The advent of modern technology has initiated rapid generation of new drug leads and formulation of novel therapeutics. Before translation into clinical settings, the drugs need to be assessed for their absorption, distribution, metabolism, and excretion (ADME) properties. The in vitro study of ADME properties predicts safety, toxicity, efficacy, and behavior of drugs for in vivo systems. Hepatocytes were commonly employed for these studies, until the subcellular fractions were discovered. Research on these fractions provides deeper insights on the drug metabolism with respect to drug stability, clearance rates, the underlying mechanisms of drug metabolism, and drug-drug interactions. The retrieved data indicates the half-life and bioavailability of the drug, thus aiding in the evaluation of therapeutic dosage. ADME studies are conducted on liver and intestine, but pulmonary tissue should also be incorporated for inhaled drugs and analysis of extra-hepatic metabolism.
Why the Need for The Lung Metabolism Study?
Respiratory diseases such as asthma, bronchitis, emphysema, COPD, etc., mandate the use of inhaled drugs. The inhalation route of drug enables tissue-specific drug delivery and action in the lungs, thereby enhancing the effectiveness as compared to other administration routes. The route also increases the bioavailability of the drug in the pulmonary tissue while potentially minimizing the drug-induced side effects. Studies have shown that inhaled corticosteroids and beta-2 agonists undergo biotransformation in pulmonary tissue. The hydrolysis of beclomethasone dipropionate, fatty acid conjugation of budesonide, glucuronidation of formoterol, sulfation of salbutamol, and methylation of cimaterol have been researched. Thus, the preclinical study on the metabolism of the inhaled drugs in the lungs is crucial. There are three Human lung subcellular fractions with their own repertoire of metabolic enzymes.
Human Lung Microsomes
The isolation of microsomes follows tissue homogenization and centrifugation at 9000 x g which pellets down cell debris (Fig 1). The supernatant is ultracentrifuged at 100,000 x g, yielding the pellet containing microsomes. Microsomes are small vesicles that retain the enzymes of the endoplasmic reticulum. These are phase I enzymes that transform the drug by oxidation, reduction, and hydrolysis reactions. Therefore, microsomes require exogenous NADPH cofactor for the oxidative enzymes. The biotransformation can lead to the activation of a prodrug, or drug modification for altered pharmacological activity. In hepatocytes, cytochrome P450 enzyme predominantly executes the phase I activity. Recent studies have revealed that cytochrome P450, flavin monooxygenase, and carboxyl esterases are the most metabolically active phase I enzymes in lung microsomes. Lung microsomes also demonstrate a higher metabolic rate, in comparison to other lung subcellular fractions.
Human Lung Cytosol
The extraction of cytosolic fraction undergoes the same procedure as microsomal isolation. The centrifugation of tissue homogenate is followed by ultracentrifugation, resulting in cytosolic fraction in the supernatant (Fig 1). It contains the cytosolic content comprising phase II metabolic enzymes, predominantly N-acetyl transferase and glutathione S-transferase. Phase II enzymes are responsible for the conversion of drugs as well as phase I metabolites. These enzymes increase the water solubility of the compound by conjugating it to different groups such as acetyl, glutathione, glycine, cysteine, sulfate, glucuronic acid, etc. Phase II enzymes are found in the cytosol except UDP-glucosyl transferase (UGT), which is located in microsomes. The cytosolic fraction requires addition of two cofactors: UDPGA for glucuronation and PAPS for sulfation. The cytosolic fraction is generally studied to gain understanding of the phase II enzymatic reactions. The most metabolic active enzymes in lung cytosol are glutathione S-transferase, sulfotransferase, and N-acetyl transferase.
Human Lung S9 Fraction
The differential centrifugation of the tissue homogenates at 9000xg separates cell debris and supernatant (Fig 1). The supernatant contains the S9 fraction. The S9 fraction consists of both microsomes and cytosolic fractions, which can be obtained by further ultracentrifugation. With a set of both phase I and phase II enzymes, S9 fractions offer a complete representation of cellular metabolic enzymes. It is particularly beneficial for studying both enzymes together, instead of separately in microsomes and cytosolic fractions. Therefore, it requires all the cofactors-NADPH for phase 1 enzymes as well as UDPGA and PAPS for phase II enzymatic activity.

