CYP3A4 and CYP3A5: the crucial roles in clinical drug metabolism and the significant implications of genetic polymorphisms

Transcriptional regulation and post-transcriptional regulation

In cells, CYP3A4/5, as part of the cytochrome P450 enzyme family, metabolizes diverse compounds. Precise control mechanisms include transcriptional and post-transcriptional regulation, adjusting enzyme activities to physiological and environmental conditions by modulating gene expression and protein stability.

Regulation of enzyme activity and influencing factors

The intracellular activity of CYP3A4 and CYP3A5, critical drug-metabolizing enzymes, is intricately regulated and influenced by a myriad of factors, including drug interactions, hormone levels, genetic factors, dietary and environmental elements, disease states, and age and gender (Fernandez-Teruel et al., 2024; Peruzzi et al., 2024). Notably, drug interactions significantly impact CYP3A4/5 activity, with certain drugs capable of acting as inducers or inhibitors, directly influencing other drugs’ metabolic rates. Moreover, hormonal changes, particularly elevated cortisol levels during pregnancy, have been shown to directly affect CYP3A4/5 enzyme activity, leading to an increased rate of drug metabolism (Mlugu et al., 2022), which underscores the importance of considering hormonal effects on CYP3A enzyme activity during pregnancy, which can guide drug dose adjustments. Genetic factors, such as single nucleotide polymorphisms, also play a pivotal role in CYP3A4/5 expression levels and activity. For instance, CYP3A4*4 (A13871G) and CYP3A4*1B (A392G) have been found to decrease enzyme activity, resulting in slower metabolism. Wang et al. (2005) reported that CYP3A4*4 mutants reduce enzyme activity and drug metabolism, enhancing the efficacy of simvastatin. At the same time, Rodríguez-Antona et al. (2005) found that CYP3A4*1B mutants decrease the enzyme’s transcription rate, reducing its activity and increasing quinine metabolism. Dietary components, including lycopene, a naturally occurring carotenoid, and environmental chemicals can also significantly affect CYP3A4/5 enzymatic activity. Studies have demonstrated that lycopene can increase the metabolic activities of CYP2B, CYP2D, and CYP3A, rat homologous enzymes to CYP3A4/5, particularly at high doses (Nosková et al., 2020). Furthermore, disease states, such as diabetes, can influence CYP3A4/5 activity in the liver due to accompanying insulin resistance and metabolic disorders (Carr, Turner & Pirmohamed, 2021; Grandl et al., 2019; Lamba et al., 2002; Neuvonen, Niemi & Backman, 2006). Research using a diabetic rat model has shown that CYP3A4 activity decreases in the short term and increases in the long term (Wang et al., 2023), highlighting the complex effects of diabetes on CYP450 enzyme system activity and the need for precise medication use in diabetic patients. Lastly, age and gender are physiological factors that may alter CYP3A4/5 activity. Changes in drug metabolism rates in children and older people may result from the maturity or functional decline of the metabolic enzyme system (Hunt, Westerkam & Stave, 1992). Research indicates that aging male rats exhibit decreased activity of major CYP isoforms (Konstandi & Johnson, 2023), reflected in lower metabolism and higher drug substrate levels in the blood, emphasizing age as a crucial factor in CYP gene regulation. Additionally, gender differences, with women potentially having higher CYP3A4/5 activity due to elevated estrogen levels, may result in more rapid drug metabolism compared to men (Khatri et al., 2021). These multifaceted factors necessitate a comprehensive understanding and personalized approach to drug therapy, considering the complex interplay of influences on CYP3A4/5 activity.

