mind that in the diffractogram, in relation to the polymer, crystalline reflections appeared at 19.30° and 23.43°, while in the SDs they were found at 18.95° and 23.07°.

**Figure 5.** Powder X-ray diffraction (PXRD) of systems (**a**) IVS320-HPMC, (**b**) IVS320-PEG, and (**c**) **Figure 5.** Powder X-ray diffraction (PXRD) of systems (**a**) IVS320-HPMC, (**b**) IVS320-PEG, and (**c**) IVS320-PVP.

#### IVS320-PVP. *3.5. Scanning Electron Microscopy (SEM)*

with the polymer, composing a uniform matrix.

*3.5. Scanning Electron Microscopy (SEM)* The micrograph of the IVS320 displays very fine crystals with varying sizes and a predominance The micrograph of the IVS320 displays very fine crystals with varying sizes and a predominance of the pyramidal shape and some crystals in the form of needles (Figure 6), corroborating the PXRD results. HPMC exhibited an irregular cylindrical filament structure, while PEG exhibited a block-shaped

of the pyramidal shape and some crystals in the form of needles (Figure 6), corroborating the PXRD structure, and the PVP particles showed irregular shapes with a smooth surface [20,30].

results. HPMC exhibited an irregular cylindrical filament structure, while PEG exhibited a block-shaped structure, and the PVP particles showed irregular shapes with a smooth surface [20,30]. Images obtained for the IVS320-polymer systems by the physical mixture (PM) method showed the deposition of fragments of the IVS320 on the polymeric surface of the carriers, not verifying a uniform structure or surface that proves the interaction of the IVS320 in the polymeric network.

Images obtained for the IVS320-polymer systems by the physical mixture (PM) method showed the deposition of fragments of the IVS320 on the polymeric surface of the carriers, not verifying a uniform structure or surface that proves the interaction of the IVS320 in the polymeric network. On the other hand, the micrographs for the IVS320-HPMC, IVS320-PEG and IVS320-PVP systems that were obtained through the kneading (KN) and rotary evaporation (RE) methods presented particles with irregular shapes and morphologies that were different from both components that originated it.

On the other hand, the micrographs for the IVS320-HPMC, IVS320-PEG and IVS320-PVP systems that were obtained through the kneading (KN) and rotary evaporation (RE) methods presented particles with irregular shapes and morphologies that were different from both components that originated it. Images of the SDs of IVS320-HPMC/KN and IVS320-HPMC/RE showed rough surfaces, while Images of the SDs of IVS320-HPMC/KN and IVS320-HPMC/RE showed rough surfaces, while the SDs of IVS320-PEG/KN and IVS320-PVP/KN exhibited images with amorphous structures of agglomerates. The IVS320-PEG/RE and IVS320-PVP/RE images suggest an association of the IVS320 with the polymer, composing a uniform matrix.

*Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 10 of 15

**Figure 6.** Scanning electron microscopy of IVS320, polymers (HPMC, PEG, and PVP), physical mixtures (PMs), and SDs obtained by kneading (KN) and rotary evaporation (RE). **Figure 6.** Scanning electron microscopy of IVS320, polymers (HPMC, PEG, and PVP), physical mixtures (PMs), and SDs obtained by kneading (KN) and rotary evaporation (RE).

#### *3.6. In Vitro Antichagasic Activity*

*3.6. In Vitro Antichagasic Activity* The antichagasic activity of the pure IVS320 is practically comparable to benznidazole (reference drug) at concentrations of 50 to 10 µg/mL, as shown in Figure 7. However, the IVS320-polymer SDs showed better activity, with a higher percentage of inhibitory growth (%) when compared to the pure The antichagasic activity of the pure IVS320 is practically comparable to benznidazole (reference drug) at concentrations of 50 to 10 µg/mL, as shown in Figure 7. However, the IVS320-polymer SDs showed better activity, with a higher percentage of inhibitory growth (%) when compared to the pure IVS320, which presented a value of 37% for the highest concentration evaluated (50 µg/mL).

IVS320, which presented a value of 37% for the highest concentration evaluated (50 µg/mL). Figure 7 reports the inhibitory growth (%) as a function of the concentration of pure IVS320 and its equivalent concentrations in the dispersions. Figure 7 reports the inhibitory growth (%) as a function of the concentration of pure IVS320 and its equivalent concentrations in the dispersions.

The IVS320-HPMC/RE dispersion showed an activity higher to pure IVS320 for all concentrations assessed in the range of 50 to 2.5 µg/mL, presenting an inhibitory growth rate around 62% for the highest concentration. The IVS320-HPMC/RE dispersion showed an activity higher to pure IVS320 for all concentrations assessed in the range of 50 to 2.5 µg/mL, presenting an inhibitory growth rate around 62% for the highest concentration.

Analyzing the IVS320-PEG system at concentrations of 50 and 25 µg/mL, the IVS320-PEG/PM and IVS320-PEG/RE dispersions exhibited a greater inhibitory effect, when compared to pure IVS320 at the same concentrations, showing inhibitory activity around approximately 55% for a concentration of 50 µg/mL. Analyzing the IVS320-PEG system at concentrations of 50 and 25 µg/mL, the IVS320-PEG/PM and IVS320-PEG/RE dispersions exhibited a greater inhibitory effect, when compared to pure IVS320 at the same concentrations, showing inhibitory activity around approximately 55% for a concentration of 50 µg/mL.

*Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 11 of 15

**Figure 7.** In vitro antichagasic activity of IVS320-polymer systems: (**a**) IVS320-HPMC, (**b**) IVS320-PEG, and (**c**) IVS320-PVP against epimastigotes of *T. cruzi* Y strain for 24 h. Values refer to % of control (untreated). \*\*\* *p* < 0.0001, \*\* *p* < 0.001 and \* *p* < 0.05 compared with pure IVS320 at the same concentration. Statistical analysis was performed using ANOVA followed by Dunnett's *t*-test for multiple comparisons. **Figure 7.** In vitro antichagasic activity of IVS320-polymer systems: (**a**) IVS320-HPMC, (**b**) IVS320-PEG, and (**c**) IVS320-PVP against epimastigotes of *T. cruzi* Y strain for 24 h. Values refer to % of control (untreated). \*\*\* *p* < 0.0001, \*\* *p* < 0.001 and \* *p* < 0.05 compared with pure IVS320 at the same concentration. Statistical analysis was performed using ANOVA followed by Dunnett's *t*-test for multiple comparisons.

Similar to the IVS320-HPMC system, the results obtained for the IVS320-PVP system showed a better performance of the inhibitory activity for the SD of IVS320-PVP/RE, with an inhibitory rate around 82% and 64%, respectively, for the concentrations of 50 and 25 µg/mL. Additionally, it exhibited inhibitory activity higher than pure IVS320, even at the lowest concentrations, showing values of 15% inhibitory growth at the concentration of 2.5 µg/mL of IVS320 when incorporated in the SDs. Similar to the IVS320-HPMC system, the results obtained for the IVS320-PVP system showed a better performance of the inhibitory activity for the SD of IVS320-PVP/RE, with an inhibitory rate around 82% and 64%, respectively, for the concentrations of 50 and 25 µg/mL. Additionally, it exhibited inhibitory activity higher than pure IVS320, even at the lowest concentrations, showing values of 15% inhibitory growth at the concentration of 2.5 µg/mL of IVS320 when incorporated in the SDs.

The SDs of the IVS320 with the HPMC and PVP polymers presented the best results for antichagasic activity when obtained by the rotary evaporation (RE) method. However, among the results, the IVS320-PVP/RE system stood out due to the 82% antichagasic activity rate and the improvement in inhibitory activity in relation to all the concentrations evaluated, when compared to pure IVS320. The SD of IVS320-PVP/KN also displays significant inhibitory activity, showing a rate of around 65% at a 50 µg/mL concentration. The SDs of the IVS320 with the HPMC and PVP polymers presented the best results for antichagasic activity when obtained by the rotary evaporation (RE) method. However, among the results, the IVS320-PVP/RE system stood out due to the 82% antichagasic activity rate and the improvement in inhibitory activity in relation to all the concentrations evaluated, when compared to pure IVS320. The SD of IVS320-PVP/KN also displays significant inhibitory activity, showing a rate of around 65% at a 50 µg/mL concentration.

