Microsomes in Metabolic Stability Assays: Role in Phase I Metabolism and Phase II Metabolism

IPHASE Biosciences Products

Microsome

Microsomes are subcellular vesicles derived from the endoplasmic reticulum of disrupted cells, primarily hepatocytes (liver cells). They are rich in drug-metabolizing enzymes, notably the cytochrome P450 (CYP) family, which play a crucial role in the oxidative metabolism of various compounds. Metabolic stability assays using microsomes are integral to early drug development because they help predict in vivo pharmacokinetics. By measuring the rate of metabolism in vitro, researchers can estimate the intrinsic clearance and extrapolate these findings to anticipate how a drug might behave in humans. Such assays not only facilitate the screening of numerous compounds in a high-throughput manner but also aid in the identification of metabolic pathways and potential drug–drug interactions. The combination of microsomes from various tissues allows for a comprehensive understanding of both hepatic and extrahepatic metabolism, which is crucial for optimizing drug design and ensuring safety before clinical trials. The common microsomes in metabolism stability assay includes: liver microsome, intesine microsome/intestinal microsome, lung microsome, kidney microsome.

Liver Microsomes

Liver microsomes are particularly rich in cytochrome P450 enzymes and related oxidoreductases. Their high enzymatic content makes liver microsomes the preferred model for evaluating metabolic stability. During an assay, a drug candidate is incubated with liver microsomes in the presence of an essential cofactor such as NADPH, and the rate at which the parent compound is metabolized is monitored over time. The information gleaned from these experiments is used to calculate intrinsic clearance, an important parameter that helps predict how quickly a drug may be eliminated in vivo. Because liver microsomes can be pooled from multiple donors, they provide a reliable and reproducible system that minimizes the variability inherent in biological systems.

Intestinal Microsomes/ Intestine Microsomes

Intestinal Microsome, sometimes also known as intestine microsome, although less abundant in metabolic enzymes compared to their hepatic counterparts, are equally important in the context of first-pass metabolism. After oral administration, a drug must pass through the intestinal wall where it may undergo significant enzymatic transformation before reaching systemic circulation. The metabolic activity in the intestinal microsomes can greatly influence a drug’s bioavailability, and data obtained from these assays are essential in developing strategies to overcome pre-systemic metabolism.

Skin Microsomes

Skin microsomes are prepared from skin tissue and exhibit drug-metabolizing activities, including those of CYP enzymes. While the specific enzymatic activity in skin is typically less than 10% of that found in the liver, skin plays a significant role in the biotransformation of transdermal xenobiotics. Utilizing skin microsomes in assays can provide insights into the metabolism of compounds that are topically applied or absorbed through the skin.

Lung Microsomes

lung microsomes are prepared from pulmonary tissue and are used to investigate the metabolism of compounds that are administered via inhalation or that exert their effects within the respiratory system. While the concentration of cytochrome P450 enzymes in the lung is lower than in the liver, the lung remains a critical site for the metabolism of environmental toxins and inhaled medications. This model is particularly valuable in assessing tissue-specific metabolic transformations and potential local toxicities.

Kidney Microsomes

Kidney microsomes are isolated from renal tissues and provide insights into the metabolic processes that occur in the kidney. Since the kidney is not only an organ of excretion but also one that contributes to the metabolic clearance of certain drugs, the use of kidney microsomes in stability assays allows researchers to evaluate the formation of metabolites that could be linked to nephrotoxicity. In this way, kidney microsomes complement data from liver and intestinal studies, giving a broader perspective on a compound’s metabolic profile.

Testis Microsomes 

Testis microsomes are derived from testicular tissue and contain enzymes responsible for metabolizing endogenous and exogenous compounds. While less commonly used than liver microsomes, they can be relevant in studying the metabolism of substances affecting male reproductive health. Specific details on their use in metabolic stability assays are limited and may vary depending on the research focus.

