Toxicity of orally inhaled drug formulations at the alveolar barrier: parameters for initial biological screening

Figures & data

Figure 1. Presentations of surfactant. Alveolar type II cells contain surfactant as intracellular lamellar bodies (a). In the alveolar hypophase lamellar bodies with different numbers of myelin layers (b) and membranes arranged as tubular myelin (c) are seen. On top of the hypophase the surfactant is arranged as multilayer (double arrow) or bilayer (one arrow) (d). Examples were taken from (Schurch et al., 1995; Walski et al., 2009).

Figure 2. Illustration of cell mobility as result of speed and persistence.

Figure 3. Dissolution and enzymatic degradation of drug-loaded particles by alveolar type I cells (AT1) with indication of the main degrading and metabolizing enzymes. Particle dissolution is slow because particles are only partly immersed in the alveolar lining fluid (hypophase). AT1 cells secrete proteases (EP24.15) in the hypophase. AT1 cells possess various membrane-associated proteases (CPM, APA, APB, APN, EP24.11, and γ-GT), and the lysosomal protease cathepsin D (CatD). The metabolizing enzymes CYP1A1 and CYP2B1 are located in the endoplasmic reticulum. The main transporter MDR-1/P-pg is located at the apical plasma membrane. Abbreviations: CPM: carboxypeptidase M; APA: aminopeptidase A; APB: aminopeptidase B; APN: aminopeptidase N; EP24.11: endopeptidase 24.11; γ-GT: gamma-glutamyltransferase; Cyt: cytoplasm; N: nucleus; P-pg: P-glycoprotein.

Figure 4. Fate of API formulations in the alveoli as scheme (a) and flow diagram (b). a: Particles can dissolve and diffuse across the alveolar epithelium. Alveolar epithelial cells (ACs) actively ingest small particles while larger particles are phagocytized by AMs. Abbreviations: EC: endothelial cell; P: particle. b: When API particles dissolve fast, either therapeutic levels can be reached in the blood or degradation in alveolar epithelial cells occurs leading to insufficient activity. When dissolution is insufficient small API particles can be taken up by the alveolar epithelial cells and be degraded. AMs can ingest and degrade larger particles that persist at the alveolar barrier. Degradation may result in low systemic drug levels and be counteracted by increase of the applied dose. Persistent particles may also activate AMs and cause inflammation and tissue transformation.

Table 1. Differences between rodent and human lungs.

Parameter	Rodent	Human
Pleura	Thin with few lymphatic vessels	Thick with many lymphatic vessels
Lung architecture	• Left lung with one lobe • Separation of lobes by little connective tissue • Monopodial airway branching pattern • Smooth muscle fibers do not extent past the bronchiole-alveolar duct junction	• Left lung with two lobes • Separation of lobes by large amount of connective tissue • Dichotomous branching pattern • Smooth muscle fibers extent into the first generation of alveolar ducts
Cell composition	• Serous cells present • Club cells in terminal bronchioles	• No serous cells • No club cells in terminal bronchioles
Mucus clearance	Slower velocity than in humans	Higher velocity than in rats
Metabolization	Higher enzyme content than humans	Lower enzyme content than rats

Table 2. Overview on dissolution methods used for orally inhaled formulations.

Particle collection	Dissolution	Reference
Impactor, polycarbonate membrane stainless steel collection base	USP Type II paddle apparatus	Son and McConville (2009); Son et al. (2010)
Powder sealed in membrane	USP Type I basket apparatus	Jaspart et al. (2007)
Impactor, regenerated cellulose membranes	USP Type II paddle apparatus, USP Type IV flow through cell, Franz diffusion cell	May et al. (2012)
Impactor, paper filter	USP Type IV flow-through, Franz diffusion cell, modified Franz cell, beaker method (stirrer)	Wang et al. (2016)
Impactor, glass fiber filter	USP Type IV flow through cell	Davies and Feddah (2003)
Impactor, regenerated cellulose membrane	USP Type IV flow through cell, Franz cell	Jensen et al. (2011)
Impactor, polyvinylidene difluoride membrane	Transwell system	Arora et al. (2010)
Impactor, nitrocellulose membrane	USP Type IV flow through cell, Franz cell	Salama et al. (2008); Salama et al. (2009)

Table 3. Composition of simulated lung fluids used for dissolution studies.