The activity of the IVS320 incorporated in the dispersions was increased when compared to the pure IVS320, which can probably be explained as being due to the increment in the solubility of the compound in these polymeric matrices. Also, linked to this behavior, it can have the influence of the polymer. Despite the low inhibitory activity found in each of the polymers, even at higher concentrations, there may be a synergism of the inhibitory effect between the two constituents of the polymeric matrix (IVS320 and polymer), and it may also be another factor that explains the increase in the antichagasic activity of the SDs in relation to pure IVS320. Studies carried out by Lima et al. [38] show that a reduction in particle size, an increase in surface The activity of the IVS320 incorporated in the dispersions was increased when compared to the pure IVS320, which can probably be explained as being due to the increment in the solubility of the compound in these polymeric matrices. Also, linked to this behavior, it can have the influence of the polymer. Despite the low inhibitory activity found in each of the polymers, even at higher concentrations, there may be a synergism of the inhibitory effect between the two constituents of the polymeric matrix (IVS320 and polymer), and it may also be another factor that explains the increase in the antichagasic activity of the SDs in relation to pure IVS320.

area, and the interaction between polymeric carriers can result in the increase in the solubility of drugs. Studies carried out by Lima et al. [38] show that a reduction in particle size, an increase in surface area, and the interaction between polymeric carriers can result in the increase in the solubility of drugs.

#### **4. Conclusions 4. Conclusions**

Using different polymeric materials, it was possible to evaluate the interactions of carriers with the compound IVS320 to obtain SDs. The results showed changes in vibrational modes, as well as Using different polymeric materials, it was possible to evaluate the interactions of carriers with the compound IVS320 to obtain SDs. The results showed changes in vibrational modes, as well as alterations in the thermal behavior and IVS320 crystallinity pattern, which in turn, led to an improvement of the IVS320 antichagasic activity, when present in polymeric matrices. The results indicated that the inhibitory growth (%) improved gradually with the increase in the concentration of the samples, and IVS320-polymer SDs showed potentiated activity in comparison with IVS320 in some concentrations. It was observed that the SD obtained by the rotary evaporation method with the PVP polymer resulted in the best antichagasic activity of the IVS320 incorporated in the SDs. This behavior shows that the addition of polymers alters the effectiveness of IVS320 in terms of its antichagasic activity, probably due to the improved solubility and bioavailability of IVS320.

**Author Contributions:** Conceptualization: Á.A.N.d.L., V.d.S.O., and E.D.D.; methodology: V.d.S.O., E.D.D., A.T.d.S.Q., J.W.d.F.O., M.d.S.d.S., P.G.F., F.d.C.d.S., and V.F.F.; formal analysis: Á.A.N.d.L. and V.d.S.O.; data curation: V.d.S.O. and E.D.D.; writing—original draft preparation: Á.A.N.d.L. and V.d.S.O.; writing—review and editing: Á.A.N.d.L., V.d.S.O., E.D.D., A.T.d.S.Q., J.W.d.F.O., M.d.S.d.S., P.G.F., F.d.C.d.S., and V.F.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES)-Finance Code 001.

**Acknowledgments:** The authors wish to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for grants and fellowships. V.d.S.O. and J.W.d.F.O. acknowledge the financial support (PhD and Postdoctoral fellowships) provided by CAPES, Brazil. M.d.S.d.S. acknowledges CNPq, Brazil, for the Research Grant. This work was partially funded by Global Health and Tropical Medicine and Fundação para a Ciência e Tecnologia, FCT, Portugal (Grant number IHMT-UID/multi/04413/2013 and Grant number PTDC/CVT/CVT/28908/2017).

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

#### **References**


et al. Amorphous solid dispersions of hecogenin acetate using different polymers for enhancement of solubility and improvement of anti-hyperalgesic effect in neuropathic pain model in mice. *Biomed. Pharmacother.* **2018**, *97*, 870–879. [CrossRef]


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### *Review* **Pulmonary Drug Delivery of Antimicrobials and Anticancer Drugs Using Solid Dispersions**

**Hisham Al-Obaidi \*, Amy Granger, Thomas Hibbard and Sefinat Opesanwo**

The School of Pharmacy, University of Reading, Reading RG6 6AD, UK; a.granger@student.reading.ac.uk (A.G.); t.hibbard@pgr.reading.ac.uk (T.H.); s.t.opesanwo@student.reading.ac.uk (S.O.)

**\*** Correspondence: h.al-obaidi@reading.ac.uk; Tel.: +44-1183-786-261

**Abstract:** It is well established that currently available inhaled drug formulations are associated with extremely low lung deposition. Currently available technologies alleviate this low deposition problem via mixing the drug with inert larger particles, such as lactose monohydrate. Those inert particles are retained in the inhalation device or impacted in the throat and swallowed, allowing the smaller drug particles to continue their journey towards the lungs. While this seems like a practical approach, in some formulations, the ratio between the carrier to drug particles can be as much as 30 to 1. This limitation becomes more critical when treating lung conditions that inherently require large doses of the drug, such as antibiotics and antivirals that treat lung infections and anticancer drugs. The focus of this review article is to review the recent advancements in carrier free technologies that are based on coamorphous solid dispersions and cocrystals that can improve flow properties, and help with delivering larger doses of the drug to the lungs.


**Citation:** Al-Obaidi, H.; Granger, A.; Hibbard, T.; Opesanwo, S. Pulmonary Drug Delivery of Antimicrobials and Anticancer Drugs Using Solid Dispersions. *Pharmaceutics* **2021**, *13*, 1056. https://doi.org/10.3390/ pharmaceutics13071056

Academic Editor: Carsten Ehrhardt

Received: 14 June 2021 Accepted: 6 July 2021 Published: 10 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Keywords:** cocrystals; coamorphous; solubility; dry powder inhalers; pulmonary drug delivery

#### **1. Introduction**

The respiratory tract is susceptible to a range of conditions, such as viral, bacterial and fungal infections [1], which in turn can result in an exacerbation of other existing conditions through inflammation [2]. Lower respiratory tract infections which include bronchitis, tuberculosis and pneumonia, are classed as two of the leading causes of death, while pneumonia is a leading cause of death in children globally [3]. While inflammation can affect the entire respiratory system, different pathogens will inhabit different parts of the respiratory tract. For example, tuberculosis causative microorganism *Mycobacterium tuberculosis* colonises the lungs, deep within, and also inside the lung's alveolar surfaces [4]. In cystic fibrosis (CF) patients, the bacterium, *Pseudomonas aeruginosa*, inhabits the conducting and respiratory zones of the lungs and is associated with recurrent infections. This is mainly caused by bacterial transformation to the biofilm producing mucoid strain, which exhibits increased resistance to both antibiotics and natural lung defence mechanisms, such as phagocytosis.

In addition to respiratory tract infections, lung cancer is a major cause of death. It is estimated that 1.6 million people die every year from lung cancer, making it one of the most fatal cancers [5]. Lung cancer remains difficult to cure using chemotherapy, as evident by the low long-term survival rate of patients [6]. Apart from the frequent low efficacy, cancer treatments have been associated with significant side effects. A study that included 449 cancer patients, revealed 86% of patients experienced at least one side effect from cancer chemotherapy, with 67% having experienced six or more side effects [7]. This calls for the exploration of new approaches, aimed at reducing the side effects, and in turn, improving patient tolerance [8], and further improve the quality of care provided to patients.

The pulmonary route has been used to treat different lung conditions, such as asthma and chronic obstructive pulmonary disease (COPD). Drugs delivered to the lungs are typically formulated in low doses; however, there is an increasing clinical need to deliver higher doses. For example, when antimicrobials are delivered directly to the lungs, higher doses ensure optimum lung concentration for tackling the infection [9]. Hence, a major challenge is to formulate these drugs with efficient deposition, while maximising the delivered dose. Current technologies are based on formulating the required doses of the active pharmaceutical ingredient (API) with the addition of excipients (known as carrier particles) [10]. These carrier particles serve to minimise undesired deposition into the oropharyngeal region, as well as reducing loss in the inhalation device itself. Among approaches to deliver larger doses of the drug to the lungs is solid dispersions. These are molecular mixtures of the drug and a miscible carrier by which properties can be tailored to achieve optimum outcomes, such as improved solubility [11]. The carrier can be a small molecule known as a coformer or a polymeric carrier leading to the formation of crystalline or amorphous dispersions.