Epididymis Microsomes

Epididymis microsomes are obtained from epididymal tissue and, like testis microsomes, are involved in the metabolism of certain compounds. Their application in metabolic stability assays is less prevalent, but they may be utilized in studies examining the metabolism of substances impacting male fertility and reproductive function. Detailed protocols and usage would depend on the specific objectives of the research.

Phase I Metabolism & NADPH regenoration system

Phase I metabolic reactions are primarily driven by CYP enzymes, and these reactions require a constant supply of reducing equivalents in the form of NADPH. To ensure that NADPH is available throughout the incubation period, a NADPH regeneration system is added to the assay. The NADPH regeneration system usually includes NADP⁺, glucose-6-phosphate, and the enzyme glucose-6-phosphate dehydrogenase, which together continuously convert NADP⁺ back to NADPH. This regeneration is essential because it sustains the redox reactions catalyzed by the CYP enzymes, allowing the microsomes to maintain their metabolic activity over extended periods.

Phase II Metabolism & UGT Incubation System

While liver microsomes are most commonly associated with Phase I metabolism, they can also be adapted to study Phase II metabolism such as glucuronidation. Glucuronidation is a conjugative process mediated by uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes, which are capable of attaching glucuronic acid to drugs or their Phase I metabolites. To facilitate glucuronidation in a microsomal assay, an UGT incubation system with the activated cofactor UDP-glucuronic acid (UDPGA) is added. The UGT incubation system usually consist UDPGA, protein of procymidin and D-glucuronosyl-1.4-lactone. Since UGT enzymes are membrane-bound and may be less accessible in the intact microsome, a pore-forming agent like alamethicin is sometimes included. Alamethicin increases the permeability of the microsomal membranes, thereby enhancing the access of UDPGA to the UGT enzymes and improving the efficiency of the glucuronidation reaction.

Buffer System

Throughout the entire process, the 0.1M PBS buffer plays a critical role in maintaining the stability and activity of the enzymes. This buffer system provides a stable pH and consistent ionic environment, which is crucial for preserving the structural integrity of both CYP and UGT enzymes. The consistent conditions afforded by the 0.1M PBS ensure that the reactions occur in a controlled manner, facilitating reliable measurement of metabolic stability and clearance.

Various Species Microsomes

Human Microsome

Human microsomes are arguably the most relevant in metabolic stability assays for drug development, as they closely mimic the human liver's metabolic environment. Human liver microsomes contain a high concentration of cytochrome P450 enzymes, which are responsible for the phase I metabolism of many drugs. These microsomes are extensively used to assess human drug metabolism, including enzyme-mediated drug interactions, metabolic stability, and the identification of potential toxic metabolites. Their use is crucial in early-phase drug development to ensure that a compound will have favorable pharmacokinetics in humans, with an eye toward avoiding liver toxicity or other adverse effects.

Non-human Primates Liver Microsomes

Non-human primate liver microsomes, typically rhesus monkey liver microsomes, marmosets monkey liver microsomes, or cynomolgus monkeys liver microsomes, are used in metabolism stability assays to assess how compounds are metabolized by enzymes in the liver. These microsomes contain cytochrome P450 enzymes and other metabolic proteins that facilitate phase I drug metabolism. Non-human primates are particularly valuable in preclinical studies because their liver enzyme profiles closely resemble those of humans, making them an important tool for evaluating the pharmacokinetics, metabolic stability, and potential toxicity of new drug candidates before human trials. They provide more relevant data on human-like metabolism compared to rodents, improving the accuracy of drug development predictions

Dog Liver Microsome

Dogs, particularly Beagle dogs, are commonly used in toxicology and pharmacokinetic studies. Dog microsomes, especially those derived from the liver, are valuable tools for understanding how a drug might be metabolized in a non-rodent mammal. Canine Liver Microsomes are often employed in preclinical safety testing to evaluate metabolic stability and the potential for drug-drug interactions. These microsomes can also help predict how drugs will be absorbed and processed in humans, providing insights into possible differences between humans and dogs in terms of drug metabolism.