Substrate categories of CYP3A4/5 metabolism

As indispensable members of the cytochrome P450 enzyme family, CYP3A4/5 are integral to the metabolism of a vast array of substrates, which can be categorized as follows: Steroid hormones, such as cortisol (Van Keulen et al., 2020), testosterone (Wu et al., 2022), and estradiol (Cheng & Zhou, 2000). Additionally, numerous lipophilic drugs necessitate CYP3A4/5 for metabolism, including benzodiazepines (Rodriguez-Antona et al., 2022), calcium channel blockers (Fuhr et al., 2022), antibiotics (Lenard et al., 2023), and specific antiepileptic medications (Zhao et al., 2021a), all of which are vital in the treatment of a myriad of diseases. While CYP3A4 and CYP3A5 share a broad substrate specificity, there are notable differences in their affinity and capacity to metabolize certain drugs. For instance, CYP3A4 is the primary enzyme involved in the metabolism of the immunosuppressant cyclosporine, which is crucial in post-organ transplant therapy (Zhai et al., 2024). In the metabolism of the immunosuppressant tacrolimus, where CYP3A5 is more efficient than CYP3A4 (Hannachi et al., 2024). Furthermore, the metabolism of drugs like nifedipine and erythromycin is primarily attributed to CYP3A4 due to its higher expression levels in the liver and intestine (Khatri et al., 2021; Szabó et al., 2024). In conclusion, the substrate categories metabolized by CYP3A4/5 encompass a broad spectrum of drugs and bioactive molecules with extensive clinical applications, significantly influencing individual drug metabolism and the potential for drug interactions.

Mechanisms of drug interactions

Since CYP3A4/5 is involved in the metabolism of a multitude of drugs, they play crucial roles in mediating drug interactions. The primary mechanisms of these interactions include competitive and non-competitive inhibition, as well as induction (Salvatorelli, Minotti & Menna, 2023; Lin et al., 2001). Competitive inhibition, a key mechanism at the microscopic level, occurs when a drug binds to CYP3A4/5 and competes for the same binding site as substrates, which are the drugs requiring metabolism (Wright, Chenge & Chen, 2019). This competition prevents substrates from binding to the enzyme, reducing their metabolic rate. Warfarin is metabolized by CYP3A4/5, when co-administered with another CYP3A4/5 substrate fluconazole, the metabolism of warfarin is significantly inhibited due to competition for enzyme active sites. The binding of inhibitors to the enzyme can be either reversible or irreversible, a characteristic that depends on the drug’s specific properties and binding mode (Ozawa, 2002). In addition to competitive inhibition, non-competitive inhibition also significantly impacts CYP3A4/5 activity. Tofacitinib is metabolized mainly by CYP3A4 and myricetin has a strong non-competitive inhibition on the metabolism of tofacitinib in CYP3A4 (Ye et al., 2024). Here, inhibitors bind to alternative sites on the enzyme, causing conformational changes or alterations in the enzyme’s active site structure, diminishing the enzyme’s catalytic efficiency (Hlavica, 2023). It is notable that the impact of the inhibitor on the enzyme also can be reversible or irreversible, depending on the specific properties of the drug and its binding mode (Fontana, Dansette & Poli, 2005; Varghese et al., 2015). Reversible inhibition of CYP3A4/5 typically involves non-covalent interactions such as hydrogen bonds or electrostatic interactions between the enzyme and the inhibitor (Scaglione, 2013). This type of inhibition is characterized by the ability of the enzyme to regain its activity once the inhibitor concentration changes. For example, midazolam, a substrate for CYP3A4/5, can be competitively inhibited by natural product goldenseal, which binds to the same active site and competes for enzyme binding, reducing the metabolic rate of midazolam. However, upon withdrawal of goldenseal, the enzyme activity can be restored, demonstrating the reversible nature of this inhibition (Nguyen et al., 2023). In contrast, irreversible inhibition results from covalent modification of the enzyme, leading to permanent inactivation. This can occur through various mechanisms, such as the formation of a covalent bond between the inhibitor and the enzyme’s active site, which cannot be reversed even after the inhibitor is removed. Understanding the reversibility of these inhibition mechanisms is crucial for predicting drug-drug interactions and their potential clinical implications. For instance, time-dependent inhibition (TDI) such as the famous antihypertensive drug mibefradil, which is a form of irreversible inhibition, can lead to more severe drug interactions as the enzyme activity remains suppressed even after the inhibitor is removed (Rougée et al., 2023). This is particularly relevant for drugs that are long-term medications or when drugs with TDI properties are co-administered. Clinical CYP3A4/5 Mediated Drug Interactions (Hohmann et al., 2024). At a microscopic level, these inducers may interact with transcription factors within the cell nucleus, initiating or enhancing the transcription of CYP3A4/5 genes. This process results in increased expression of the enzymes, which subsequently leads to elevated substrate metabolism rates. Moreover, drugs can also directly interact with CYP3A4/5 proteins, affecting their activity. Such interactions can induce conformational changes in the enzyme or obstruct substrate binding, influencing the overall metabolism of the substrate (Denisov et al., 2021). Collectively, these mechanisms highlight the multifaceted nature of drug interactions involving CYP3A4/5 and underscore the importance of understanding these processes to optimize drug therapy and minimize adverse effects.