Although oral formulations have a more enhanced therapeutic profile when it comes to treating systemic diseases, inhaled drug formulations are better at targeting lung conditions, such as infections and cancer. The aim of this review article is to explore the respiratory route for drug delivery, highlighting its advantages and challenges to deliver larger doses. While there have been different approaches to maximise the dose that can be delivered, the focus of this review is on recent advancements in the delivery of particulates prepared as solid dispersions. The use of solid dispersions represents a novel approach to deliver larger amounts of the drug while maintaining enhanced physicochemical properties. These physical molecular complexes (i.e., solid dispersions) can be engineered to modify properties, such as adhesion, aerodynamic diameter and morphology, allowing enhanced pulmonary drug delivery.

#### *Physiology of the Lungs and Factors Affecting Particles Deposition*

The pulmonary alveoli exhibit a large surface area of over 100 m<sup>2</sup> and thin walls of less than 1 µm, allowing fast absorption of drugs into the rich blood supply for systemic effect [12]. This would be beneficial for a number of drugs as the fast absorption would result in a more rapid onset of action compared to other administration routes, such as oral administration. As well as being advantageous for drugs acting both locally and systemically, it also has reduced systemic side effects that are common with other administration routes [13]. It is considered a non-invasive form of drug administration and often require lower doses compared with other systemic drug delivery routes [14]. The local administration also avoids first pass liver metabolism, which is detrimental for some drugs. Thus, the pulmonary route may be favoured over other parenteral routes, or when absorption via the gastrointestinal (GI) tract is inappropriate or ineffective [12,15].

The respiratory system comprise 23 generations (G0–G23) which have varying sizes, structures and functions [16]. Each generation splits into two smaller daughter branches to give the next generation. Altogether, these generations are divided into two zones: Conducting and respiratory. The conducting zone includes the structures from the trachea to the bronchioles from G0 to G16. The main role of the conducting zone is to carry the air into the lungs. The respiratory zone includes structures from the respiratory bronchioles to the alveolar ducts and alveoli from G17 to G23. This zone contains functional tissues where gaseous exchange occurs [17].

As a result of the varying size and structure of the lungs, inhaled particle size affects how deep into the lungs the drug and excipients can penetrate. For example, particles with a mass median aerodynamic diameter (MMAD) of 10 µm or larger tend to be deposited in the oropharyngeal region, whereas smaller particles with an MMAD of less than 3 µm can penetrate much deeper through the lungs into the alveoli [18]. MMAD is a measurement used to define the size of aerosol particles. The aerodynamic diameter of a particle relates to a sphere with the same density as water (1 g/cm<sup>3</sup> ) which settles at the same velocity as the particle of interest in still air [19]. Using the mass median value, the aerosol size distribution is divided in half [20].

Particles intended for pulmonary drug delivery are categorised according to their size. The ideal particle size for inhalation is said to be <5 µm [21], with <3 µm having an 80% chance of reaching the lower airways and 50–60% chance of reaching the alveolar regions [22]. Particles > 5 µm are identified as coarse particles; fine particles range from 0.1–5 µm, and ultrafine particles are <0.1 µm. A monodisperse aerosol is said to be highly desirable for maximum deposition and specific targeting in the lungs [23]. However, there have been reports showing minimum differences in deposition between polydisperse and monodisperse aerosols [24]. The deposition of inhaled particles within the regions of the lungs is dependent on a number of physiological and pharmaceutical factors, such as particle shape, particle size, surface morphology, the breathing rate of the patient, lung volume and health condition of the patient. The physicochemical factors that affect particles deposition in the respiratory system are shown in Table 1. Particle deposition can occur through the following mechanisms: sedimentation, impaction and diffusion. Diffusion is a fundamental mechanism of particle deposition for particles < 0.5 µm. The process is influenced by Brownian motion—in other words, motion increases with decreasing particle size and particles move from a higher concentration to a lower concentration leading to the deposition upon contact with the airway walls. This mechanism heavily influences deposition in the lower regions of the lungs and the alveoli. Gravitational sedimentation can happen at a later region, typically within the tracheobronchial region (approximately the last six generations), as a result of the relatively low air velocity within this region [25]. In fact, as the residence time is longer, a combination of both sedimentation and free diffusion can occur. It is vital for drug absorption to occur, that the particles are deposited before exhalation takes place. Typical size depends on the aerodynamic diameter, which is approximately above 0.5 µm for sedimentation and below that for diffusion [26,27]. The bigger the particle and the lower the airflow rate, the faster the sedimentation in which inertial impaction plays a significant role for particles > 5 µm [28].


**Table 1.** Physicochemical factors to be considered when designing inhaled formulations. Each parameter influences both aerosolisation and deposition. Adapted from the work by the authors of [29–31].

Note—\* Factors specifically affecting aerosolisation.

#### **2. The Necessity to Deliver Larger Doses to Treat Lung Infections and Cancer**

There are a variety of inhaled products for the treatment of conditions affecting the respiratory system (Table 2). As can be seen in Table 2, the maximum dose that has been successfully formulated for inhalation, thus far, is colistimethate sodium, with doses ranging from 80 to 125 mg, formulated as inhalation powder. Many of the higher inhalable doses are administered via the use of nebulisers, which may require guidance and can also be complicated for patients to use [32]. Desgrouas and Ehrmann reviewed the available evidence and called for developing inhaled antibiotics, especially for mechanically ventilated patients [33]. However, the majority of inhalable drug formulations are available in the dose range of micrograms and are indicated for conditions, such as asthma

and COPD. In addition, there is a limited number of inhaled medications available as combinations, which are again mostly limited for use in asthma and COPD.


**Table 2.** Examples of currently available inhaled products in the market.

There are two major lung conditions that require high doses of medications, these being infections and malignancy. For lung infections, the causative bacteria and viruses can be found throughout the different structures of the lungs, which will alter the efficacy of treatment. The conducting zone of the respiratory system, which consists of the trachea, bronchi and bronchioles, plays a role in trapping microbes in mucus produced by goblet cells. The mucus is then transported by ciliated cells located on the epithelium to the oropharynx, where the mucus is either swallowed or removed via coughing [43]. However, in cases where the microbes go on to develop into infections, it is important to determine the specific site in the lungs where the infection has developed to effectively target and deliver the drug directly to the causative microbes. Table 3 lists common loci of prevalent microorganisms within the respiratory tract noting that several pathogens found in the respiratory tract are associated with the development of biofilms.

Upper respiratory tract infections typically occur in the conducting zone, examples of which include common cold, sinusitis and pharyngitis, whereas lower respiratory tract infections happen in the respiratory zone and include pneumonia and bronchitis. In addition, some microorganisms, such as *Haemophilus influenzae*, that are normally found in the conducting zone can go on to migrate to the respiratory zone, subsequently causing lower respiratory tract infections [44]. Some studies suggest that the conducting zone of the respiratory tract is the more common zone where bacteria and viruses are contained during infections. One study found that for *Pseudomonas aeruginosa* biofilms in cystic fibrosis patients, the majority of the bacteria were present in the conducting zone, which acted as a reservoir for the bacteria to multiply and form biofilms [45]. Consequently, it is important for inhaled formulations of antibacterials and antivirals to reach and deposit in the conducting zone of the respiratory tract to effectively treat the infections. With the ability to locally deliver antimicrobial drugs for treatment, there could also be a further advantage of reducing antimicrobial resistance. With oral and intravenous administration, there is a risk of an accumulation in infection-free sites, leading to the development of antimicrobial resistance. In addition, a number of antimicrobials, such as tobramycin, can be toxic when given in repeated high doses systemically, which can occur when antimicrobials are prescribed for recurrent infections [46]. Hence, utilising local drug delivery directly into the lungs could reduce the risk of toxicity.


