CYP2C19 polymorphism has no influence on rabeprazole-based hybrid therapy for Helicobacter pylori eradication.

AIM: To evaluate the impact of cytochrome P450 2C19 (CYP2C19) and interleukin-1β (IL-1β) polymorphisms on the efficacy of Helicobacter pylori (H. pylori) eradication by using rabeprazole-based hybrid therapy.
METHODS: A total of 88 H. pylori-infected patients were recruited to receive 14-d of hybrid therapy from March 2013 to May 2014. Three patients were excluded from analysis because of incomplete compliance. Either a follow-up endoscopy or 13C-urea test was performed to determine the results of H. pylori eradication therapy. The genotypes of CYP2C19 and IL-1β were analyzed to investigate the impact on treatment effect.
RESULTS: The total eradication rate of H. pylori was 92.94% (79/85). According to the CYP2C19 genotypes, the rates of H. pylori eradication were 89.19% in extensive metabolizers (EM) and 95.83% in non-EM. The H. pylori eradication rates regarding the IL-1β genotypes were 92.59% in the normal acid secretion group and 93.10% in the low acid secretion group. After multivariable logistic regression analysis, both the genotypes of CYP2C19 and IL-1β had no significant influences on the eradication rates of H. pylori.
CONCLUSION: The CYP2C19 and IL-1β polymorphisms are not significantly independent factors of H. pylori eradication using rabeprazole-based hybrid therapy.

Core tip: In this study, we investigated the efficacy of hybrid therapy as a first-line treatment for Helicobacter pylori eradication and evaluated the independent predictor associated with eradication efficacy, including cytochrome P450 2C19, interleukin-1β (IL-1β)-511 polymorphism and antibiotic resistance. This study is pilot work investigating the impact of the IL-1β-511 polymorphism on the eradication rate of hybrid therapy.

INTRODUCTION

The global infection rate of Helicobacter pylori (H. pylori) is more than 50%. The infection of H. pylori is associated with gastric cancer, gastric ulcer, duodenal ulcer, non-ulcer dyspepsia, chronic gastritis, and mucosa-associated lymphoid tissue lymphoma[1-4]. The efficacy of standard proton pump inhibitor (PPI)-clarithromycin-amoxicillin triple therapy for H. pylori eradication has decreased to an unacceptable level (< 80%) due to rising rates of antibiotic resistance in most countries, especially clarithromycin[4-7]. Therefore, several novel first-line regimens have been proposed, including sequential, concomitant and hybrid therapies.

The hybrid therapy, first proposed by Hsu et al[8], is an effective treatment method, and a 14-d hybrid therapy can achieve a > 95% eradication rate of H. pylori in their study. Hybrid therapy is composed of PPI and amoxicillin for 14 d, and clarithromycin and metronidazole/tinidazole for the final 7 d. This is similar to the hybrid form of the sequential (the first 7 d) and concomitant therapies (the last 7 d). Two recent reports of systematic review and meta-analysis showed that hybrid therapy can achieve similar eradication rates of H. pylori compared with sequential or concomitant therapies[9,10].

The PPI is metabolized by the hepatic cytochrome P450 system, especially CYP2C19[11]. Three different genotypes of CYP2C19, including extensive metabolizers (EM), intermediate metabolizers and poor metabolizers (PM), will have different degrees of PPI metabolism. The PM genotype will result in higher intragastric pH levels and higher effectiveness in H. pylori eradication due to the low pH level of the stomach that may affect the stabilization of acid-labile antibiotics, such as clarithromycin[12]. Therefore, the EM genotype of CYP2C 19 may result in treatment failure for H. pylori eradication[13]. Since rabeprazole is mainly metabolized by a non-enzymatic reaction[14], the CYP2C19 polymorphism may have less influence on the efficacy of rabeprazole-based H. pylori eradication treatment[15].