Rat Liver Microsome

Rats are one of the most widely used laboratory animals for pharmacological and toxicological research, and their liver microsomes are crucial in metabolic stability assays. Rat microsomes are commonly used in early-stage drug development to evaluate the metabolism of experimental compounds, as their metabolic processes are well-understood. Although rats share several metabolic pathways with humans, there are notable differences in enzyme activity, particularly with respect to certain cytochrome P450 enzymes. Rat microsomes are frequently used to test a compound’s general metabolic stability and to assess potential pharmacokinetic issues, such as clearance rates and bioavailability.

Mouse Liver Microsome

Similar to rats, mice are extensively used in biomedical research, and mouse microsomes play a key role in metabolic stability assays. Mice are particularly valuable for studying genetic variations in drug metabolism due to their well-characterized genome. Mouse liver microsomes contain a range of cytochrome P450 enzymes, which makes them useful for evaluating how a drug might be metabolized across different genetic backgrounds. A particular strain of mice, BALB/c Nude, is known as a strain that lacks the thymus, making them immunodeficient. By using BALB/c nude liver microsomes, researchers can assess how a drug or compound is metabolized, the rate of its biotransformation, and its potential stability in the liver, which is crucial for predicting pharmacokinetics in humans. However, mice have some distinct metabolic pathways compared to humans, meaning that data from mouse microsomes should be interpreted with caution when predicting human metabolism. Mouse microsomes are often used in high-throughput screening to evaluate a large number of compounds quickly.

Hamster Liver Microsome

Hamsters, particularly Golden Syrian hamsters, are often used in metabolic studies due to their unique physiological characteristics. Hamster liver microsomes are helpful in evaluating drug metabolism and toxicology, particularly for compounds that may show species-specific metabolic profiles. Hamster microsomes are often used to study how drugs are metabolized in small mammals, offering insights into metabolic pathways that may not be fully understood in other rodent models.

BALB/c Nude Liver Microsomes

BALB/c nude mice are a strain characterized by a genetic mutation that results in a compromised immune system, making them valuable in research requiring immunodeficient models. Liver microsomes derived from these mice can be used in metabolic stability assays to study drug metabolism. However, specific information on the use of BALB/c nude liver microsomes in such assays is limited.

Gerbillinae Liver Microsomes

Gerbillinae liver microsomes are derived from gerbils, a small mammal species commonly used in toxicology and pharmacology studies. In metabolism stability assays, Gerbillinae liver microsomes are employed to evaluate how a compound is metabolized by the liver enzymes present in the microsomal fraction, particularly cytochrome P450 enzymes. These assays help determine the metabolic stability of drugs or chemicals, assessing their potential for biotransformation and elimination. Gerbils are sometimes used for these studies due to their specific metabolic profile, which can offer insights into species-specific differences in drug metabolism.

Minipig Liver Microsome

Minipigs are gaining increasing popularity in pharmacokinetic and toxicology studies due to their physiological similarities to humans, particularly in terms of liver metabolism. Minipig microsomes are often used in metabolic stability assays to provide data that more closely resembles human drug metabolism compared to rodent models. These microsomes are especially useful for studying drug absorption, distribution, metabolism, and excretion (ADME) in a model organism with a more human-like metabolic profile. Minipigs are particularly valuable in evaluating compounds that require a more accurate prediction of human metabolic responses.

Guniea Pig Liver Microsomes

Unlike other rodents, guinea pigs lack certain drug-metabolizing enzymes, such as cytochrome P450 2D, which can affect the way they process specific compounds. This makes guinea pig liver microsomes particularly useful for studying species-specific differences in drug metabolism. Their unique enzyme profile can offer insights into how a compound might behave in a species with limited metabolic pathways, and it can highlight potential risks or variations in drug metabolism that might not be observed in other models. This makes guinea pigs valuable for comparative toxicology and pharmacokinetic studies.

