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To identify the distribution of CYPs families and subfamilies within the Chinese population, a total of 600 blood samples, including 300 HCC patients and 300 healthy people were collected and DNA was extracted. We utilized allele-specific PCR and long-fragment gene sequencing technique (Supplementary Figures S1 and S2 available in www.besjournal.com). The results revealed that in the Chinese population, the most common member of CYP3A4 family was the wildtype with normal function. In both CYP3A5 and CYP2D6 families, two mutant types with weaker function had the highest expression level. In CYP2C19 family, the wild type and one mutant type was found in about 50% in Chinese population. This indicates that the function of CYP2C19 was evidently decreased in half of the population (Figure 1).
Figure Supplementary Figure S1. Allele-specific PCR for the detection of CYP3A5 gene polymorphism. A. Comparison of allele-specific PCR gel electrophoresis results and sequencing results of different allele loci. B. Allele fluorescence quantitative PCR test results.
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Since CYP450 plays a crucial role in the metabolic process of sorafenib, we assumed that predominantly expressing a mutant CYP genotype might closely influence drug efficacy and frequency of adverse effects. We planned to confirm our hypothesis using animal models. The rat chow was mixed with aflatoxin and fed to rats for 3 months. AFP levels (15 ± 2.5 ng/mL after 22 weeks) in peripheral blood increased significantly with the extension of feeding time compared to the control group (6 ± 0.9 ng/mL). However, after the administration of sorafenib on the 22th week, AFP levels stopped climbing and remain constant (Figure 2A). After comparing ultrasonography images to those from the control group, the HCC rat models were grouped into an early stage group (Ⅰ), an intermediate stage group (Ⅱ) and a terminal stage group (ⅢA and Ⅳ). After 22 weeks and before sorafenib treatment, 105 of 120 rats were induced into HCC successfully with an incidence rate of 87.5%. There were 38, 41, and 26 rats in early stage group (Ⅰ), intermediate stage group (Ⅱ) and terminal stage group (ⅢA and Ⅳ), respectively. However, after sorafenib treatment, most tumors responded to the drug and shrank significantly. After 6 weeks of treatment, only 19 rats remained in the terminal stage group (Figure 2B).
Figure 2. The therapeutic effect of sorafenib in aflatoxin-induced HCC rat model. (A) The changes of AFP levels (ng/mL) in aflatoxin-feeding group and control group before and after sorafenib administration. Sorafenib is administered at the 22th week. Two groups are compared at indicated times. Data are presented as mean ± SEM (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001. (B) Tumor stage progression in aflatoxin-induced HCC rat before and after sorafenib administration. Sorafenib is administered at the 22th week.
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To determine the metabolic efficacy of certain CYP genotypes on sorafenib, we assigned cancer-induced rats into four groups based on their predominant CYP genotype expression (Supplementary Figure S3 available in www.besjournal.com). After receiving sorafenib treatment for eight weeks, the level of sorafenib plasma concentration varied significantly among the aflatoxin-induced HCC model rats with different genotypes of CYPs (Figure 3A). The rat models with the polymorphic form of CYP3A4*1 had the lowest levels of sorafenib plasma concentration (8 ± 2.5 ng/mL), while the rat models with the polymorphic form of CYP3A5*3 had the highest levels (67 ± 4.8 ng/mL) (Figure 3B).
Figure 3. Different CYP genotypes greatly affect the concentration of sorafenib in the blood of rats. (A) Analysis of blood sorafenib concentration change in four different CYP genotype groups. Data are presented as mean ± SEM (n = 10). *P < 0.05; **P < 0.01; ***P < 0.001. CYP3A4*1 is compared with CYP3A5*3 at indicated times. (B) The blood sorafenib concentration of four groups at the 8th week. CYP3A4*1 is compared with the rest 3 groups. Data are presented as mean ± SEM (n = 10). *P < 0.05; **P < 0.01; ***P < 0.001.
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To explore the relationship between polymorphic forms of CYPs and variations in response to sorafenib therapy on rat models, we inspected damage to hepatic and renal function. Parameters related to hepatic injury, ALT and AST showed markedly different trends in different CYP genotypes. Rats in the CYP3A5*3 group showed the most severe liver injury, while rats in the CYP3A4*1 group showed the least severe injury (Figure 4A, 4B, 4F). Consistent with hepatic and renal injury-related parameters, BUN and Cr increased most quickly in the CYP3A5*3 group and slowest in the CYP3A4*1 group (Figure 4C, 4D, 4E).
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In HCC rat models, we confirmed that CYP genetic polymorphism plays an important role in sorafenib-induced organ damage and drug efficacy. To test the clinical significance of our findings, we recruited 51 advanced HCC patients the basic characteristics of the patients were shown in Table 1, 24 of which showed CYP3A5*3 expression dominance. The other 27 showed CYP3A4*1 expression dominance. After sorafenib administration, ALT levels increased quickly in the CYP3A5*3 group, with only little fluctuations in the CYP3A4*1 group (Figure 5A). AST levels exhibited the same tendency (data not shown). However, BUN and Cr showed no marked difference between the two different groups (data not shown), which is not concordant with other results. Intriguingly, rats in the CYP3A4*1 group showed improved survival compared to rats in the CYP3A5*3 group (Figure 5B).
Variables Data Age* 53.88 ± 13.67 Gender (Female, %) 3 (5.88) Preoperative AFP, n (%)* > 20 ng/mL 35 (68.63) ≤ 20 ng/mL 16 (31.37) HBV infection, n (%) 51 (100) HBV DNA, n(%) 32 (62.75) Total bilirubin (μmol/L)* 18.78 ± 8.63 Albumin (g/L)* 42.39 ± 4.20 ALT (U/L)* 72.02 ± 148.19 AST (U/L)* 83.74 ± 133.01 Portal hypertension, n (%) 14 (27.45) Cirrhosis, n (%) 20 (39.22) Tumor size (cm)* 6.90 ± 4.57 Vascular invasion, n (%) 13 (25.49) Tumor capsule, n (%) 20 (39.22) Note. AFP, alpha fetoprotein; HBV, hepatitis B virus; ALT, alanine aminotransferase; AST, aspartate aminotransferase. *Data were not available for all patients. Table 1. Baseline Characteristics of the Patients with HCC (n = 51)
Figure 5. The classification of CYPs in sorafenib treated HCC patients might have great clinical significance. (A) Analysis of blood ALT level in two patient groups with predominant CYP3A4*1 and CYP3A5*3 expression respectively. Data are presented as mean ± SEM (n = 10). *P < 0.05; **P < 0.01; ***P < 0.001. (B) Survival analysis of CYP3A4*1 and CYP3A5*3 patient group.