Clinical CYP3A4/5 mediated drug interactions

Ketoconazole is a broad-spectrum antifungal agent known to inhibit CYP3A4 activity (Wen et al., 2024). Consequently, when patients are co-administered ketoconazole and pioglitazone, a medication used for diabetes management, the metabolic rate of pioglitazone is slowed down by ketoconazole (Melillo et al., 2023). This interaction increases pioglitazone’s blood concentrations and a heightened risk of adverse reactions (Wang et al., 2022). Similarly, red yeast rice, a popular herbal remedy for cholesterol reduction, can inhibit CYP3A4 activity. This inhibition is primarily attributed to the component monacolin K, which is structurally analogous to lovastatin. This inhibition impacts the metabolism of statin drugs, such as atorvastatin and simvastatin, leading to elevated blood concentrations of these statins and a potential risk of adverse effects, including myopathy (Raguzzini et al., 2021). Another example of a significant drug interaction involving CYP3A4 is the co-administration of rifampicin, an antibiotic medication, and warfarin, an anticoagulant. Rifampicin induces CYP3A4 activity, which, when combined with warfarin, accelerates the metabolism of the anticoagulant (Fahmi, Abdelsamad & Elewa, 2016). This induction effect reduces warfarin’s blood concentration, potentially diminishing its efficacy and increasing the risk of thrombosis (Mar et al., 2022). In addition, tacrolimus C0/D (concentration/dose) increased 1.8-fold in CYP3A5 expressing individuals (CYP3A5*1/*1 and *1/*3) when used in combination with diltiazem, while it increased 1.3-fold in CYP3A5 non expressing individuals (CYP3A5*3/*3), These individual differences in tacrolimus metabolism, distribution, and responsiveness affect therapeutic efficacy (Yang et al., 2024). These examples illustrate the critical role of CYP3A4 and CYP3A5 in drug metabolism. Clinicians should adjust drug dosages based on a patient’s genotype particularly for drugs with narrow therapeutic indices, select alternative medications with fewer interactions, and implement strategies to monitor patients more closely for adverse effects during concurrent therapy in practical applications. Management strategies could involve the use of clinical decision support systems that incorporate pharmacogenomic data to predict and mitigate potential drug-drug interactions. By integrating these practical applications and management strategies, clinicians can improve drug efficacy and ensure patient medication safety.

Effect of CYP3A4 genetic polymorphisms on drug metabolism

Through in-depth research on CYP3A4 enzymatic activity, alterations in the relevant encoding genes have been found to affect its metabolic capacity for drugs significantly, demonstrating individual differences in drug response. Against this backdrop, a joint research team from the First Affiliated Hospital of Wenzhou Medical University and Wenzhou Medical University explored the impact of CYP3A4 gene polymorphism on the effects of various drugs using methods such as gene screening, gene recombinant expression, and enzymatic kinetic studies, providing a reference for clinical medication.

Chemotherapeutic drugs

Acalabrutinib’s metabolism exhibits significant variability with CYP3A4 variants, such as *3, *11, *29, and *33, as shown in Fig. 2, showing 166% to 599.86% higher activity than the wild type, while *9, *14, *16, *19, *23, *24, *28, and *32 have 9.32–23.91% lower activity (Han et al., 2021a). Osimertinib metabolism is elevated in variants *29, *32, and *33 by 223.43% to 284.52%, contrasting with *2, *7, *8, *12, *13, *14, *15, *16, *17, and *18, which have 25.68% to 48.25% less activity (Gao et al., 2022). Cabozantinib’s metabolism is notably reduced in *6, *20, and *30, with the other 24 variants displaying considerable shifts (Han et al., 2021b). Midostaurin metabolism is increased in *4, *5, *10, *15, *16, *19, *23, *28, *29, *31, *32, and *33 by 101.61% to 253.31%, and decreased in others like *2, *14, *18, and *34 (Xu et al., 2023). Regorafenib metabolism varies, with *20 inactive and *5, *16, *19, *24, and *29 similar to wild type, and the other 20 variants showing significant differences. Ibrutinib is metabolized by variants *28 and *29 at 39–46% of wild type, with *3, *4, *9, *19, and *34 at 117.49% to 291.30% higher activity. Artemether-lumefantrine metabolism is mostly decreased by variants, with artemether clearance reduced to 6.7% to 66.1% and lumefantrine increased to 1.73 to 3.96 times wild type (Zhang et al., 2023). Quinine metabolism is diminished in *8, *12, *13, *17, *20, and *21 to less than 10%, while *15 and *29 have 131.1% and 316.5% higher activity. Vandetanib metabolism is absent in *6 and *30, with *4 at 95.81% of wild type and other variants showing increased or decreased activity.