The interleukin (IL)-1 family of cytokines comprises 11 members, including seven pro-inflammatory agonists (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ) and four defined or putative antagonists [IL-1R antagonist (IL-1Ra), IL-36Ra, IL-37, and IL-38] that exert anti-inflammatory activities[16]. The proinflammatory cytokine IL-1β is a strong inhibitor of gastric acid secretion and is highly expressed in the gastric mucosa of H. pylori-infected patients[17]. Different genotypes of IL-1β have been reported to have different influences on gastric acid secretion[18]. The IL-1β-511 C/T or T/T genotype has low gastric acid secretion and the IL-1β-511 C/C genotype has normal gastric acid secretion. Therefore, the efficacy of H. pylori eradication may be affected by the particular IL-1β-511 genetic polymorphism. One study reported that CYP2C19 genotype-dependent differences in eradication rates of one-week triple therapy were only observed in patients with the IL-1β-511 C/C type[19].

In the initial study of hybrid therapy, no significant factors related to treatment failure were found; however, the CYP2C19 polymorphism was not analyzed[8]. The one study addressing the influence of CYP2C19 by using hybrid therapy as a first-line treatment for H. pylori eradication found that resistance to clarithromycin or metronidazole and poor compliance were the independent factors of treatment failure[20]. Thus, the CYP2C19 polymorphism was not the significant predictor. The goals of our study were to investigate the efficacy of hybrid therapy as a first-line treatment for H. pylori eradication and to evaluate the independent predictor associated with eradication efficacy, including CYP2C19, IL-1β-511 polymorphisms and antibiotic resistance. This study is pilot work investigating the impact of IL-1β-511 polymorphisms on the eradication rate of hybrid therapy.

MATERIALS AND METHODS

Patients

At the out-patient Department of Gastroenterology in Taipei City Hospital, Ren-Ai Branch, patients without a history of H. pylori eradication were recruited consecutively from March 2013 to May 2014. All of the patients received endoscopic examinations, and biopsies of the gastric mucosa was evaluated by rapid urease test, histology and tissue cultures. The infection of H. pylori was defined as either (1) a positive result of culture; or (2) positive results of both the rapid urease test and histological examination. The following patients were excluded: (1) previously treated for H. pylori infection; (2) use of antibiotics within the preceding 30 d; (3) regular use of a PPI (> 3 times per week) in the 30 d before enrollment; (4) allergy to any medication in this study; (5) known to interact with study medication; (6) use of concomitant antibiotics; (7) previous surgery of the stomach; (8) presence of Zollinger–Ellison syndrome; (9) presence of a serious medical condition; and (10) pregnancy or lactation. The study was approved by the Institutional Review Board and ethics committee of Taipei City Hospital (TCHIRB-1011111). Written informed consents were provided by all participants.

Interventions

A total 88 patients with H. pylori infections were included in our study and treated with 14 d of hybrid therapy (20 mg rabeprazole and 1000 mg amoxicillin twice daily for 7 d, followed by 20 mg rabeprazole, 1000 mg amoxicillin, 500 mg clarithromycin and 500 mg metronidazole twice daily for 7 d). A written handout with instructions about how to take the drugs correctly was given to the patients. Medical history and demographic data were obtained by a well-trained interviewer who interviewed the patients by using a standardized questionnaire. Patients were arranged to return to evaluate the drug compliance and adverse events 2 wk after the start of drug administration. Endoscopic examination with biopsy for histology, rapid urease test, and culture was repeated to assess the status of H. pylori infection 8 wk after the completion of H. pylori eradication. If the patient refused an endoscopy during follow-up, the 13C-urea test was alternatively used at least 4 wk after the completion of therapy. H. pylori eradication was defined as (1) a negative result of the 13C-urea test; or (2) negative results of both the rapid urease test and histological examination.

Questionnaires

The questionnaires contained questions regarding personal medical histories and demographic data, including systemic disease, age, gender, alcohol, smoking, tea and coffee consumption. Drinkers were defined as drinking more than one cup of alcoholic beverage per day, and smokers were defined as consuming more than one pack of cigarettes per week. The adverse events included bitter taste, headache, dizziness, nausea, vomiting, anorexia, abdominal pain, diarrhea, constipation, fatigue and skin rash.

Diagnosis of H. pylori infection

Rapid urease test: The results of the rapid urease test (Delta West Bently, Western, Australia) were interpreted as positive if the color of the gel turned pink or red six hours after examination at room temperature.