Composition	Gamble solution 1	Gamble solution 2	Gamble solution 3	Gamble solution 4	Gamble solution 5	Modified Gamble solution	Pseudo alveolar fluid	Simulated lung fluid	Artificial interstitial fluid	Synthetic serum	SELF	SLF (mM)
MgCl2⋅6H2O (mg L^−1)	95	203	203				212	212	203		200
NH4Cl (mg L^−1)				535	118	5300				535		10
NaCl (mg L^−1)	6019	6019	6019	6786	6400	6800	6415	6400	6193	6786	6020	11,6
KCl (mg L^−1)	298
CaCl2 (mg L^−1)				22						22
CaCl2⋅2H2O (mg L^−1)	368	368	368		225	290	255	255	368		256	0,2
Na2SO4 (mg L^−1)	63	71	71				79		71		72
H2SO4 (mg L^−1)				45		510				45		0,5
Na2SO4⋅10H2O (mg L^−1)								179
Na2HPO4 (mg L^−1)	126				150		148	148	142		150
NaH2PO4 (mg L^−1)		142	142	144						144
NaH2PO4⋅H2O (mg L^−1)						1700						1,2
H3PO4 (mg L^−1)						1200
NaHCO3 (mg L^−1)	2604	2604	2604	2268	2700	2300	2703	2700	2604	2268	2700	27
Na2CO3 (mg L^−1)						630
NaHC4H4O6⋅2H2O (sodium hydrogen tartrate dihydrate) (mg L^−1)							180	180
H2C6H5O7Na⋅2H2O (sodium dihydrogen citrate dihydrate) (mg L^−1)	97	97	97				153	153				0,2
CH3CHOHCOONa (sodium citrate) (mg L^−1)	574			52	160		175			52
Citric acid⋅H2O (mg L ^1)						420
NaOCOCOCH3 (sodium pyruvate) (mg L^−1)							0,72	172
NH2CH2COOH (glycine) (Gly) (mg L^− 1)				375	190	450	118	118		450	376	5
L-Cysteine (C3H7NO2S) (mg L^−1)				121							122	1
DPPC (dipalmitoyl phosphatidyl choline) (C40H80NO8P) (mg L^−1)			200		200				200		100
CH3COONa⋅3H2O (sodium acetate trihydrate) (mg L^−1)		953	953						952
Sodium acetate (CH3COONa) (mg L^−1)						580
HOC (COONa) (CH2COONa)2⋅2H2O (sodium citrate dihydrate) (mg L^−1)						590			97
C3H5NaO3 (sodium lactate) (mg L^−1)								290
KCl (mg L^−1)		298	298						298		298
Potassium hydrogen phthalate (C8H5KO4) (mg L^−1)						200
C14H23N3O10 (DTPA) (pentetic acid) (mg L^−1)				79								0,2
C21H38NCl (ABDAC) (mg L^−1) (benzalkonium chloride)				50								50
Ascorbic acid (mg L^−1)											18
Uric acid (mg L^−1)											16
Glutathione (mg L^−1)											30
Albumin (mg L^−1)											260
Mucin (mg L^−1)											500
pH (adjustment with HCl)	7.4			7.3		7.4	7.6		7.4	7.3	7.4
Reference	Colombo et al. (2008)	Moss (1979); Ungaro et al. (2009)	Yang et al. (2008)	Wragg and Klinck (2007)	Julien et al. (2011)	Gray et al. (2010)	Takaya et al. (2006)	Taunton et al. (2010)	Stopford et al. (2003)	Kanapilly et al. (1973)	Boisa et al. (2014)	Cheng et al. (1997)

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