Endocrine therapy drugs

Azelastine, an antihistamine for allergies, is metabolized by CYP3A4 to its active metabolite, with variants *3, *11, *18, *23 showing 1.98–3.85 times higher activity and *17 showing 17.36% lower activity shown in Fig. 3. Marcitentan, used for pulmonary hypertension, is also metabolized by CYP3A4, with variants *2, *3, *4, *5, *8, *10, *15, *16 having 102–504% higher activity, *7, *14, *18, *28–*32 having 50%-91% lower activity, and *9, *11-*13, *17, *20–*24, *33, *34 showing 5–45% lower activity (Li et al., 2019). Saxagliptin, an anti-diabetic medication, is metabolized by CYP3A4 to 5-hydroxy saxagliptin, with variants *11, *12, *17, *20, *23 having 1.91–7.90% lower clearance, *2 showing 10% lower 5-hydroxy saxagliptin formation, and *3, *4, *7, *8, *9, *13, *16, *18, *24, *29, *31, *32, *34 having 24.16–45.38% lower clearance, and *5, *10, *14, *19, *28, *33 classified as a mild reduction group (Liu et al., 2022).

Central therapeutic drugs

Lidocaine, an antiarrhythmic for ventricular arrhythmias and local anesthesia, is metabolized by CYP3A4 to MEGX, as shown in Fig. 4, with variants *11, *14, *15, *18, *19, *23, *29, *31, *32, *34 showing 123.27–213.61% higher intrinsic clearance, and *2, *5, *9, *16, *24 having 27.93–67.93% lower clearance (Fang et al., 2017). Istradefylline, an A2A receptor antagonist for Parkinson’s, is metabolized by CYP3A4, with *29 showing increased activity, *2, *3, *14, *31, *32 having 50–90% of wild-type activity, and *33, *34 having 35–38%, while *17 is nearly inactive (Hu et al., 2022). Brexpiprazole, an antipsychotic, is metabolized by CYP3A4 to DM-3411, with *2, *3, *7, *8, *9, *10, *11, *12, *13, *16 having 16.54–59.06% lower clearance than the wild type, *17 and *20 nearly inactive, and *14, *15 showing slightly higher, non-significant clearance (Chen et al., 2020). These CYP3A4 polymorphisms can significantly impact drug metabolism, efficacy, and safety, emphasizing the importance of personalized medicine.

Circulatory system drugs

Sildenafil, a PDE5 inhibitor for pulmonary arterial hypertension and erectile dysfunction, is metabolized by CYP3A4 to N-desmethyl sildenafil. As shown in Fig. 5, variants *3, *10, *14, *15, *19, *32, *33 have 110–140% higher metabolic activity, while *24 has significantly reduced activity, nearly inactive, and *2, *5 have about 50% decreased activity (Tang et al., 2020). Amiodarone, a class III antiarrhythmic, is primarily metabolized by CYP3A4 to desethylamiodarone. Variants *17 and *24 have significantly reduced activity (11.07% and 2.67% of wild type), while *34, *15, *31, *29, *32, *18, *14, *10, *23, *19, *2, and *11 have increased activity (155.20~435.96% of wild type), with *2 and *11 showing a 289.48% and 435.96% increase, respectively. The remaining seven alleles, *4, *28, *5, *33, *3, *9, and *16, have similar activity to wild type (Yang et al., 2019).