Histological examination and culture: We performed biopsies from the lesser curvature site of the antrum and corpus of gastric mucosa for histological examination. The biopsy specimens were smeared on the surface of a Columbia blood agar plate and then incubated at 35°C under microaerobic conditions for 5 d. When a curvy, gram-negative bacterium was found on the smear, the Gram stain was defined as a positive result. The pathologists were blinded to the results of the laboratory or genotypic tests as well as to the therapies each patient received. If one or more colonies of Gram-negative bacilli with positive urease, oxidase, and catalase tests were found, the result of the H. pylori culture was defined as positive.

Seventy-five mg 13C-Urea mixed with 100 mL of fresh water was used as the test drink. The 13C-urea was manufactured by the Institute of Wagner Analysen Technik Vertriebs GmbH, Germany.

Analysis of CYP2C19 and IL-1β-511 genotypes

Peripheral blood was drawn in an EDTA vacutainer, and a commercially available kit (Qiagen K.K., Tokyo, Japan) was used to isolate DNA from the leukocytes. The method of polymerase chain reaction–restriction fragment length polymorphism established by de Morais et al[21,22] with minor modifications was performed to analyze the wild-type (wt) gene and the two mutated alleles, CYP2C19 m1 and CYP2C19 m2[21-23]. Homozygous EM (i.e., wild-type) was defined as wt⁄wt; heterozygous EM as wt⁄m1 and wt⁄m2; and PM as m1⁄m1, m2⁄m2 and m1⁄m2, respectively. We also used the method of polymerase chain reaction–restriction fragment length polymorphism with allele-specific primers to identify the C-to-T single nucleotide polymorphism of IL-1β-511[24].

Analysis of antibiotics resistance

To culture H. pylori, we rubbed one antral gastric biopsy specimen on the surface of a Campy-BAP agar plate (Brucella agar, Difco, Sparks Maryland) + IsoVitalex (Gibco, Grand Island, New York) + 10% whole sheep blood. The agar plate then was incubated at 37 °C under microaerobic conditions (5%O2, 10% CO2 and 85%N2) for 4-5 d. Antibiotic susceptibility for the H. pylori strain was tested for clarithromycin, metronidazole and amoxicillin by using an E-test (AB Biodisck, Solna, Sweden). Resistance to clarithromycin, metronidazole, and amoxicillin was defined as a minimal inhibitory concentration value of 1 μg/mL, 8 μg/mL, and 0.5 μg/mL, respectively.

Statistical analysis

Data were summarized as mean ± SD. Data were compared between groups on the basis of H. pylori eradication results. Categorical variables were compared with the chi-square test or Fisher’s exact test as required. Continuous variables were compared between groups by using the unpaired t-test. The Mann-Whitney test was used when appropriate. Multivariable logistic regression analysis was used to identify the independent predictors related to the eradication of H. pylori. A P value < 0.05 was statistically significant.

RESULTS

Baseline demographic data of patients

A total of 88 H. pylori-infected patients were treated with hybrid therapy. Three patients were excluded from analysis because of poor compliance. According to the treatment outcome, baseline demographic data from the 85 patients with complete therapy of H. pylori eradication are shown in Table 1. A total of 79 patients had successful eradication of H. pylori and the eradication rate was 92.94% using 14-d of hybrid therapy.

Table 1

Univariable analysis of the clinical factors and genotyped polymorphisms n (%)

Variable	Eradication (n = 79)	No eradication (n = 6)	P value
Age (yr)	51.95 ± 13.48	53.17 ± 12.06	0.831
Sex (male:female)	35:44	2:4	0.693
Smoking	14 (17.72)	0 (0)	0.583
Alcohol	19 (24.05)	1 (16.67)	1.000
Betel	1 (1.27)	0 (0)	1.000
Coffee	56 (70.89)	4 (66.67)	1.000
Tea	59 (74.68)	4 (66.67)	0.646
NSAID user	7 (8.86)	1 (16.67)	0.458
Steroid user	3 (3.80)	0 (0)	1.000
Anticoagulant user	4 (5.06)	1 (16.67)	0.316
CYP2C19 genotype			0.380
HomoEM	33 (41.77)	4 (66.67)
HeteroEM	36 (45.57)	1 (16.67)
PM	10 (12.66)	1 (16.67)
IL-1β-511 genotype			0.934
CC	25 (31.65)	2 (33.33)
CT	32 (40.51)	2 (33.33)
TT	22 (27.85)	2 (33.33)
Resistance (n = 65)	(n = 61)	(n = 4)
Amoxicillin	0/61 (0)	0/4 (0)
Clarithromycin	8/61 (13.11)	0/4 (0)	1.000
Metronidazole	25/61 (40.98)	1/4 (25.00)	0.644

Data are expressed as mean ± SD or n (%). NSAID: Non-steroid anti-inflammatory drug; CYP2C19: Cytochrome P450 2C19; EM: Extensive metabolizer; PM: Poor metabolizer; IL-1β: Interleukin-1β.