**Table 3.** Location of infections causative microorganisms within the respiratory tract.

Similar to infectious conditions, lung cancer is another serious condition that requires large doses of drugs for treatment. Current survival rates for all stages of lung cancer include 40% of patients surviving for one year or more after diagnosis, 15% of patients surviving for five years or more after diagnosis and only 10% of patients surviving for 10 years or more after diagnosis [50]. The nature of lung cancer suggests that it is more effectively treated by direct delivery to the lungs. Sardeli et al., suggested the need to use inhaled immunotherapy as opposed to intravenous administration to avoid systemic side effects and achieve a localised effect [51]. Hence, pulmonary drug delivery provides a novel opportunity to avoid unwanted drug distribution and could achieve maximum deposition of the drug at the site of action. As such, there is the potential for higher concentrations of the drugs to reach the lungs compared with oral and intravenous administration. Lung cancer cells are prone to rapidly developing resistance to anticancer drugs, so higher doses are given to combat this challenge [52]. When administered systemically, chemotherapy drugs can cause toxic side effects affecting healthy organs, especially when administering large doses, which may be prevented by local administration of the drugs [53]. By targeting the lungs directly, this also means there is potential for an accumulation of the drug to build up in the tumour cells, rather than in the kidneys, liver and spleen, which is observed with systemic drug use [54]. This has the potential to beneficially impact patients' treatment and increase the likelihood of remission and survival.

#### **3. Challenges Associated with Drug Delivery to the Lungs**

Whilst pulmonary drug delivery has its advantages, to ensure advantageous deposition in the lungs, drug particles must be within the optimum size for lung deposition (Table 4 and Figure 1). If the particle size is relatively large, the deposition will occur in the larynx causing irritation, and if the particle size is relatively small, the particles will be immediately exhaled from the lungs and will not be deposited [55]. It is important to note that drug particles should be deposited in the lungs in a high enough proportion for the API to be absorbed and produce a therapeutic effect. Unsurprisingly, it has been shown that higher drug deposition in the lungs leads to enhanced clinical benefits [56]. As mentioned above, a common approach to improve the flow properties of powder for inhalation is to use carrier particles (such as lactose monohydrate). This is based on combining the drug with the larger lactose particles, which strip from the drug particles inside the device in response to the high velocity created by inhalation [57,58]. Any escaped lactose will deposit in the throat, leaving the drug particles to carry on with their journey towards the alveoli. Hence, the main challenge would be to deliver high doses of the API without significant loss in the oropharynx region. Another challenge is that inhalation devices do not have the capacity to accommodate high masses of powders [59].

As shown by the data collated and presented in Table 4, two different studies used a combination of beclomethasone dipropionate with formoterol labelled with technetium-99. The first dissolved the particles in hydrofluoroalkane to give an MMAD of 1.3 µm via pressurised metered-dose inhaler (pMDI) [60], the second delivered the solid particles with MMAD of 1.5 µm via a NEXThaler® dry powder inhaler (DPI) [61]. The lung deposition for the pMDI was 34.08% (standard deviation (SD) = 9.3%), and the DPI had a lung deposition of 55.2% (SD = 3.7%). This shows that even though the same APIs were used, and the particle sizes were similar, the way that the API is formulated plays a significant role in the lung deposition of the particles. It is apparent that the type of the API has an effect by comparing the lung deposition of albuterol and beclomethasone dipropionate with formoterol in the pMDI considered previously [62]. They both have an MMAD of 1.5 µm, are labelled with technium-99 and are administered using an pMDI; however, the lung deposition of albuterol was 56.3% (SD = 9.2%), which is considerably higher than the beclomethasone, which has a lung deposition of 34.08% (SD = 9.3%). This suggests that API type and formulation must also be carefully considered when formulating drugs for pulmonary drug delivery.

Furthermore, Table 4 shows that within the same administration device, the preparation method of the particle has an impact on the deposition within the lungs. One study compared two suspensions of beclomethasone dipropionate labelled with technetium-99 in a metered-dose inhaler, by which the first formulation was dissolved in hydrofluoroalkane, and the other was dissolved in chlorofluorocarbon [63], it demonstrated that the different preparation methods led to a difference in both MMAD and lung deposition. Therefore, this suggests that the type of excipients used must be carefully considered for optimum lung deposition.


**Table 4.** MMAD, lung deposition and preparation method of different inhaled APIs.

4.

*Pharmaceutics* **2021**, *13*, 1056 7 of 28

**Figure 1.** A scatter graph, comparing MMAD with corresponding lung deposition values in Table **Figure 1.** A scatter graph, comparing MMAD with corresponding lung deposition values in Table 4.

#### **4. Impact of Drug Delivery Devices on the Extent of Pulmonary Drug Delivery**

**4. Impact of Drug Delivery Devices on the Extent of Pulmonary Drug Delivery**  There are currently three major categories of delivery devices that are used for inhalation therapy: pressurised metered-dose inhalers (pMDIs), nebulisers and dry powder inhalers (DPIs). pMDIs are widely available, portable inhalers designed to releases a specific dose of the drug, following depression of an actuator, which the patient then inhales in a single inspiration or via a spacer device. Studies have found that many patients have difficulty in using these devices, due to the required coordination of pressing down on the actuator and inhaling the resultant mist simultaneously [66]. If patients do not have the required coordination to self-administer their medication using a pMDI, some of the drug is deposited in the mouth and at the back of the throat. As a result, less of the API will be inhaled than intended, so the correct dose will not be administered. This could potentially result in substandard treatment, leading to exacerbation or worsening of a patient's condition. pMDIs are formulated as a suspension or solution with one or more APIs along with a propellant. The propellant is used to generate the pressure required to form micron-scaled droplets for inhalation [67]. Furthermore, pMDIs deposit less of the drug into the lungs, due to the high particle exit velocity with the actuations [68]. This means that it is more difficult to formulate higher There are currently three major categories of delivery devices that are used for inhalation therapy: pressurised metered-dose inhalers (pMDIs), nebulisers and dry powder inhalers (DPIs). pMDIs are widely available, portable inhalers designed to releases a specific dose of the drug, following depression of an actuator, which the patient then inhales in a single inspiration or via a spacer device. Studies have found that many patients have difficulty in using these devices, due to the required coordination of pressing down on the actuator and inhaling the resultant mist simultaneously [66]. If patients do not have the required coordination to self-administer their medication using a pMDI, some of the drug is deposited in the mouth and at the back of the throat. As a result, less of the API will be inhaled than intended, so the correct dose will not be administered. This could potentially result in substandard treatment, leading to exacerbation or worsening of a patient's condition. pMDIs are formulated as a suspension or solution with one or more APIs along with a propellant. The propellant is used to generate the pressure required to form micron-scaled droplets for inhalation [67]. Furthermore, pMDIs deposit less of the drug into the lungs, due to the high particle exit velocity with the actuations [68]. This means that it is more difficult to formulate higher drug doses for use with this type of inhaler device.

drug doses for use with this type of inhaler device. DPIs are portable inhalers that operate by breath actuation, thus removing the need for coordination of inhalation with actuation required for pMDIs. Breath actuation involves the powder being released from the inhaler when the patient inhales with enough force, known as inspiratory flow. The inhalers are formulated to contain powder forms of drugs, and this solid-state gives the drug improved stability compared to pMDIs formulations [67]. The use of powders also allows for greater potential when formulating higher dose medications. Dry powder inhalers are available in three types: single-unit dose, multi-dose reservoir and multi-unit dose. The powder is stored in either capsules or sealed blisters that are broken during actuation. In general, patients tend to find that DPIs are portable inhalers that operate by breath actuation, thus removing the need for coordination of inhalation with actuation required for pMDIs. Breath actuation involves the powder being released from the inhaler when the patient inhales with enough force, known as inspiratory flow. The inhalers are formulated to contain powder forms of drugs, and this solid-state gives the drug improved stability compared to pMDIs formulations [67]. The use of powders also allows for greater potential when formulating higher dose medications. Dry powder inhalers are available in three types: single-unit dose, multi-dose reservoir and multi-unit dose. The powder is stored in either capsules or sealed blisters that are broken during actuation. In general, patients tend to find that multi-dose DPIs are preferable to single-unit dose DPIs, due to the convenience of not having to replace the capsule or blisters with every use of the inhaler.

multi-dose DPIs are preferable to single-unit dose DPIs, due to the convenience of not having to replace the capsule or blisters with every use of the inhaler. Nebulisers are less popular inhalation devices compared to the other two devices and are available in two forms. The most common nebuliser is the jet nebuliser, which works by passing a liquid formulation along with compressed air through a narrow tube into a wide chamber, which causes a reduction in pressure. This reduction in pressure then forms micron-sized droplets, which can then be inhaled. The alternative type of nebuliser Nebulisers are less popular inhalation devices compared to the other two devices and are available in two forms. The most common nebuliser is the jet nebuliser, which works by passing a liquid formulation along with compressed air through a narrow tube into a wide chamber, which causes a reduction in pressure. This reduction in pressure then forms micron-sized droplets, which can then be inhaled. The alternative type of nebuliser is the ultrasonic nebuliser, which breaks down the solution of the drug into inhalable droplets by piezoelectric vibrations [68]. Both types of nebulisers require a face mask or

is the ultrasonic nebuliser, which breaks down the solution of the drug into inhalable

mouthpiece, which are bulkier and less portable than the other two devices, and therefore, less convenient for frequent and repeated use [69]. The way nebulisers are formulated are unsuitable for some drugs, particularly for drugs that are unstable when in solution [70]. Nebulisers also take longer to administer the drug when compared to other drug delivery devices, due to the time taken for the drug to pass through the chamber into the mouthpiece or mask, and with inhalation over a prolonged period. The use of nebulisers can also result in the administration of less precise doses as there can be deposition of the API particles in the chamber and mouthpiece or face mask. In general, nebulisers tend to be used by patients who cannot use the other devices, due to difficulties with coordination associated with pMDIs and/or the inability to produce adequate inspiratory flow associated with DPIs. The main patient groups these affect are young children, elderly populations, patients with COPD and patients with CF [71].