Effect of CYP3A5 genetic polymorphisms on drug metabolism

CYP3A5 shares 84.1% amino acid homology with CYP3A4, but its expression level in the liver is much lower than that of CYP3A4. To date, 34 variants of CYP3A5 have been identified, of which CYP3A5*6, which arises from a splice variant of 14690G > A, is a nonfunctional protein and is predominantly found in the African American population (Ranasinghe et al., 2024), and CYP3A5*8 and CYP3A5*9 are defective coding alleles that result in a reduction of CYP3A5 activity. CYP3A5*3 is the most studied and common variant of the CYP3A5 gene, which is predominantly found in European populations, and CYP3A5*3 may result in loss of CYP3A5 functional activity (*3/*3), partial deletion (*1/*3) or normal expression (*1/*1). The following pharmacokinetic differences in drugs affected by polymorphisms in the CYP3A5 gene will be explored below.

Atorvastatin is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, which can stabilize atherosclerotic plaques and lower lipid indices by inhibiting HMG-CoA reductase and decreasing cholesterol synthesis in the liver and can lower LDL-C by increasing the number of LDL receptors on the surface of hepatocytes and accelerating cholesterol Atorvastatin is used for the treatment of patients with primary hypercholesterolemia, as it reduces triacylglycerol (TG) and raises HDL-C by increasing the number of LDL receptors on the surface of hepatocytes, and accelerates cholesterol catabolism. Atorvastatin is mainly hydroxylated by CYP3A4 and CYP3A5 as well as CYP2C8 to produce the two main active metabolites, 2-hydroxy (2-OH) atorvastatin and 4-hydroxy atorvastatin. It was shown that the CYP3A5*3 genotype significantly affects the pharmacokinetics of both atorvastatin and 2-OH atorvastatin, and this was confirmed by comparing the CYP3A5*3/*3, CYP3A5*3, and CYP3A5*3 genotypes. *3, CYP3A5*1/*1, and CYP3A5*1/*3 carriers, it was found that the CYP3A5*3/*3 mutase enzyme activity was reduced compared to CYP3A5*1/*1 and that carriers of CYP3A5*3/*3 exhibited more than two-fold higher Cmax and AUCIN of atorvastatin than the wild type (Park et al., 2022).

Fentanyl is an opioid receptor agonist commonly used for all kinds of pain and surgery and other postoperative and surgical procedures of analgesia. It also can be used as an anesthesia adjuvant, but too large of a dose can cause addiction; in May 2019, the Chinese government formally merged fentanyl into the whole class of listed substances.

Fentanyl is metabolized to desmethyl fentanyl and hydroxydesmethylfentanyl primarily through CYP3A4 and CYP3A5.2022. Williams et al. (2022) investigated the effect of CYP3A5 gene polymorphisms on the pharmacokinetics of fentanyl use in children, and found that clearance was not associated with the CYP3A4*1G gene variant but was not associated with CYP3A5*3 or CYP3A5*6 allele variants. There was evidence of loss of physiological function effects for both CYP3A5*3 and CYP3A5*6 variants, and the data indicated that the clearance of fentanyl was lower in carriers of CYP3A5*3/*3, CYP3A5*3/*6, and CYP3A5*6/*6 compared to carriers of CYP3A5*1/*3 and CYP3A5*1/*6, so the study demonstrated that fentanyl was more effective in children than in those with CYP3A5*1/*3 and CYP3A5*1/*6. This study demonstrates that CYP3A5 nonfunctional variants affect fentanyl clearance in the pediatric population (Williams et al., 2022).

Vincristine is an alkaloid extracted from periwinkle that targets microtubules and stops tumor cells from dividing by preventing spindle microtubule formation through inhibition of polymerization of microtubule proteins. It is mainly used for the treatment of childhood lymphoblastic leukemia and non-Hodgkin’s lymphoma. Vincristine metabolism is linked to CYP3A5, and in 2018, Skiles et al. (2018) found that the majority of Kenyan children with a high CYP3A5-expressing genotype (*1/*1) had 58% less exposure to vincristine than Kenyan children with a low-expressing genotype (*3, 6, or 7/*3, 6, or 7), which may lead to the development of child drug resistance, and carriers of the CYP3A5 high-expressing genotype had a significantly reduced chance of developing vincristine-induced peripheral neuropathy (VIPN); therefore, genetic variation in CYP3A5 affects the metabolism of vincristine and susceptibility to toxicity.