Genotypes of CYP2C19 and single nucleotide polymorphisms of the IL-1β gene (SNP-511)

Three different patterns of CYP2C19 polymorphisms were analyzed in our study, including homozygous EMs, heterozygous EMs and PMs. The prevalence of CYP2C19 homEM, hetEM, PM was 41.77%, 45.57%, 12.66% in patients with eradicated H. pylori and 66.67%, 16.67%, 16.67% in patients without eradicated H. pylori, respectively. In addition, three different allelic patterns of the IL-1β-511 gene were examined, including C/C, C/T, and T/T. The CC/CT/TT genotype frequency was 31.65%, 40.51%, 27.85% in patients with eradicated H. pylori and 33.33%, 33.33%, 33.33% in patients without eradicated H. pylori, respectively (Table 1).

Factors associated with H. pylori eradication

No significant clinical or genetic factors were found to be associated with successful eradication of H. pylori by univariable analysis, including age, gender, coffee/tea drinking, alcohol drinking, betel using, use of steroid, anticoagulant or non-steroid anti-inflammatory drug, antibiotic resistance, or CYP2C19 and IL-1β-511 polymorphisms (Table 1). The rates of H. pylori eradication were 89.19% in EM and 95.83% in non-EM (Table 2). Patients were classified into two groups according to IL-1B-511 genetic polymorphisms. The normal acid secretion group was defined as those with the alleles (C/C), and the low acid secretion group was defined as those with either the alleles (T/T) or (C/T). The cure rates of each IL-1β-511 genetic polymorphism in relation to the CYP2C19 genotype are showed in Table 2. The rates of H. pylori eradication were 92.59% in the normal acid secretion group and 93.10% in the low acid secretion group. There was no statistically significant difference in the eradication rates of H. pylori between the two CYP2C19 genotype subgroups (EM and non-EM) for both normal acid (IL-1β-511 C/C) and low acid (IL-1β-511 C/T and T/T) secretion groups. After multivariable analysis, both CYP2C19 and IL-1β-511 genetic polymorphisms were not significant factors of H. pylori eradication by using 14-d of hybrid therapy (Table 3).

Table 2

Eradication rates according to cytochrome P450 2C19 and interleukin-1β genotypes n (%)

Hybrid therapy	(n = 85)	IL-1β-511 C/C (normal gastric acid) (n = 27)	IL-1β-511 C/T, T/T (low gastric acid) (n = 58)
	79/85 (92.94)	25/27 (92.59)	54/58 (93.10)
CYP2C19 EM (n = 37)	33/37 (89.19)	13/14 (92.86)	20/23 (86.96)
CYP2C19 PM and hetero EM (n = 48)	46/48 (95.83)	12/13 (92.31)	34/35 (97.14)
P value	0.649	1.000	0.556

CYP2C19: Cytochrome P450 2C19; IL-1β: Interleukin-1β; EM: Extensive metabolizer; PM: Poor metabolizer.

Table 3

Multivariable logistic regression analysis of independent predictors of Helicobacter pylori eradication rates

Variable	Odds ratio	95%CI	P value
EM vs PM and hetero EM	0.359	0.062–2.075	0.252
IL-1β-511 C/C vs IL-1β-511 C/T, T/T	1.047	0.175–6.251	0.960

EM: Extensive metabolizer; PM: Poor metabolizer; IL-1β: Interleukin-1β.

DISCUSSION

The failure of H. pylori eradication is mainly related to antibiotic resistance, poor compliance of patients, and duration of therapy[25]. The present study was performed to investigate the eradication rate of H. pylori by using 14-d of hybrid therapy. In addition, the main purpose of our study was to further explore the influence of CYP2C19 and IL-1β-511 genotypes on the outcome of hybrid therapy. Therefore, patients with poor compliance were excluded from analysis.