There is a limited number of licensed inhaled antimicrobials that are only available for patients suffering from recurrent infections as in CF (Table 5). Other antibiotics, such as gentamicin and amikacin, can be nebulised for pulmonary drug delivery by using the solution for injection if required; however, these cases are for unlicensed use in CF patients only and not for use for respiratory infections [72]. As can be seen in Table 5, nebuliser solutions and inhalation powders can be used to formulate higher doses of drugs, and hence, these are the devices currently used on the market. Tobramycin, colistimethate sodium and zanamivir are all available as dry powders for inhalation. Tobramycin inhalation powder is produced via an oil-in-water emulsion-based spray-drying process [69], and each particle consists of an amorphous tobramycin sulphate, and a gel-phase phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Tobramycin inhalation powder comes in a device known as a TOBI® Podhaler®, which consists of the engineered dry powder, the hard capsule, which is the primary packaging of the powder, and the device to administer the powder [70]. Other excipients used in the capsules include calcium chloride, which is used to stabilise the emulsion droplets during the spray drying and sulphuric acid for pH adjustment [71,73]. Each inhalation delivers 28 mg of tobramycin from the capsule to the lungs; however, four capsules are required for each dose, and therefore, eight inhalations per day. Colistimethate sodium is an inactive prodrug of colistin (polymyxin E), and is formulated as such to reduce the toxicity, typically nephrotoxicity, that is often experienced by patients when taking colistin. It is formulated to be administered using a Turbospin® inhaler, under the brand name Colobreathe® [74]. The powder is micronised and filled in polyethylene glycol (PEG)-gelatin hard capsules, which also contain purified water and sodium lauryl sulphate as excipients [75,76]. Zanamivir is an antiviral drug used for the treatment and prevention of influenza. It is formulated under the brand name Relenza® for its inhalation powder, and is produced via air-jet milling to contain 5 mg of zanamivir and 20 mg of lactose monohydrate particles as carrier particles in each double-foil blister [77].


**Table 5.** Licensed inhaled antibacterials currently available on the market.

#### **5. Pulmonary Drug Delivery Using Carrier Free Technology**

Large carrier particles, such as alpha-lactose monohydrate, are added to the API to form an inhaled powder with enhanced formulation properties. These properties include

the flowability and aerosol dispersion of the powder to maximise the number of API particles reaching the site of action. Lactose is available in a variety of grades for inhalation, with median diameters ranging from <5 µm to 250 µm [82]. During formulation, micronised API particles with an MMAD of between 1 µm and 5 µm are attached to the surface of these much larger carrier particles. Physical interactions hold the API in place, which prevents agglomeration of micronised API, due to cohesive forces. On inhalation, API is detached from the carrier and aerosolised, due to force exerted by the inhaler design in the form of friction, inertia and drag [83]. By retaining micronised API particle size, maximum dispersion of powder into the lungs is seen, due to increased aerosolisation [84]. This influences the drug administration in addition to preventing problems during the manufacturing process of the powder [85].

Carrier-based DPIs are the most prevalent formulation of DPIs and tend to be used to deliver relatively higher doses of API compared to pMDIs. However, certain mass ratios of API to carrier particles must be maintained to prevent agglomeration, as for example these can vary depending on the type of the drug, ranging from 1:2 to 1:4 in some cases [86]. For example, Relenza® requires a 25 mg powder to be inhaled so that to deliver 5 mg of zanamivir (i.e., a mass ratio of 1:5). These ratios create a maximum practical dose that can be delivered to the lungs through dry powder inhalation, making high dose delivery even more challenging. Even with carrier particles enhancing aerosolisation, deposition in the lungs is still relatively low, and as a result, the amount of API to reach the site of action is low. Fine particle fraction (FPF) refers to the fraction of particles with a size smaller than the respirable size divided by the total emitted dose of the inhaled API [87]. DPIs that do not contain carrier particles, therefore, have greater potential for delivering higher doses to the lungs.

Carrier free DPIs are a new-generation system that use special excipients and technologies, such as crystal engineering, to formulate micronised API particles whilst addressing the limitations explained above. These methods can increase the amount of lung deposition, with some formulations reporting FPF values of 63% [88]. One of the benefits of developing carrier free DPI formulations is that there can be an increased mass of API without having to retain large masses of the carrier particles. Therefore, this can maximise the dose available in each inhalation through maximising the amount of API that is deposited in the lungs. There are several dry powder formulations that have been or are currently being investigated to produce carrier free dry powder inhalers, as shown below in Table 6. As can be seen, different doses have been tested with some formulations delivering doses up to 50 mg, as displayed in Figure 2.

Many different methods have been used to develop dry powders for inhalation, also shown in Table 6, and each method produces variation to yield, cost of production and manufacture time. This data shows the difference in the size of the doses able to be formulated in DPIs for inhalation, compared to current doses available for inhalation (Figure 2). This also suggests that the use of carrier free inhalation technology can play a key factor in addressing the challenge of increasing doses of drugs for pulmonary drug delivery. Also noted is the wide range of drug classes that are being considered with regards to delivery via dry powder inhalation, including non-steroidal anti-inflammatory drugs (NSAIDs, i.e., ibuprofen and indomethacin), antimicrobials (i.e., tobramycin and netilmicin), antihistamines (i.e., ketotifen), and phosphodiesterase type 5 inhibitors (i.e., sildenafil). It was also noted that for many antimicrobials, there is the possibility of creating drug combinations within the same device—a useful tool for patients who require complex antimicrobial treatment.


Netilmicin 30 mg Cospray drying [102]

#### **Table 6.** Carrier free DPIs and the technology used to produce the powders.

#### **6. Pulmonary Drug Delivery on the Nanoscale**

Nanoparticulate formulations maintain nanoscale particles of the drug through encapsulation within inhalable size particles to prevent aggregation and ensure deposition of nanoparticles in the lung. There are several particle engineering methods to achieve nanoparticle formulation, such as the use of liposomes, solid lipids and polymers, which have been reviewed elsewhere [103–105]. It is common for these formulations to be stored as suspensions then delivered to the lung through a nebuliser; however, carrier free, dry powder nanoparticle formulations are also being developed.

Zhu et al., described the formulation of poorly soluble ivacaftor (Iva) as bovine serum albumin nanoparticles, which were then spray freeze-dried with different ratios of soluble colistin (Co) matrix and L-leucine [106]. All subsequent formulations showed high emitted dose following inhalation for both APIs (>90%); however, a range of FPF values (%) were seen corresponding to initial solid content in the formulations. Dissolution rates of the best performing nanoformulation and a jet-milled physical mixture of Iva and Co were then compared. After 3 h, the nanoformulation showed three times greater dissolution of Iva compared to the physical mixture control, with the concentration of Iva dissolved equivalent to highly soluble Co. Enhanced dissolution of Iva was attributed to the albumin maintaining Iva nanoscale particle size and amorphous form. The use of Co as a watersoluble matrix both enhanced the dispersion of Iva nanoparticles, as well as broadening the formulation clinical scope for patients with cystic fibrosis.