Complications in Lung Subcellular Fraction
Enzyme Dilution
As compared to other tissues, pulmonary tissue is more heterogeneous in nature, thus necessitating the use of tissue models, multiple cells, or subcellular fractions. But the amount of metabolic activity varies among different pulmonary cells. The S9 fractions from whole tissue lysate provide a complete tissue representation. However, it dilutes the overall concentration of highly active metabolic enzymes, affecting the in vivo translation of the results. Therefore, the protein levels of the fraction are adjusted before use.
Overestimation of Clearance Rates
There has been concerns that increased substrate concentration in the study on subcellular fractions might overestimate the clearance rate. The fractions also don’t account for the drug absorption stage present in the tissues. Several studies have compared the results from cells to that of subcellular fractions and demonstrated equivalent results by both.
Application of Subcellular Fractions over Other Models
Several research projects have employed lung slices or perfused lung model in animals for evaluating drug metabolism in lungs. However, there is a significant difference in the metabolic activity of animals and humans. Primary cells have also been utilized, but they are difficult to culture, whereas cell lines do not exhibit the metabolic profile similar to cells despite the ease of their culture process. In such cases, subcellular fractions provide a better alternative. There are more advantages to the use of lung subcellular fractions than other models.
Heterogeneous Cells:
Over 90% of the liver is composed of a single cell type, that is, hepatocyte. But pulmonary tissue comprises almost 40 different cell types belonging to epithelial, endothelial cell, mesenchymal, and immune cells. These cells have their own set of metabolic enzymes, rendering the cellular model inaccurate. Subcellular fractions from whole tissue accurately represent the tissue’s metabolic enzyme profile.
High-Throughput Application:
ADME studies require assays in high-throughput format to allow screening of large numbers of compounds in short time span. The prompt acquisition of data can accelerate the discovery of new leads and the modification process of the tested drugs. Tissue and cellular models are labor-intensive and don’t provide the needed efficiency and speed. But subcellular fractions are amenable to high throughput screening of drugs. The use of subcellular fractions also has chances of automating the process, eliminating the human interventions.
Metabolite Identification:
In comparison to other models, lung subcellular fractions offer additional knowledge on the enzymatic reaction of the drugs and the resultant metabolites. This data is especially useful for quantitative structure-activity relationships (QSAR), which can adequately optimize the drugs for therapeutic use.
Availability:
Tissues, animal models, and cells are expensive to procure and maintain. On the other hand, subcellular fractions provide a cost-effective alternative. Genetic variability evident in other models, is absent in these fractions. Moreover, these fractions can be frozen and stored at -80˚C for long terms while also retaining enzymatic activity, which makes it cost-effective.
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Conclusion
ADME studies on lung tissue hold importance for inhaled drugs. Although tissue and cell models have been employed, they exhibit genetic variation and are rather expensive to obtain and maintain. Subcellular fractions of the liver and intestine have already been extensively studied, employed in the preclinical research. Thus, the subcellular fractions from lungs are also applicable for such studies. The heterogeneous nature of the pulmonary cells deems subcellular fractions derived from whole tissue as a more appropriate model for preclinical investigations. Kosheeka provides S9, microsomes, and cytosolic fraction enriched with metabolic enzymes derived from human as well as mice to accelerate your ADME studies and drug discovery process. We also offer matching (from same donor) pulmonary subcellular fractions i.e. microsomes, S9 and cytosol. These undergo robust quality checks for functionality, to provide high-quality subcellular fractions.
FAQs
What are subcellular fractions?
Subcellular fractions contain the different cellular components remaining after tissue or cell homogenization. They have found applications in the drug metabolism studies during preclinical research owing to their repertoire of metabolic enzymes. The subcellular fractions are categorized into microsomes, cytosol, and S9 comprising the enzymes of the endoplasmic reticulum, cytosol, and both endoplasmic reticulum and cytosol, respectively.
How are subcellular fractions isolated?
The isolation process requires centrifugation of a tissue or cell homogenate. The supernatant obtained contains S9 fraction. Further ultracentrifugation of the S9 fraction separates microsomes in the pellet and cytosol fraction in the supernatant.
Are lung subcellular fractions better than cells?
Research has demonstrated equivalent results with cells and subcellular fractions. But lungs possess heterogeneity in its cell type, deeming the one cell type or a mixture of cell types an inaccurate model for study. Subcellular fractions from whole lung tissue provide a better representation of tissue metabolic enzymes. Hence, lung subcellular fractions are better models than pulmonary cells.