Apatinib is a VEGFR2 inhibitor that prevents the generation of new blood vessels in tumor tissues by inhibiting tyrosine kinase activity and is the world’s first orally available small molecule targeted drug that can be safely and effectively used in the treatment of advanced gastric cancer, as well as for the treatment of solid tumors, such as breast, gynecological and lung cancers. Apatinib is metabolized to inactive E-3-hydroxyapatite primarily through CYP3A4 and CYP3A5. The disposition process of apatinib in vivo has large individual differences Yang et al. (2023) established a pharmakinetic (PK) model of adult cancer patients treated with apatinib, which was found that the apparent clearance and apparent volume of distribution of CYP3A5-expression (CYP3A5*1/*1 and CYP3A5*1/*3) were lower than that of CYP3A5-non-expression (CYP3A5*3/*3), while the dose should be 33.33–50.00% lower than in CYP3A5 non-expression (Yang et al., 2023). Therefore, the latter would require a higher dose of the drug to achieve a therapeutic effect during treatment.

Sorafenib is a multi-targeted tyrosine kinase inhibitor that exerts antitumor effects by inhibiting tumor angiogenesis and cell proliferation and is used in the treatment of patients with advanced hepatocellular carcinoma who are not suitable for surgery. CYP3A4 and CYP3A5 mainly metabolize sorafenib in the human body. Its main metabolite exists in the form of pyridine N-oxide and a small number of people may experience adverse reactions such as hypertension, edema, and hand-foot syndrome. Individuals with the CYP3A5*3 variant are less able to metabolize sorafenib compared to the wild type (Guo et al., 2018). Song et al. (2022) found that the enzyme catalytic activities of the CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 variants were all lower than those of the wild-type so that the intrinsic clearance of sorafenib was reduced, while the CYP3A5*6 allele exhibited a higher enzyme catalytic activity than the wild-type. Alleles with high catalytic activity help to inhibit the proliferation of tumor cells and induce their apoptosis (Song et al., 2022).

In addition, CYP3A5 polymorphisms help predict disease susceptibility. Some studies have reported that CYP3A5 polymorphisms are closely associated with prostate cancer, and 3A5 gene polymorphisms can be used as biomarkers to help prevent and treat prostate cancer (Zhenhua et al., 2005). For instance, a meta-analysis including six case-control studies with 2,522 cancer patients and 2,444 healthy controls showed that CYP3A5*3 polymorphisms were significantly associated with an increased risk of prostate cancer under two genetic models (GG + AG vs. AA: OR = 1.53, 95% CI [1.23–1.90], P = 0.000; GG vs. AA: OR = 1.46, 95% CI [1.14–1.87], P = 0.000). This suggests that genetic variations within the CYP3A5 gene can substantially affect an individual’s susceptibility to prostate cancer, emphasizing the importance of considering these genetic factors in clinical practices (Liang et al., 2018). Another study has shown that CYP3A5 regulates prostate cancer cell growth by facilitating nuclear translocation of AR, highlighting the enzyme’s direct role in cancer progression (Mitra & Goodman, 2015). Therefore, when administering drugs in the clinic, appropriate drug therapy should be selected for the mutation of the CYP3A5 allele to achieve individualization of drug dosage, thus improving the efficiency of drug utilization and reducing drug toxicity.