Doxorubicin nanoparticles (DNPs) were formulated as a colloid through emulsion polymerisation then spray freeze-dried with lactose to form particles of inhalable size [107,108]. DNPs showed increased survival rates compared to IV doxorubicin controls (including IV DNPs) in a rat model and much lower cardiotoxicity compared to doxorubicin DPI control. DNPs were also formulated with sodium carbonate to enhance the release of doxorubicin from the formulation. When inhaled, the effervescent formulations showed increased survival rates compared to DNP controls showing the importance of an active release mechanism from the formulation. In a slightly different approach, often utilising nanoscale dimensions to enhance inhalation properties, the use of porous particles has been investigated and was shown to improve efficacy of inhalation [109–111].

#### **7. The Design of Carrier Free Formulations Using Coamorphous Solid Dispersions (CACDs)**

Most solid APIs exist in a crystalline state held together by strong intermolecular bonds, and therefore, display good stability profiles (Figure 3). However, the crystalline state often shows poor solubility, due to the high energy required to break the crystalline lattice which creates a major problem for developing new APIs [112,113]. There has been much interest in the process of 'drug amorphisation', to address poor solubility, which involves the conversion from a crystalline state to an amorphous solid state. The amorphous solid state offers an improved solubility and dissolution rates as a result of possessing higher entropy [114–116]. The advantages associated with higher energy forms are often negated, due to recrystallisation to a more thermodynamically stable form during processing and storage [117–119]. This can limit applications, and so the production and maintenance of amorphous drugs with adequate stability remain a challenge. Formulation strategies based on solid molecular dispersions are being explored, including polymeric amorphous solid dispersions (PASDs), and more recently, coamorphous solid dispersions (CASDs).

PASDs incorporate low API loading within a compatible polymer to maintain the solubility advantage of amorphous systems through the formation of strong intermolecular attractions [120]. Examples of PASDs applications are seen in the combination of paclitaxel and polyvinylpyrrolidone (PVP), ritonavir and PVP, ciprofloxacin and polyvinyl alcohol [121–123]. However, the main limitation of this approach is the low level of API loading within the formulation, requiring increased dosing. In addition, many polymeric carriers are hygroscopic, which can lead to API recrystallisation while in some cases polymers

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can be unsuccessful in maintaining a good stability profile [123]. The applications and limitations of PASDs have been highlighted in a number of reviews [124–126].

**Figure 3.** Illustration of the crystalline state, amorphous state and coamorphous state and their characteristics. **Figure 3.** Illustration of the crystalline state, amorphous state and coamorphous state and their characteristics.

PASDs incorporate low API loading within a compatible polymer to maintain the solubility advantage of amorphous systems through the formation of strong intermolecular attractions [120]. Examples of PASDs applications are seen in the combination of paclitaxel and polyvinylpyrrolidone (PVP), ritonavir and PVP, ciprofloxacin and polyvinyl alcohol [121,122,123]. However, the main limitation of this approach is the low level of API loading within the formulation, requiring increased dosing. In addition, many polymeric carriers are hygroscopic, which can lead to API recrystallisation while in some cases polymers can be unsuccessful in maintaining a good stability profile [123]. The applications and limitations of PASDs have been highlighted in a number of reviews [124,125,126]. CASDs are a relatively new formulation approach through which the crystalline drug is amorphised thermally or mechanically and stabilised with a small molecular weight coformer. These systems are often stabilised through strong intermolecular hydrogen bonding and exhibit apparent stability, due to an increased glass transition temperature [124]. Glass transition temperature refers to the temperature at which below, the system exists in an unstable glassy state and above, the system exists in a rubbery state [125]. CASDs can be obtained through different preparation methods, such as freeze drying, quench cooling, milling and solvent evaporation methods [126,127]. Milling is an example of mechanical amorphisation where a direct mechanical impact on the drug causes a disturbance in the crystalline structure, forming an amorphous state. Quench cooling and CASDs are a relatively new formulation approach through which the crystalline drug is amorphised thermally or mechanically and stabilised with a small molecular weight coformer. These systems are often stabilised through strong intermolecular hydrogen bonding and exhibit apparent stability, due to an increased glass transition temperature [124]. Glass transition temperature refers to the temperature at which below, the system exists in an unstable glassy state and above, the system exists in a rubbery state [125]. CASDs can be obtained through different preparation methods, such as freeze drying, quench cooling, milling and solvent evaporation methods [126,127]. Milling is an example of mechanical amorphisation where a direct mechanical impact on the drug causes a disturbance in the crystalline structure, forming an amorphous state. Quench cooling and solvent evaporation are examples of thermal amorphisation where a crystalline solid state is prevented from reforming, due to molecular level interactions. Here, the drug is either in a liquid state following melting or dissolved to form a solution then rapidly cooled, or solvent removed rapidly. CASDs are believed to be able to provide an increase in drug kinetic solubility thanks to the high energy of the amorphous state driving faster dissolution [128]. In addition, improved physical stability is offered through an increase in the glass transition temperature (Tg) and the presence of intermolecular interactions between the API and coformer, as reported by several research papers [124,129]. CASDs are said to reduce hygroscopicity, as API hydrogen bonding regions are strongly bound to the coformer. Therefore, hydrogen bonding with water is less favourable, which helps to maintain stability and particle integrity.

solvent evaporation are examples of thermal amorphisation where a crystalline solid state is prevented from reforming, due to molecular level interactions. Here, the drug is either in a liquid state following melting or dissolved to form a solution then rapidly cooled, or solvent removed rapidly. CASDs are believed to be able to provide an increase in drug kinetic solubility thanks to the high energy of the amorphous state driving faster dissolution [128]. In addition, improved physical stability is offered through an increase in the glass transition temperature (Tg) and the presence of intermolecular interactions between the API and coformer, as reported by several research papers [124,129]. CASDs CASDs provide the opportunity for many types of formulations, such as drugexcipient and drug-drug combinations, and many have been reported as being successful. An example of a drug-drug combination is the spray drying of colistin with azithromycin to treat infections caused by multi-drug resistant bacteria [130]. A study conducted by Wang et al., showed an enhanced solubility profile between two APIs with low aqueous solubility: lacidipine and spironolactone [131]. Many other drug-excipient combinations are listed in Table 7, by which commonly used coformers in CASDs for pulmonary drug delivery include mannitol, sugars and amino acids, such as leucine [132,133].

are said to reduce hygroscopicity, as API hydrogen bonding regions are strongly bound to the coformer. Therefore, hydrogen bonding with water is less favourable, which helps to maintain stability and particle integrity. CASDs provide the opportunity for many types of formulations, such as drugexcipient and drug-drug combinations, and many have been reported as being successful. The spray drying technique is based on solvent evaporation which uses the processes of liquid atomisation, gas/droplet mixing and drying to create microparticles [134]. First, a liquid feed consisting of mixed components, typically pure API, excipient and a common solvent, is converted into smaller droplets via atomisation. Next, the droplets are sprayed downwards into a vertical drying chamber exposed to air or nitrogen at a temperature

higher than the solvent boiling point [135]. Within this drying chamber, the droplets rapidly shrink in size to produce dry microparticles.

Through spray drying, aspects of particle morphology, such as shape, size and surface properties, can be highly controlled, which deems the technique appropriate for formulating inhalation powders. Many papers have controlled particle morphology through variations to the inlet temperature, the spray nozzle diameter and properties of the feed solution, such as concentration and viscosity [136]. The inlet temperature (also known as the drying temperature) and the feed rate are parameters of high importance as they control the rate of evaporation and drying load, which determine particle properties and product yield [137]. It is common to see outlet temperatures ranging from 70 ◦C to 105 ◦C with inlet temperatures ranging from 100 ◦C to 210 ◦C. A faster drying rate could lead to a higher product yield, as there are fewer particles adhering to the chamber walls, which may also implicate that solvent evaporation is not complete. The spray drying process is highly applicable for large scale manufacture, where the integrity of formulation is retained despite 'upscaling' [138]. Altogether, spray drying offers a great opportunity to incorporate a number of excipients into one formulation whilst improving the physical stability of the particles.

**Table 7.** Application of coamorphous solid dispersions prepared using different preparation methods. The inclusion of PASD of ciprofloxacin was shown to identify the apparent benefits of polymeric solid dispersions compared to coamorphous solid dispersions.