In one review article, a total of 1871 patients in 12 studies received hybrid therapy[26]. The eradication rate of H. pylori was 82.6%-99.1%, and pooled analysis showed the eradication rate was 91.2% in per-protocol analyses. The other review article with meta-analysis included 2516 patients from eight studies, and the mean cure rate of hybrid therapy was 93.3% (n = 1109, range: 85.7%-99.1%) by per-protocol analyses[27]. Our study found that the eradication rate was 92.94% by using 14-d of hybrid therapy, which was comparable to the results of the two review articles. Nonetheless, one study in the population with high antibiotic resistance rates found that the eradication rate of hybrid therapy was 86.0% in per-protocol analyses and graded as an unacceptable level[20]. In addition, the study showed that resistance to clarithromycin, resistance to metronidazole and poor compliance were the significant predictors of treatment failure for hybrid therapy and that the CYP2C19 genotype was not. In our study, no independent factors were found to be associated with treatment failure. The first key factor was compliance; however, we excluded patients with poor compliance from the beginning. Secondarily, because hybrid therapy achieved a high eradication rate in our study, the number of patients with treatment failure was too small to identify the significant predictors. Two other studies also explored the influence of antibiotic resistance on the treatment outcome of hybrid therapy[8,28]. Both of the studies had not demonstrated the significance of antibiotic resistance on the eradication rate of H. pylori. This may be related to the lack of antimicrobial susceptibility of H. pylori for most patients included in the studies. The study revealed that a compliance of more than 80% was the only significant factor of successful eradication[28]. However, the first study of 14-d hybrid therapy found that no risk factors, including compliance, influenced the efficacy of H. pylori eradication[8]. Another study of 10-d hybrid therapy also showed that no clinical factors were associated with treatment failure, however antibiotic resistance and CYP2C19 genotype were not investigated in the study[29].

The key finding of our study was that CYP2C19 and IL-1β-511 genotypes had no influence on the treatment outcome of 14-d hybrid therapy. To date, only one study had examined the influence of the CYP2C19 genotype on hybrid therapy[20]. This study showed no significant effect of CYP2C19 polymorphisms on the eradication rate of H. pylori. In one study of 12-d reverse hybrid therapy, the CYP2C19 genotype also had no significant impact on the treatment outcome[30]. The limitation of our study was that the number of patients may be too small to identify the significant factors predicting eradication failure.

Both CYP2C19 and IL-1β polymorphisms had no significant impact on rabeprazole-based hybrid therapy. Our findings suggest that rabeprazole may be used as a priority in EMs of CYP2C19 to maintain the eradication rate of H. pylori.

ARTICLE HIGHLIGHTS

Research background

In the initial study of hybrid therapy, no significant factors related to treatment failure were found; however, the cytochrome P450 2C19 (CYP2C19) polymorphism was not analyzed. Only one study addressed the influence of CYP2C19 by using hybrid therapy as a first-line treatment for Helicobacter pylori (H. pylori) eradication.

Research objectives

The aims of this study were to investigate the efficacy of hybrid therapy as a first-line treatment for H. pylori eradication, and to evaluate the independent predictors associated with eradication efficacy, including CYP2C19, the interleukin (IL)-1β-511 polymorphism, and antibiotic resistance.

Research methods

About 88 H. pylori-infected patients were recruited to receive 14-d of hybrid therapy. Endoscopies or 13C-urea tests were performed to determine the results of H. pylori eradication therapy. To investigate the impact on treatment effect, the genotypes of CYP2C19 and IL-1β were analyzed.

Research results

The total eradication rate of H. pylori was 92.94%. The rates of H. pylori eradiation were 89.19% in extensive metabolizers (EM) and 95.83% in non-EM, according to the CYP2C19 genotypes. Both the genotypes of CYP2C19 and IL-1β had no significant influence on the eradication rates of H. pylori.

Research conclusions

The CYP2C19 and IL-1β polymorphisms are not significantly independent factors on rabeprazole-based hybrid therapy for H. pylori eradication.

Research perspectives

The limitation of this study was that the number of patients may be too small to identify the significant factors predicting eradication failure. In addition, the findings suggest that rabeprazole may be used as a priority in EMs of CYP2C19 to maintain the eradication rate of H. pylori.