Effect of individual differences on drug metabolism

During drug therapy, individual differences between patients, particularly with drugs that have a narrow therapeutic window, can significantly impact drug plasma concentrations, potentially leading to adverse drug reactions or therapeutic failure. These individual differences mainly encompass gender, age, race, and disease status (Eichelbaum, Ingelman-Sundberg & Evans, 2006). Gender is increasingly recognized as a determinant of pharmacokinetic (PK) and pharmacodynamic (PD) profiles. Reports indicate that females metabolize drugs faster than males, partly due to gender differences in metabolic enzyme activity, which includes a higher level of CYP3A4 protein expression in the liver of females (Yoon et al., 2021). Given that CYP3A4 and CYP3A5 account for over half of the total CYP content in the human liver and metabolize more than half of prescribed drugs (Urban et al., 2018), it is not surprising that clearance of most drugs tends to be higher in women than in men. Additionally, gender differences in renal function, often influenced by body weight, result in a higher glomerular filtration rate in males than in females (Layton & Sullivan, 2019). Age also exerts a crucial influence on drug metabolism. Hepatic levels of total CYP enzymes begin to decline around 40 years old, with a decrease in CYP3A content observed in the liver with increasing age. While substrate metabolism rates for CYP1A2 and CYP2C19 decrease with age, those for CYP2A6, CYP3A4, CYP2C9, and CYP2D6 do not show significant changes (Corton et al., 2022). Furthermore, older age is associated with a reduction in hepatic blood flow by approximately 35% and a decrease in liver size by 24–35% (Wynne, 2005). Consequently, the clearance of liver-metabolized drugs declines with the reduced liver size. Additionally, age-related declines are observed in intestinal absorption, glomerular filtration, tubular secretion, passive reabsorption, renal elimination, and hormonal levels, including gonadal hormones, thyroid hormones, insulin, glucagon, and glucocorticoids. These hormonal changes can affect CYP expression and the functionality of the hormonal system. Racial differences can result in varying capacities for drug metabolism. For instance, midazolam area under the curve (AUC) levels were observed to be 27% higher in Caucasian populations compared to South Asian populations when administered at the same dose, with clearance of midazolam being significantly higher in South Asians than Caucasians (Van Dyk et al., 2018) Genetic variations between racial groups also contribute to differences in metabolic activity. For instance, African Americans exhibit the lowest metabolic activity for warfarin among Caucasian, Asian, and African American populations (Ohara et al., 2019). Moreover, disparities in living environments and dietary habits between races can influence drug metabolism. Disease states frequently impair the ability to metabolize drugs. For example, patients with hypoalbuminemia who utilize drugs with high protein binding exhibit elevated free drug concentrations in comparison to healthy individuals. In conditions such as renal insufficiency and cirrhosis, the capacity to metabolize drugs is diminished.

In conclusion, individual differences, influenced by a multitude of factors, including gender, age, race, and disease status, play a pivotal role in drug metabolism. Consequently, it is of the utmost importance for healthcare professionals to individualize patient treatment in clinical settings, tailor drug administration protocols, and monitor blood concentrations promptly to ensure the efficacy and safety of the drugs administered.

Drug metabolism genetic testing and personalized medication

Cytochromes P450 3A4 and 3A5 are involved in the metabolism of many drugs, and there are many genetic variants of these two enzymes due to, for example, nucleotide mutations. Currently, commonly used methods for detecting polymorphisms in these two genes include DNA sequencing analysis, Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), capillary polymerase chain reaction (CPR-RFLP), and capillary polymerase chain reaction (CPCR). Phism, PCR-RFLP, Capillary Electrophoresis (CE), Amplification Refractory Mutation System PCR (ARMS-PCR), Real-time Reverse Transcriptase-Polymerase Chain Reaction (RT-qPCR) DNA sequencing is the process of chemically sequencing DNA.

DNA sequencing is the conversion of DNA chemical signals into digital signals that computers can process. DNA sequencing has been used for nearly 40 years. Currently, the most commonly used is the second-generation sequencing technology, also known as high-throughput sequencing technology, which is fast and can sequence millions to billions of DNA molecules at the same time in a single experiment through automation and parallelization; it is characterized by short read lengths, usually in the range of 50–600 bp; due to high throughput and automation, its cost per sequencing site is lower; however, its short read lengths may lead to certain regions of repetitive sequences or structural variants are difficult to analyze. Recently, third-generation sequencing (TGS) technologies have shown clear advantages in resolving highly repetitive or polymorphic regions in pharmacogenomics.

PCR-RFLP is a combination of PCR technology, RFLP analysis, and electrophoresis. PCR and the specific endonuclease digests first amplify the target DNA. It cuts the amplified product into fragments of different sizes, which are then separated by gel electrophoresis. Then, the sizes and positions of the various restriction fragments can be distinguished under ultraviolet light. This method is highly accurate and simple but requires manual operation and is not suitable for high-throughput and large-scale sample detection.