#### **8. Examples of Coformers as Components of CAMs**

L-leucine has been widely used in many studies to improve the aerosolisation properties of particles, due to its antihygroscopic effect and its ability to generate coarse particles, following surface enrichment. Momin et al., formulated spray dried kanamycin with various concentrations of L-leucine, showing the 5% L-leucine formulation to have the best improvement in aerosolisation properties [146]. Likewise, significant increases in aerosoli-

sation properties were recorded for the other ratios of L-leucine. Other papers also reported L-leucine as a well-rounded excipient for improving the aerosolisation properties of powder particles. Interestingly Mangal et al., formulated spray dried azithromycin with various concentrations of L-leucine yet reported no significant change to powder morphology or FPF (*p* > 0.05) [132]. In addition, there was no significant increase seen in the percentage emitted dose (*p* > 0.05); however, an increased in vitro dissolution rate was reported. A lack of change to FPF and emitted dose can be explained when considering particle surface morphology and composition. It is believed that the more corrugated a particle surface is, the greater the resulting aerosolisation properties. This process is attributed to the success of L-leucine in improving aerosolisation. Chen et al., reported how changes to the composition of PASDs result in differences to the surface composition of particles, and therefore, flowability and aggregation [136]. Perhaps the surface enrichment of azithromycin with L-leucine for this formulation was poor, resulting in little change to particle surface morphology when compared to the control. When using L-leucine as a coformer, it is important that drying causes the distribution to the outer regions of droplets, causing recrystallisation of L-leucine on the particle surface, creating a corrugated effect [147]. This concept has also been documented by McShane et al. [138]. The impact of spray drying conditions on particles morphologies was recently highlighted in a different study by which spray dried coamorphous ciprofloxacin tartrate salt was shown to exhibit improved properties compared to ciprofloxacin alone [148].

The distribution of the components within the droplet during spray drying is said to be dependent on physicochemical properties of the components, such as solubility, the diffusivity of components and hydrophobicity [149]. These properties ultimately result in the final composition of the surface of spray dried particles. In the example mentioned above of azithromycin with L-leucine, the solubility of azithromycin is very low, and it likely had an impact on the distribution of L-leucine within the CASD. Li et al., conducted in vitro study to assess deposition, by which, spray drying azithromycin with mannitol resulted in low FPF values ranging between 38–42% [150]. Signifying the impact of preparation method and locality of excipients, Padhi et al., suggested that by increasing the ratio of L-leucine may improve surface enrichment, which would result in a substantial increase in the FPF [151]. In a different study, it was proposed that the presence of hydrophobic azithromycin helps to control humidity to prevent reduction in FPF [130].

Hassan et al., suggested that surface enrichment of spray dried materials is dependent on solvent evaporation rate and diffusion coefficients of the solutes [152]. Based on computational fluid dynamics, it was shown that the maximum rate of solvent evaporation proportionally affected MMAD [153]. It is believed the flow of the solute equates to that of the solvent; therefore, the movement of solute within a droplet is highly linked to solvent flow. Lower evaporation rates have a more significant impact on redistribution to droplet surface for solutes with high diffusion coefficients [149,152]. Shetty et al., when using water as a solvent, concluded that a low inlet temperature (<120 ◦C) was not considered, due to the possibility of it not being sufficient for drying [141]. Chen et al., also documented lower surface enrichment occurring, due to a reduction in the inlet temperature [136]. It is believed that the inlet temperature influences the particle shape, as well as surface enrichment. The inlet temperature indirectly affects the outlet temperature, and a lower outlet temperature is believed to result in a more spherical particle shape, which is detrimental to FPF [154,155].

Benke et al., reported spray dried meloxicam potassium (MXLspd) that was compared to the carrier-based meloxicam and lactose monohydrate InhaLac® (µMXL + IH70) [21]. As expected MXLspd showed an improvement in FPF < 5 µm (59.47%) compared to µMXL + IH70 with an FPF < 5 µm (24.99%). In addition, µMXL + IH70 had an MMAD of 7.18 µm, therefore unfavourable for pulmonary drug delivery. When assessing the impact of humidity on FPF, authors observed a significant decline (*p* < 0.0001) observed in the FPF (<4.9 µm) between cospray dried ciprofloxacin with lactose when stored at 20% RH and 50% RH after 10 days. This may be due to the caking observed, which led to larger powder

particles formed. Similar observations were reported by Guenette et al., which showed that larger lactose particles led to reduced flow properties [156]. The larger particle size may have occurred, due to particle agglomeration when stored at a higher RH. These examples highlight that for highly aerosolised powder, it is necessary to maintain the stability of spray dried CASDs when hygroscopic coformers are used. This is because of the presence of the excipient such as L-leucine, which has been reported to combat moisture-related stability issues, due to its hydrophobic nature [157]. Part of the reason why CASDs are successful is that the coformer changes from being a passive carrier to an essential constituent in the formulation [138].

In a separate study, Lababidi et al., further examined the success CASDs had in pulmonary drug delivery by which azithromycin and ciprofloxacin were spray dried with L-leucine and n-acetyl cysteine (NAC), an amino acid derivative and known mucolytic [129]. Mucolytics are used in combination with antibiotics to dissolve thick mucus found in the CF lung [157,158]. When mucolytics, such as NAC, are used as CASDs coformers, the formulation benefit becomes two-fold, creating a more effective treatment with better formulation characteristics. As mentioned before, the addition of L-leucine is aimed at reducing particle cohesion and increasing aerosolisation properties. However, in the study conducted by Lababidi et al., the main aim of adding NAC was to the formulation was to dissolve mucus, enhancing antibiotic effect against the bacteria. The authors showed that azithromycin/nacetyl cysteine combination reduced *P. aeruginosa* biofilm by 25% at a concentration of 0.3 µm/mL compared to azithromycin and n-acetyl cysteine alone.

Forming amorphous dispersions composed of antimicrobials through complex formation with metal cations has been explored as a method for enhancing formulation characteristics of inhaled antimicrobials. Lamy et al., produced inhalable dry powder microparticles of ciprofloxacin, complexed with calcium or copper ions (Cip-Ca; Cip-Cu), through spray drying [159,160]. The resulting powders were amorphous, and both calcium and copper formulations showed similar shell-like morphology, characteristic of spray dried materials, and similar in vitro solution properties. When comparing pharmacokinetics in vivo, both formulations showed enhanced lung retention compared to the ciprofloxacin control, suggesting that forming metal counterion complexes can reduce the rate of transcellular diffusion from the lung fluid. It was also seen that the ratio of ciprofloxacin in epithelial lining fluid to plasma was five times greater for the Cip-Cu than Cip-Ca formulations. The authors linked this with the strength of complex which had formed, corresponding to: The enhanced stability of the Cip-Cu, a slower rate of absorption into plasma and longer elimination half-life. Thus, antibiotic lung retention can be controlled depending on the metal ion selected, leading to an optimised concentration in lung fluid.

The influence of metal salts on the inhalation properties of levofloxacin has also been analysed. Barazesh et al., cospray dried levofloxacin with four metal chloride salts with and without leucine [161]. Sodium (Na) and potassium (K) monovalent salts were compared with magnesium (Mg) and calcium (Ca) divalent salts. When measuring aerosol properties, most effects was seen for formulations containing the highest measured percentage of metal ion (i.e., 20% *w*/*w*). It was also observed that the addition of divalent salts reduced the FPF of formulations and caused higher water retention post spray drying. The authors concluded that the highly hygroscopic divalent metal salts are likely to uptake more water, leading to local crystallisation and particle aggregation. Monovalent salts at 20% *w*/*w* showed a higher FPF compared to lower concentrations. With respect to leucine formulations, FPF was significantly higher, corresponding to the characteristic corrugated effect seen when spray drying with leucine.

#### **9. The Design of Carrier Free Formulations Using Cocrystals**

The formulation of carrier free dry powders can be achieved by a variety of methods. These include the formation of salts, such as the generation of a sildenafil-citrate salt [92], forming complexes with cationic groups, such as the generation of indomethacin

and polylysine microparticles [89], and forming CASDs, such as with ciprofloxacin and colistin [101]. Another route being investigated is the generation of cocrystals and seems to be a promising method of improving physicochemical properties if other methods are not suitable. For example, with the formation of salts, if the API is neutral, and therefore, does not contain an acidic or basic group that can be ionised, salts cannot be practically formed [162]. Cocrystallisation may also be seen as a more desirable method of physicochemical enhancement than others, for example, CASDs, due to enhanced physical and chemical stability [163].