Feline Liver Microsome

Feline liver microsomes are used in metabolic studies to assess how drugs are processed in cats. Cats have unique metabolic characteristics, including limited glucuronidation activity, which can affect the metabolism of certain drugs. Because of this, cat liver microsomes are essential for studying how specific compounds behave in cats, particularly for veterinary pharmaceuticals. They are used to test for potential toxicity or metabolic issues in drugs intended for feline use and can help assess interspecies differences in drug metabolism when transitioning from human to animal studies.

Bovine Liver Microsome

Bovine microsomes, derived from cattle, are particularly useful in studying the metabolism of compounds used in livestock. Cattle have different metabolic pathways compared to humans, particularly in the activity of certain enzymes involved in phase I metabolism. Bovine liver microsomes are used to predict how veterinary drugs or agricultural chemicals will be metabolized in cattle. Additionally, these microsomes are utilized to investigate potential residues in meat and milk, helping to ensure food safety for human consumption. While bovine microsomes provide valuable data on livestock metabolism, they may not always be directly applicable to human drug development due to significant metabolic differences. Addition to bovine, horse liver microsome, sheep liver microsomes and goat liver microsomes are wildly used.

Poultry, Liver Microsome

Poultry microsomes, while less commonly used than those from mammals, can be useful in studies of avian drug metabolism. Common poultry microsomes includes duck liver microsomes, chicken liver microsomes, turkey liver microsomes and quail liver microsomes.  are employed to assess how drugs may be processed in avian species. This is particularly important in the development of veterinary drugs for poultry, as well as in environmental studies to understand the metabolism of chemicals that may enter the food chain through birds.

Fish Liver Microsome

Fish microsomes, especially Rainbow Trout Liver Microsomes, are employed in environmental and toxicological research. Fish are particularly sensitive to environmental pollutants, and their liver microsomes are valuable tools in studying the metabolic pathways involved in the detoxification of contaminants in aquatic ecosystems. Fish microsomes are also used to study the environmental impact of pharmaceuticals and industrial chemicals, helping to assess their potential to bioaccumulate and affect aquatic life.

Conclusion

Microsomes play a critical role in the early stages of drug development by providing essential insights into the metabolic stability and pharmacokinetics of drug candidates. Through the use of microsomal assays, researchers can assess phase I and phase II metabolic reactions, identify potential drug-drug interactions, and evaluate tissue-specific metabolism. The availability of microsomes from a wide range of species, including humans and animals, enables cross-species comparisons, enhancing the prediction of drug metabolism in humans and ensuring the safety and efficacy of new compounds. As the drug development process continues to evolve, microsomal models will remain a vital tool in the pursuit of safer and more effective medications.

Keywords: Metabolism stability, Phase I Metabolism, Phase II Metabolism, Phase I  Reactions, Phase II Reactions, Liver Microsomes, Intesine Microsomes, Intestinal Microsomes, Lung Microsomes, Kidney Microsomes, Skin Microsomes, Testis Microsomes, Epididymis Microsomes, NADPH Regenoration System, UGT Incubation System, UDPGA, 0.1M PBS, Tris-HCL, Human Microsomes, Cynomolgus Monkey Liver Microsomes, Rhesus Monkey Liver Microsomes, Marmoset Liver Microsomes, Dog Liver Microsomses, Canine Liver Microsomes, Rat Liver Microsomes, Mouse Liver Microsomes, Gerbillinae Liver Microsomes, BALB/c Nude Liver Microsomes, Guniea Pig Liver Microsomes Minipig Liver Microsomes, Feline Liver  Microsomes, Cat Liver Microsomes, Bovine Liver Microsomes, Duck Liver Microsomes, Fish Liver Microsomes, Rainbow Trout Liver Microsomes, Hamster Liver Microsomes, Rabbit Liver Microsomes, Turkey Liver Microsomes, Horse Liver Microsome, Sheep Liver Microsomes, Goat Liver Microsomes, Quail Liver Microsomes