CE uses a high-voltage electric field as the driving force and a capillary tube as the separation channel and realizes separation based on the different migration rates of different protein molecules in the electric field. It has a wide range of applications and can be used for genetic diseases caused by gene mutations, oncogenes, and infectious diseases. It also has the advantages of rapid, high throughput, easy quantification, automation, and suitability for analysis of micro samples. At the same time, CE also has some limitations: it is more sensitive to the initial conditions of the problem, the selection of the initial population is more demanding, and due to the complexity of the operation mechanism of CE, the computational complexity of its algorithm is correspondingly higher. It can be seen that all three methods have the disadvantages of complicated operation, longer time-consuming, high cost, low detection sensitivity, and easy to cause false positives or false negatives (Cheng, 2016; Li & Li, 2014; Ribeiro, Martins & Grazina, 2017).

ARMS-PCR technology is based on allele-specific extension reaction, which can be performed only when the 3′ end base of an allele-specific primer is complementary to the base at the mutation site. It is a new method developed on the basis of PCR, which can detect various point mutations and known mutations in DNA. It has the advantages of simple operation, short time, high sensitivity, and low cost. However, ARMS-PCR has low throughput, cannot detect unknown mutations, and is not suitable for typing SNPs with too high or too low GC content near the site.

RT-qPCR is a technique that combines reverse transcription and real-time fluorescent quantitative PCR. The most common methods are fluorescent dye and probe methods, which emit a signal when binding to amplified DNA or RNA molecules, allowing quantification of the target sequence. There is a one-to-one correspondence between the probe and the template, so its probe method has the advantages of high specificity and sensitivity. However, the quenching is difficult to thorough, the background is high, and the detection results are difficult to judge the actual amplification characteristics (Zucha, Kubista & Valihrach, 2021). The fluorescent dye method has no selection of DNA templates and may produce false positive results.

CYP450, as a hemoglobin with many isozymes and isoforms, is responsible for the metabolism of more than two-thirds of exogenous substances. With advances in sequencing technology, the main ones identified so far are CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and CYP3A7 (Naidoo, Chetty & Chetty, 2014). Single nucleotide polymorphisms (SNPs) are major variants on the genome that may lead to increased or decreased enzyme activity, resulting in different drug metabolizing abilities between individuals. The population is categorized into four groups based on the genes carrying different forms of polymorphisms: extensive metabolizers (with normal enzyme functional activity), poor metabolizers (with loss of enzyme functional activity), ultra-rapid metabolizers (with increased enzyme functional activity), and intermediate metabolizers (with decreased enzyme functional activity) (Zanger & Schwab, 2013). Each individual is unique, and therefore, drug class selection and drug dosage must be adapted to genomic diversity and other determinants of drug response in order to minimize toxic reactions and improve therapeutic efficacy.

The role of CYP3A5 in guiding clinical drug use

Genetic variation in the CYP3A5 gene can alter the pharmacokinetics and pharmacodynamics of drugs during clinical drug administration. Loss of CYP3A5 function results in decreased drug clearance and increased blood levels, while gain of CYP3A5 function results in increased drug clearance and decreased blood levels. There is a strong correlation between the expression of CYP3A5 and race, with the allele with the highest prevalence among Asian Koreans being CYP3A5*1/*3, and white patients are more likely to have a CYP3A5*3/*3 non-expressing genotype than black patients.

Tacrolimus is currently the only drug used in the clinic based on recommendations for CYP3A5 genotype guidance. Genetic polymorphisms in CYP3A5 have been reported to affect the immunosuppressive efficacy of tacrolimus in patients after liver transplantation by altering tacrolimus bioavailability and metabolism rate after liver transplantation (Birdwell et al., 2015). In 2015, the Clinical Pharmacogenetics Implementation Consortium (CPIC) published guidelines on the use of pharmacogenomic testing in tacrolimus administration. Tacrolimus is a macrolide immunosuppressive drug used primarily after solid organ and hematopoietic stem cell transplantation. The arrival of effective blood concentrations may be delayed after tacrolimus administration in CYP3A5-expressing populations compared to CYP3A5 non-expressing populations. Therefore, during clinical use of tacrolimus, in order to maintain tacrolimus blood levels at therapeutic levels, CYP3A5 non-expressors require a standard recommended starting dose, whereas CYP3A5-expressors require a higher recommended starting dose. In summary, CYP3A5 guides the clinical use of tacrolimus and makes drug use safer.