Pharmaceutical cocrystals are multicomponent crystals formed between an API and pharmaceutically acceptable coformer. API and coformer exist in a fixed stoichiometric ratio held together through [164] non-covalent and non-ionic interactions. These interactions are intermolecular in nature and include hydrogen bonding, halogen bonding, π-π stacking and van der Waal forces [165]. Hydrogen bonds are considered the 'key interaction' for cocrystallisation, due to their strength, directionality and being commonly found in organic molecules [166]. Therefore, cocrystals are distinct from polymorphs, due to multiple components, and salts, due to lack of ionisation, and CASDs, due to their crystalline nature. The formulation of APIs as cocrystals is regarded as a physical modification to improve physicochemical properties, which maintains the API pharmacodynamic profile [167]. Since the cocrystal is a unique crystalline entity, it has unique physical properties, including solubility, dissolution, flowability and stability. In general, coformers are chosen based on molecular compatibility, as well as possession of desired physical property change. For example, to improve an API's solubility, one would choose a coformer with compatible functional groups, as well as high solubility [168].

Cocrystallisation is currently attracting considerable interest, due to the improved physicochemical properties that can be introduced to the API [169,170]. For example, cocrystals of meloxicam–succinic acid were combined with PEG 4000 to enhance aqueous solubility [171]. Cocrystals have largely been studied with a focus on improving oral drug delivery, and it is only recently that studies have begun to explore how cocrystals can be used for other administration routes, including pulmonary drug delivery. It is important to focus on all changes to API physical properties rather than flowability and dispersion alone. For example, altering solubility and dissolution will influence retention and epithelium wall permeation which will determine either a local or systemic effect. Selecting the correct coformer can increase aerosolisation properties, as well as maintaining the balance between API retention in the lung and API clearance through various mechanisms [172].

M Karashima et al., produced micronised powder formulations of itraconazole for inhalation using cocrystallisation and jet-milling [163]. These cocrystal formulations showed superior aerodynamics compared with itraconazole control put through the same processing and amorphous itraconazole spray dried with mannitol corresponding with reduced particle size. Also tested were the intrinsic dissolution rates of cocrystal formulations compared to itraconazole and amorphous itraconazole in mock lung surfactant. After 1 h, the dissolved concentration of itraconazole from cocrystal formulations were between 5 and 10 times higher than itraconazole and the amorphous form. The authors reported higher plasma concentrations (Cmax) compared to itraconazole and the amorphous form of itraconazole. Comparing this finding with intrinsic dissolution data, supports the conclusion that the extent of dissolution in the lungs influences the extent of systemic partitioning.

Alhalaweh et al., produced several inhalable powders of theophylline through cocrystallisation with nicotinamide, urea and saccharin [173]. These were compared against theophylline control, but also against cocrystals formulated with lactose carrier. The authors comment that cocrystal formulations showed more favourable aerosolisation with less API loss, due to impaction. However, not all cocrystal formulations were better aerodynamically compared to the control. This highlights the unpredictable nature of coformer selection in cocrystal formation and the importance of considering all aspects of physical property changes. Tanaka et al., produced a theophylline:oxalic acid cocrystal through a combination of spray and freeze drying [174]. When compared to the control,

the resulting formulation showed resistance to hygroscopicity and polymorphic transition whilst maintaining good pulmonary delivery. The authors attribute this stability to the low recorded energy state and close intermolecular interactions within the cocrystal.

API solution properties are a key component to determining API destination following inhalation. One study investigated how the properties of 5-fluorouracil differ from three different cocrystals created with gentisic acid (FUGA), 4,5 dihydroxybenzoic acid (FUBA) and 4-aminopyridine (FUPN). This study determined that the intrinsic dissolution of all three cocrystals were increased compared to pure 5-fluorouracil in both pHs of 1.2 and 6.8, with the largest difference being between 5-fluoruracil and FUPN. 5-fluorouracil had an intrinsic dissolution rate (IDR) of 0.12 mg cm−2/s in pH 1.2 and 0.18 cm−2/s in pH 6.8, whereas FUPN had an IDR of 0.7 mg cm−2/s in pH 1.2 and 0.92 mg cm−2/s in pH 6.8, showing more rapid dissolution from the cocrystal forms of 5-fluoruracil than the pure API [175]. Increased dissolution has also been observed in three diacerein cocrystals with isonicotinamide, nicotinamide and theophylline (DA-ISO), (DA-NIC) and (DA-THE). Diacerein had an IDR of 0.065 mg cm−2/min compared to 0.216 mg cm−2/min, 0.284 mg cm−2/min and 0.137 mg cm−2/min for DA-ISO, DA-NIC and DA-THE, respectively. This again shows that each cocrystal had a higher IDR value than the pure API molecule, suggesting a faster rate of dissolution [176].

Another study demonstrated the effects that cocrystals have on the pharmacokinetic profile of a drug. Daidzein and three cocrystals, with isonicotinamide (DIS), cytosine (DCYT) and theobromine (DTB) as the coformers, were compared. It was found that all three cocrystals had higher Cmax values, than daidzein (870.5 ng/mL), with DIS possessing the highest Cmax (1848.7 ng/mL). In addition, the time taken to reach the Cmax (Tmax) values for each cocrystal was 3 h compared to 4 h for pure daidzein [177]. Table 8 summarises a variety of cocrystal formulations, the method of manufacture used and the difference between API and coformer molecular weight. Shown is that cocrystallisation applied to a great variety of different APIs produced through a wide range of different production methods. Of note are the studies which use methods applicable for producing inhalable dry powders, such as spray drying, spray freeze drying and jet milling. It also gives an example of the wide variety of successful coformers available for cocrystallisation, suggesting a great variety in physical property variations even for the same API. Examples of drug:drug cocrystals are listed to show the possibility of creating synergistic therapies through a single phase crystalline powder, i.e., cocrystals. As shown in Table 8 and Figure 4, all coformers have molecular weights lower than alpha-lactose monohydrate (342.3 g/mol) and either much smaller or similar molecular weight to the API. This demonstrates how formulations containing coformers take up less volume in comparison to carrier-based delivery systems. As discussed earlier, the volume which lactose particles take up in formulations restricts the quantity of API that can be practically delivered. By avoiding the use of carrier particles, through coformer based methods, greater amounts of API particles can be formulated within a DPI device, possibly increasing the dose of API per actuation.


Sulfaguanidine Thiobarbutaric acid Slow evaporation 214.24 144.15 [193] 1,10-phenanthroline 180.20

**Table 8.** Cocrystalline APIs, their coformers, technology used and molecular weights.

**Figure 4.** A scatter graph of the data from Table 8 comparing the molecular weights of the APIs and their coformers. **Figure 4.** A scatter graph of the data from Table 8 comparing the molecular weights of the APIs and their coformers.

**10. Conclusions** 

#### **10. Conclusions**

Coamorphous solid dispersions and cocrystals are attractive physical structures that have been used to improve the physicochemical properties of inhaled APIs to target infections and lung cancer. When developing pharmaceutical cocrystals/coamorphous for inhalation, careful selection of the coformer should be followed to improve the efficacy to deliver drugs to the lungs. It is likely that by creating cocrystals/coamorphous dispersions, the need for carrier particles in DPIs can be eliminated, and therefore, a greater amount of the API can be delivered. This would result in an increased dose in each inhalation, allowing lower potency drugs that require higher doses to be formulated for inhalation. It is important to understand the complexities of pulmonary drug delivery when considering the formulation of drugs for inhalation to ensure the drug particles have adequate aerosolization properties. This is necessary to ensure the particles can reach deep into the lungs at the site of action, without being aggregated or immediately exhaled from the lungs. It is important to consider all steps throughout drug development process, including the excipients that are used and the devices to administer the drug, because some of the devices have shown clear advantages when it comes to overcoming the challenges with formulating inhaled APIs. The complexity of diseases imposes greater challenges demanding more studies to optimise particles properties for better deposition. Ultimately, it is crucial to match the properties of the particles to the desired site of action, such as the case when targeting bacterial biofilms and cancer.

**Author Contributions:** Conceptualization, H.A.-O.; investigation, H.A.-O., A.G., T.H., S.O.; data curation, H.A.-O., A.G., T.H., S.O.; writing—H.A.-O., T.H.; writing—review and editing, H.A.-O.; supervision, H.A.-O.; project administration, H.A.-O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

#### **References**

